Neutralizing molecules to influenza viruses

Abstract

The present invention concerns methods and means for identifying, producing, and engineering neutralizing antibodies against influenza A viruses, and to the neutralizing antibodies produced. In particular, the invention concerns neutralizing antibodies against various influenza A virus subtypes, and methods and means for making such antibodies.

1 . A binding molecule that comprises one, two, or three hypervariable region sequences from a heavy chain selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, or a functionally active fragment thereof. 
     
     
         2 . A binding molecule that comprises one, two, or three hypervariable region sequences from a heavy chain selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, or a functionally active fragment thereof. 
     
     
         3 . The binding molecule of  claim 1  which comprises all hypervariable region sequences from SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, or a functionally active fragment thereof. 
     
     
         4 . The binding molecule of  claim 2  which comprises all hypervariable region sequences from SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12, or a functionally active fragment thereof. 
     
     
         5 . The binding molecule of  claim 1  which is capable of binding a target when associated with a light chain. 
     
     
         6 . The binding molecule of  claim 2  which is capable of binding a target when associated with a light chain. 
     
     
         7 . The binding molecule of  claim 5  wherein the light chain comprises one, two or three hypervariable sequences of the polypeptide sequence of SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15, or a functionally active fragment thereof. 
     
     
         8 . The binding molecule of  claim 5  wherein the light chain comprises one, two or three hypervariable sequences of the polypeptide sequence of SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18, or a functionally active fragment thereof. 
     
     
         9 . The binding molecule of  claim 5  wherein the light chain comprises one, two or three hypervariable sequences of the polypeptide sequence of SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21. 
     
     
         10 . The binding molecule of  claim 6  wherein the light chain comprises one, two or three hypervariable sequences of the polypeptide sequence of SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24, or a functionally active fragment thereof. 
     
     
         11 . The binding molecule of  claim 7  which comprises all hypervariable region sequences SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15, or a functionally active fragment thereof. 
     
     
         12 . The binding molecule of  claim 7  which comprises all hypervariable region sequences SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18, or a functionally active fragment thereof 
     
     
         13 . The binding molecule of  claim 7  which comprises all hypervariable region sequences SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21, or a functionally active fragment thereof. 
     
     
         14 . The binding molecule of  claim 8  which comprises all hypervariable region sequences SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24, or a functionally active fragment thereof. 
     
     
         15 . The binding molecule of  claim 1  or  2  further comprising a polypeptide comprising a VpreB sequence and/or a λ5 sequence, or a functionally active fragment thereof. 
     
     
         16 . The binding molecule of  claim 1  or  2 , further comprising a polypeptide comprising a VpreB sequence fused to a λ5 sequence, or a functionally active fragment thereof. 
     
     
         17 . The binding molecule of  claim 1  or  2 , further comprising a κ-like surrogate light chain (SLC) construct comprising a Vκ-like and/or a JCκ sequence, or a functionally active fragment thereof. 
     
     
         18 . The binding molecule of  claim 1  or  2  which is an antibody or a functionally active fragment thereof. 
     
     
         19 . A binding molecule comprising a heavy chain, the heavy chain comprising the amino acid sequence shown as SEQ ID NO:1, or a functionally active fragment thereof. 
     
     
         20 . A binding molecule comprising a heavy chain, the heavy chain comprising the amino acid sequence shown as SEQ ID NO: 2, or a functionally active fragment thereof. 
     
     
         21 . The binding molecule of  claim 19 , further comprising a light chain comprising the amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, or a functionally active fragment thereof. 
     
     
         22 . The binding molecule of  claim 20 , further comprising a light chain comprising the amino acid sequence shown as SEQ ID NO:6, or a functionally active fragment thereof. 
     
     
         23 . The binding molecule of  claim 19 , or functionally active fragment thereof, which (i) neutralizes more than one subtype and/or more than one isolate of an influenza A virus, (ii) binds to a hemagglutinin (HA) antigen of the virus, and (iii) inhibits hemagglutination. 
     
     
         24 . The binding molecule of  claim 20 , or functionally active fragment thereof, which (i) neutralizes at least one subtype and/or at least one isolate of an influenza A virus, and (ii) binds to a hemagglutinin (HA) antigen of the virus. 
     
     
         25 . The binding molecule of  claim 19  or  20 , or functionally active fragment thereof, which binds to an epitope of an H1 subtype of the HA antigen. 
     
     
         26 . The binding molecule of  claim 19  or  20 , or functionally active fragment thereof, which binds to an epitope of an H3 subtype of the HA antigen. 
     
     
         27 . The binding molecule of  claim 19  or  20 , or functionally active fragment thereof, which binds to an epitope of an H1 subtype of the HA antigen and to an epitope of an H3 subtype of the HA antigen. 
     
     
         28 . The binding molecule of  claim 19  or  20 , or functionally active fragment thereof, which binds to an epitope of an H9 subtype of the HA antigen. 
     
     
         29 . The binding molecule of  claim 20 , or functionally active fragment thereof, which binds to an epitope of an H5 subtype of the HA antigen. 
     
     
         30 . The binding molecule of any one of  claims 25 - 29 , or functionally active fragment thereof, wherein the epitope is displayed on the surface of an influenza A virus. 
     
     
         31 . The binding molecule of  claim 19  or  20 , or functionally active fragment thereof, which is cross-reactive with an H1 HA antigen and an H3 antigen. 
     
     
         32 . The binding molecule of  claim 19 , or functionally active fragment thereof, which neutralizes at least one of the H1 and H3 influenza A virus subtypes. 
     
     
         33 . The binding molecule of  claim 19 , or functionally active fragment thereof, which neutralizes the H1 and H3 influenza A virus subtypes. 
     
     
         34 . The binding molecule of  claim 20  which neutralizes the H1 HA antigen, or functionally active fragment thereof. 
     
     
         35 . The binding molecule of  claim 20  which neutralizes the H5 HA antigen, or functionally active fragment thereof. 
     
     
         37 . The binding molecule of  claim 20  which neutralizes the H1 and H5 influenza A virus subtypes, or functionally active fragment thereof. 
     
     
         38 . The binding molecule of  claim 25  wherein said H1 subtype is, or the HA is from, a New Calcdonia/20/99 isolate of the H1 virus, or functionally active fragment thereof. 
     
     
         39 . The binding molecule of  claim 25  wherein said H1 subtype is, or the HA is from, a Solomon Islands/3/06 isolate of the H1 virus, or functionally active fragment thereof. 
     
     
         40 . The binding molecule of  claim 26  wherein said H3 subtype is, or the HA is from, a Wisconsin/67/05 isolate of the H3 virus, or functionally active fragment thereof. 
     
     
         41 . The binding molecule of  claim 26  wherein said H3 subtype is, or the HA is from, a Hong Kong/68 isolate of the H3 virus, or functionally active fragment thereof. 
     
     
         42 . The binding molecule of  claim 28  wherein said H9 subtype is, or the HA is from, a Hong Kong/1073/99 isolate of the H9 virus, or functionally active fragment thereof. 
     
     
         43 . The binding molecule of  claim 29  wherein said H5 subtype is, or the HA is from, a Vietnam/1203/04 isolate of the H5 virus, or functionally active fragment thereof. 
     
     
         44 . The binding molecule of  claim 23 , which prevents the globular head region of the influenza A virus from binding the surface of a cell, or functionally active fragment thereof. 
     
     
         45 . The binding molecule of  claim 23 , which prevents the influenza A virus from attaching to a cell to be infected, or functionally active fragment thereof. 
     
     
         46 . The binding molecule of  claim 23  or  24  wherein at least one of said viruses has the ability to infect humans, or functionally active fragment thereof. 
     
     
         47 . The binding molecule of  claim 23  or  24  wherein at least one of said isolates has been obtained from a human subject, or functionally active fragment thereof. 
     
     
         48 . The binding molecule of  claim 23  or  24  wherein at least one of said isolates has been obtained from a non-human animal, or functionally active fragment thereof. 
     
     
         49 . The binding molecule of  claim 45  wherein said non-human animal is a bird, or functionally active fragment thereof. 
     
     
         50 . The binding molecule of  claim 46  wherein said bird is a wild-fowl or a chicken, or functionally active fragment thereof. 
     
     
         51 . The binding molecule of  claim 45  wherein said non-human animal is a pig, or functionally active fragment thereof. 
     
     
         52 . The binding molecule of  claim 19  which binds to an H1 HA antigen, or functionally active fragment thereof. 
     
     
         53 . The binding molecule of  claim 52  which binds to at least one additional HA antigen, or functionally active fragment thereof. 
     
     
         54 . The binding molecule of  claim 53  wherein said additional HA antigen is H3, or functionally active fragment thereof. 
     
     
         55 . An antibody which binds essentially the same epitope as the epitope for an antibody comprising a heavy chain polypeptide comprising an amino acid sequence shown as SEQ ID NO:1; or a consensus or variant sequence based upon said amino acid sequence. 
     
     
         56 . The antibody of  claim 55  which binds essentially the same epitope as the epitope for an antibody comprising a light chain polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5; or a consensus or variant sequence based upon said amino acid sequence. 
     
     
         57 . An antibody which binds essentially the same epitope as the epitope for an antibody comprising a heavy chain polypeptide comprising an amino acid sequence shown as SEQ ID NO:2; or a consensus or variant sequence based upon said amino acid sequence. 
     
     
         58 . The antibody of  claim 57  which binds essentially the same epitope as the epitope for an antibody comprising a light chain polypeptide comprising an amino acid sequence shown as SEQ ID NO:6; or a consensus or variant sequence based upon said amino acid sequence. 
     
     
         59 . The antibody of  claim 55  or  56  which (i) neutralizes more than one subtype and/or more than one isolate of an influenza A virus, (ii) binds to a hemagglutinin (HA) antigen of the virus; and (iii) inhibits hemagglutination. 
     
     
         60 . The antibody of  claim 57  or  58 , which (i) neutralizes more than one subtype and/or more than one isolate of an influenza A virus, and (ii) binds to a hemagglutinin (HA) antigen of the virus. 
     
     
         61 . A composition comprising a binding molecule or an antibody according to any one of  claims 1 - 60 . 
     
     
         62 . A method for the prevention and/or treatment of an influenza A virus infection in a subject comprising administering to said subject an effective amount of a composition of  claim 61 . 
     
     
         63 . A method for treating influenza A virus infection in a subject comprising administering to said subject an effective amount of a neutralizing antibody or binding molecule of any one of  claims 1 - 60 . 
     
     
         64 . The method of  claim 62  or  63  wherein said subject is a human patient. 
     
     
         65 . A vaccine effective against influenza A virus comprising a peptide or polypeptide functionally mimicking a neutralization epitope bound by an antibody or binding molecule according to any one of  claims 1 - 60 . 
     
     
         66 . The vaccine of  claim 65  which is a synthetic vaccine. 
     
     
         67 . The vaccine of  claim 65  which comprises an attenuated influenza A virus, or a part thereof. 
     
     
         68 . The vaccine of  claim 65  which comprises a killed influenza A virus, or part thereof. 
     
     
         69 . The vaccine of  claim 65 , wherein said antibody or binding molecule is selected from the group consisting of
 (a) an antibody or binding molecule which binds essentially the same epitope as the epitope for an antibody comprising a heavy chain polypeptide comprising an amino acid sequence shown as SEQ ID NO:1; or a consensus or variant sequence based upon said amino acid sequences;   (b) an antibody comprising a heavy chain polypeptide comprising a heavy chain polypeptide comprising an amino acid sequence shown as SEQ ID NO:1; or a consensus or variant sequence based upon said amino acid sequence;   (c) an antibody or binding molecule which binds essentially the same epitope as the epitope for an antibody comprising a heavy chain polypeptide comprising an amino acid sequence shown as SEQ ID NO:2; or a consensus or variant sequence based upon said amino acid sequences, or a fragment thereof; and   (d) an antibody comprising a heavy chain polypeptide comprising an amino acid sequence shown as SEQ ID NO:2; or a consensus or variant sequence based upon said amino acid sequences, or a fragment thereof.   
     
     
         70 . The vaccine of  claim 65  wherein said antibody or binding molecule binds an HA antigen. 
     
     
         71 . The vaccine of  claim 70  wherein said HA antigen is an H3 subtype. 
     
     
         72 . The vaccine of  claim 70  wherein the HA antigen is an H1 subtype. 
     
     
         73 . The vaccine of  claim 70  wherein the HA antigen is an H1 subtype and an H3 subtype. 
     
     
         74 . The vaccine of  claim 70  wherein the HA antigen is an H5 subtype. 
     
     
         75 . The vaccine of  claim 70  wherein the HA antigen is an H9 subtype. 
     
     
         76 . The vaccine of  claim 70  wherein the antigen is displayed on the surface of an influenza A virus. 
     
     
         77 . The vaccine of any one of  claims 65  to  76  wherein said peptide or polypeptide comprises antigenic determinants that raise neutralizing antibodies. 
     
     
         78 . The vaccine of  claim 65  suitable for oral administration. 
     
     
         79 . The vaccine of  claim 65  suitable for transmucosal delivery. 
     
     
         80 . The vaccine of  claim 79  wherein the transmucosal delivery is intra-nasal administration. 
     
     
         81 . The vaccine of  claim 65  suitable for parenteral administration. 
     
     
         82 . The vaccine of  claim 65  suitable for transdermal administration. 
     
     
         83 . The vaccine of  claim 65  wherein the vaccine is for childhood immunization. 
     
     
         84 . A neutralizing antibody or binding molecule binding to a hemagglutinin of an influenza A virus having the ability to infect humans neutralizing at least one isolate of an influenza A virus comprising a length-modified heavy chain loop. 
     
     
         85 . The antibody or binding molecule of  claim 84 , wherein the extended heavy chain loop is a CDR3 loop. 
     
     
         86 . The antibody or binding molecule of  claim 85 , wherein the CDR3 loop comprises an amino acid sequence SEQ ID NO:9. 
     
     
         87 . The antibody or binding molecule of  claim 84 , wherein the extended heavy chain loop is a CDR1 loop. 
     
     
         88 . The antibody or binding molecule of  claim 87 , wherein the CDR1 loop comprises an amino acid sequence SEQ ID NO:7. 
     
     
         89 . The antibody or binding molecule of  claim 84 , wherein the length modification comprises an extended heavy chain loop. 
     
     
         90 . The antibody or binding molecule of  claim 84 , wherein the length modification comprises a deletion in the heavy chain loop. 
     
     
         91 . An engineered antibody or binding molecule with reduced oxidative potential binding to a hemagglutinin of an influenza A virus having the ability to infect humans neutralizing at least one isolate of an influenza A virus comprising one or more substitutions for methionine in a heavy chain variable domain. 
     
     
         92 . The engineered antibody or binding molecule of  claim 91  wherein the heavy chain variable domain is a CDR3 region. 
     
     
         93 . The engineered antibody or binding molecule of  claim 91  wherein the substitution for methionine is at position 96 according to Kabat numbering system. 
     
     
         94 . The engineered antibody or binding molecule of  claim 91  wherein the substitution for methionine is at position 98 according to Kabat numbering system. 
     
     
         95 . The engineered antibody or binding molecule of  claim 91  wherein the substitution for methionine is at positions 96 and 98 according to Kabat numbering system. 
     
     
         96 . The engineered antibody or binding molecule of any one of  claims 91  to  95  wherein the methionine is substituted with a leucine. 
     
     
         97 . The engineered antibody or binding molecule of  claim 91 , wherein the heavy chain variable domain comprises an amino acid sequence SEQ ID NO:29.