# Patent

## Title
RSV G protein specific antibodies

## Abstract

The disclosure relates to human isolated, synthetic or recombinant antibodies or functional parts thereof, specific for the RSV G protein. Antibodies specific for the RSV G protein are particularly suitable for counteracting RSV and symptoms, such as inflammation, resulting from an RSV infection. The disclosure further relates to the use of such RSV G-specific antibodies for diagnosis of an RSV infection and as a medicament and/or prophylactic agent for, at least in part, treating or alleviating symptoms of a Respiratory Syncytial Virus infection and/or a Respiratory Syncytial Virus related disorder.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a national phase entry under 35 U.S.C. §371 of International Patent Application PCT/NL2012/050812, filed Nov. 16, 2012, designating the United States of America and published in English as International Patent Publication WO2013/095091 A2 on Jun. 27, 2013, which claims the benefit under Article 8 of the Patent Cooperation Treaty to European Patent Office Application Serial No. 11189613.0, filed Nov. 17, 2011, the disclosure of each of which is hereby incorporated herein in its entirety by this reference.
STATEMENT ACCORDING TO 37 C.F.R. §1.821(c) or (e)—SEQUENCE LISTING SUBMITTED AS A TXT AND PDF FILES
Pursuant to 37 C.F.R. §1.821(c) or (e), files containing a TXT version and a PDF version of the Sequence Listing have been submitted concomitant with this application, the contents of which are hereby incorporated by reference.
TECHNICAL FIELD
The disclosure relates to the fields of biology, immunology and medicine.
BACKGROUND
Respiratory Syncytial Virus (RSV) is a common cold virus belonging to the family of paramyxovirus. RSV is virulent, easily transmissible and the most common cause of lower respiratory tract disease in children of less than 2 years of age. Up to 98% of children attending day care will be infected in a single RSV season. Between 0.5% and 3.2% of children with RSV infection require hospitalization. Approximately 90,000 hospital admissions and 4500 deaths per year were reported in United States. Major risk factors for hospitalization due to RSV are premature birth, chronic lung disease, congenital heart disease, compromised immunity, and age younger than 6 weeks in otherwise healthy children.
Two subtypes of RSV have been identified, subtype A and subtype B. RSV has two major surface glycoproteins, the fusion protein (F protein) and the attachment protein (G protein). The F protein of RSV is a viral membrane protein and responsible for fusion of the virion with a host cell after attachment. In addition, infection of neighboring cells through the formation of syncytia is promoted by the F protein and its function is thought to depend on the original oligomeric structure of the protein. The G protein is a 89 kD protein, which is also known as the attachment protein. The G protein differs considerably between the two RSV subtypes, whereas the F protein is more conserved. Approximately 53% homology is present within a G protein from subtypes A and B. Although G protein is not required for infection of host cells, anti-RSV G antibodies have shown to improve symptoms in animal models and can induce virus neutralization in the presence of complement.
Antibodies against the F or G protein of RSV have been described. Palivizumab is a genetically engineered, humanized monoclonal antibody against the F protein. WO 2008/147196 discloses sequences of human RSV F protein binding molecules. A mouse monoclonal antibody (131-2G) against the G protein has been described, which is thought to bind to a CX3C (fractalkine) motif in the RSV G protein, which motif is capable of binding to the CX3CR1 (Fractalkine) receptor on NK cells, T cells and monocytes. This antibody was demonstrated to reduce migration of PBMCs towards RSV G glycoprotein (Tripp et al., 2001, Nat. Immunol. 2001, 2(8):732-8). Antibody 131-2G does not neutralize RSV in vitro, however, in an in vivo mouse model dosing at 300 mg/mouse resulted in reduced RSV A2 recovery from lungs, reduced pulmonary inflammation, and lowered IFN-gamma levels in a mouse model. Human monoclonal antibodies against RSV G protein have been described in U.S. 2010-0285022, WO 2009/055711 and Collarini et al. (Journal of Immunology, 2009, 183: 6338-6345). The antibodies bind to a conserved epitope in the G protein close to the CX3C domain, which is located in a region of the G protein corresponding to amino acid positions 164-172.
No effective treatment of RSV positive bronchiolitis beside supportive care in the form of adequate nutrition and oxygen therapy is currently available. Antiviral therapies such as Ribavirin have not been proven to be effective in RSV infection. Only monoclonal antibody palivizumab (also called Synagis), is registered for prophylaxis against RSV infection. However, palivizumab is not always effective. It is only useful and approved for prophylactic treatment of premature infants up to 4 KG body weight. Thus, palivizumab cannot be used to treat an established RSV infection. Furthermore, palivizumab is only partly effective as it reduces hospitalization of infants by approximately 50%.
Therefore, there is a need for additional antibodies and therapies against RSV.
BRIEF SUMMARY
It is an object of the present disclosure to provide additional antibodies against the G protein of RSV, or functional equivalents of such antibodies and compositions comprising antibodies. Preferably, antibodies are provided which recognize a different epitope as compared to known RSV antibodies. It is a further object to provide antibodies against the G protein of RSV, which are able to potentiate neutralizing activity of an antibody capable of binding an F protein of RSV.
The disclosure, therefore, provides a human isolated, synthetic or recombinant antibody or functional part thereof, or immunoglobulin chain or functional equivalent thereof, capable of binding an epitope of a G protein of Respiratory Syncytial Virus, which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of the G protein, wherein the numbering of amino acids is based on the RSV G protein of subtype A2 and B1 as depicted in FIG. 1.
A “functional part of an antibody” is defined as a part which has at least one shared property as the antibody in kind, not necessarily in amount. The functional part is capable of binding the same antigen or epitope as the antibody, albeit not necessarily to the same extent. A functional part of an antibody preferably comprises a single domain antibody, a single chain antibody, a nanobody, an unibody, a single chain variable fragment (scFv), a Fab fragment or a F(ab′)2 fragment.
A functional part of an antibody is also produced by altering an antibody such that at least one property, preferably an antigen-binding property, of the resulting compound is essentially the same in kind, not necessarily in amount. This is done in many ways, for instance, through conservative amino acid substitution, whereby an amino acid residue is substituted by another residue with generally similar properties (size, hydrophobicity, etc.), such that the overall functioning is likely not to be seriously affected.
A “functional equivalent of an immunoglobulin chain” is defined herein as an artificial binding compound, comprising at least one CDR sequence of an immunoglobulin chain.
The term “a human isolated, synthetic or recombinant antibody or functional part thereof, or immunoglobulin chain or functional equivalent thereof” includes isolated human antibodies or functional parts or immunoglobulins or functional equivalents thereof, as well as synthetic or recombinant antibodies or functional parts or immunoglobulins or functional equivalents thereof, the sequence of which is derived from the sequence of human antibodies.
Isolated, synthetic or recombinant antibodies or functional parts thereof, or immunoglobulin chains or functional equivalents thereof, capable of binding to a G protein of Respiratory Syncytial Virus, described herein, are also referred to as “RSV G-specific antibodies, according to the disclosure.”
An RSV G-specific antibody, according to the disclosure, is preferably a human antibody. The use of human antibodies for prophylaxis and therapy in humans diminishes the chance of side-effects due to an immunological reaction in a human individual against non-human sequences. In another embodiment, an RSV G-specific antibody, according to the disclosure, is a humanized antibody. Humanized antibodies are made by incorporating non-human hypervariable domains into human antibodies and, therefore, immunogenic properties are diminished as compared to fully non-human antibodies. In another embodiment, an RSV G-specific antibody, according to the disclosure, is a chimeric antibody. In a chimeric antibody, sequences of interest, such as, for instance, a binding site of interest, are included into an RSV G-specific antibody, according to the disclosure. FIG. 1 shows the amino acid sequence of RSV G protein of subtypes A2 and B1. If a part of RSV G protein is indicated herein by the amino acid residues of which the part consists, the numbering is based on the numbering shown in FIG. 1 and includes corresponding amino acid residues in G proteins of other RSV strains.
In one embodiment, RSV G-specific antibodies, according to the disclosure, are capable of binding an epitope of a G protein of RSV, which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of the G protein. This provides the advantage that they bind to a different epitope as compared to previously disclosed RSV G antibodies. For instance, RSV G antibodies disclosed in U.S. 2010-0285022 bind to multiple but different epitopes of the G protein of RSV, which epitopes are located between amino acids 160-176 of the G protein. The CX3C motif is located between amino acids 182-186, as is shown in FIG. 1. Thus, several preferred RSV G-specific antibodies, according to the disclosure, bind a different epitope of the G protein than the epitope of antibodies disclosed in U.S. 2010-0285022 and the CX3C motif. RSV G-specific antibodies, according to the disclosure, that bind to a different epitope as compared to known RSV G antibodies are thus advantageously combined with such known antibodies in order to improve the treatment with antibodies. Such RSV G-specific antibodies, according to the disclosure, and such known antibodies do not compete for the same epitope in the G protein.
An RSV G-specific antibody, according to the disclosure, capable of binding an epitope of a G protein of RSV, which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of the G protein is particularly suitable for combination with one or more known RSV G binding antibodies. Such preferred RSV G-specific antibodies, according to the disclosure, are also particularly suitable for combination with one or more other RSV G-specific antibodies, according to the disclosure, that are capable of binding other epitopes of RSV G protein, such as, for instance, a conformational epitope or an epitope, which comprises the CX3C motif of the RSV G protein.
RSV G-specific antibodies, according to the disclosure, which are capable of binding an epitope of a G protein of RSV, which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of the G protein, and thus are particularly preferred, are the antibodies designated AT35, AT37, AT39, AT43, AT51, AT47, AT32, AT33, AT36 and AT50, which have heavy chain sequences of SEQ ID NOS:133, 115, 116, 119, 125, 122, 110, 111, 114 and 124 as depicted in table 1, respectively, and light chain sequences of SEQ ID NOS:131, 133, 134, 137, 143, 140, 128, 129, 132 and 142 as depicted in table 1, respectively. The heavy and light chain CDR sequences of these preferred antibodies are also depicted in table 1, namely SEQ ID NOS:2, 3, 5, 6, 7, 8, 11, 14, 16 and 17 being the heavy chain CDR1 sequences of these antibodies, SEQ ID NOS:20, 21, 23, 24, 25, 26, 29, 32, 34 and 35 being the heavy chain CDR2 sequences of these antibodies, SEQ ID NOS:38, 39, 41, 42, 43, 44, 47, 50, 52 and 53 being the heavy chain CDR3 sequences of these antibodies, SEQ ID NOS:56, 57, 59, 60, 61, 62, 65, 68, 70 and 71 being the light chain CDR1 sequences of these antibodies, SEQ ID NOS:74, 75, 77, 78, 79, 80, 83, 86, 88 and 89 being the light chain CDR2 sequences of these antibodies, and SEQ ID NOS:92, 93, 95, 96, 97, 98, 101, 104, 106 and 107 being the light chain CDR3 sequences of these antibodies.
The disclosure thus provides an isolated, synthetic or recombinant antibody or functional part thereof, or immunoglobulin chain or functional equivalent thereof comprising:

    
    
        a heavy chain CDR1 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO:2, 3, 5, 6, 7, 8, 11, 14, 16 and 17, and/or
        a heavy chain CDR2 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 20, 21, 23, 24, 25, 26, 29, 32, 34 and 35, and/or
        a heavy chain CDR3 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 38, 39, 41, 42, 43, 44, 47, 50, 52 and 53, and/or
        a light chain CDR1 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 56, 57, 59, 60, 61, 62, 65, 68, 70 and 71, and/or
        a light chain CDR2 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 74, 75, 77, 78, 79, 80, 83, 86, 88 and 89, and/or
        a light chain CDR3 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 92, 93, 95, 96, 97, 98, 101, 104, 106 and 107. Preferably, the antibody or functional part or immunoglobulin chain or functional equivalent comprises heavy chain CDR1, CDR2 and/or CDR3 sequences and/or light chain CDR1, CDR2 and/or CDR3 sequences that are at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably 100% identical to these sequences.
    
    


Of course, the six CDR sequences of one given antibody of interest (or sequences at least 70% identical thereto) are typically combined. An antibody, functional part, immunoglobulin or functional equivalent, according to the disclosure, thus preferably comprises CDR sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably 100%, identical to the heavy chain CDR1, CDR2 and CDR3 sequences and the light chain CDR1, CDR2 and CDR3 sequences of the same antibody provided by the disclosure (as depicted in table 1).
The terms “AT35,” “AT37,” “AT39,” “AT43,” “AT51,” “AT47,” “AT32,” “AT33,” “AT36,” and “AT50, as used herein, encompass all antibodies and functional equivalents with the indicated heavy chain and light chain sequences, for instance, isolated and/or purified or recombinantly produced. The indicated particularly preferred antibodies do not compete with antibody 3D3 described in US 2010-0285022 or monoclonal antibody 131-2G, which binds to the CX3C motif in the G protein, which is located at amino acid positions 173-186 of the G protein. Thus, the indicated preferred antibodies are advantageously combined with these known antibodies.
In another embodiment, the disclosure provides a human isolated, synthetic or recombinant antibody or functional part thereof, or immunoglobulin chain or functional equivalent thereof, capable of binding to a G protein of Respiratory Syncytial Virus (RSV), which antibody or functional part, or immunoglobulin chain or functional equivalent is capable of potentiating RSV neutralizing activity of an antibody capable of binding an F protein of RSV.
“An antibody capable of binding an F protein of RSV” is herein also called an RSV F-specific antibody. With the term “potentiating RSV neutralizing activity of an antibody capable of binding an F protein of RSV” is meant that the RSV neutralizing activity of the antibody capable of binding an F protein of RSV is increased if an RSV G-specific antibody, according to the disclosure, is also present. An RSV G-specific antibody, according to the disclosure, is itself not capable of neutralizing RSV in the absence of complement factors. However, it was surprisingly found that the neutralizing activity of a RSV F-specific antibody is nevertheless increased if such RSV G-specific antibody, according to the disclosure, is present. The neutralizing activity can be neutralizing activity in vitro or in vivo. An antibody capable of binding a F protein of RSV of which RSV neutralizing activity is potentiated by an RSV G-specific antibody, according to the disclosure, is preferably palivizumab, AM14, AM16, AM23 or D25, which are described in WO 2008/147196, or AM22, described in WO 2011/043643 and of which the heavy and light chain and CDR sequences are depicted in table 1.
Preferred RSV G-specific antibodies capable of potentiating RSV neutralizing activity of an RSV F-specific antibody are AT46, AT32, AT33 and AT35, which have heavy chain sequences of SEQ ID NOS:109, 110, 111 and 113 as depicted in table 1, respectively, and light chain sequences of SEQ ID NOS:127, 128, 129 and 131 as depicted in table 1, respectively. The heavy and light chain CDR sequences of these preferred antibodies are also depicted in table 1, namely SEQ ID NOS:1, 2, 3 and 5, being the heavy chain CDR1 sequences of these antibodies, SEQ ID NOS:19, 20, 21 and 23 being the heavy chain CDR2 sequences of these antibodies, SEQ ID NOS:37, 38, 39 and 41 being the heavy chain CDR3 sequences of these antibodies, SEQ ID NOS:55, 56, 57 and 59 being the light chain CDR1 sequences of these antibodies, SEQ ID NOS:73, 74, 75 and 77 being the light chain CDR2 sequences of these antibodies, and SEQ ID NOS:91, 92, 93 and 95 being the light chain CDR3 sequences of these antibodies.
The disclosure thus provides an isolated, synthetic or recombinant antibody or functional part thereof, or immunoglobulin chain or functional equivalent thereof comprising:

    
    
        a heavy chain CDR1 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO:1, 2, 3 and 5, and/or
        a heavy chain CDR2 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO:19, 20, 21 and 23, and/or
        a heavy chain CDR3 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO:37, 38, 39 and 41, and/or
        a light chain CDR1 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO:55, 56, 57 and 59, and/or
        a light chain CDR2 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO:73, 74, 75 and 77, and/or
        a light chain CDR3 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO:91, 92, 93 and 95. Preferably, the antibody or functional part or immunoglobulin chain or functional equivalent comprises heavy chain CDR1, CDR2 and/or CDR3 sequences and/or light chain CDR1, CDR2 and/or CDR3 sequences that are at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably 100% identical to these sequences.
    
    


As described before, the six CDR sequences of one given antibody of interest (or sequences at least 70% identical thereto) are typically combined. Since AT46, AT32, AT33 and AT35 are preferred examples of antibodies capable of potentiating the RSV neutralizing activities of antibodies capable of binding an F protein of RSV, the disclosure thus provides a human isolated, synthetic or recombinant antibody or functional part thereof, or immunoglobulin or functional equivalent thereof, capable of binding to a G protein of Respiratory Syncytial Virus (RSV), which antibody or functional part or immunoglobulin or functional equivalent is capable of potentiating RSV neutralizing activity of an antibody capable of binding an F protein of RSV, the antibody or functional part or immunoglobulin or functional equivalent having a combination of CDR sequences selected from the group consisting of:

    
    
        SEQ ID NO: 1 (heavy chain CDR1 of AT46) and SEQ ID NO: 19 (heavy chain CDR2 of AT46) and SEQ ID NO: 37 (heavy chain CDR3 of AT46) and SEQ ID NO: 55 (light chain CDR1 of AT46) and SEQ ID NO: 73 (light chain CDR2 of AT46) and SEQ ID NO: 91 (light chain CDR3 of AT46); and
        SEQ ID NO: 2 (heavy chain CDR1 of AT32) and SEQ ID NO: 20 (heavy chain CDR2 of AT32) and SEQ ID NO: 38 (heavy chain CDR3 of AT32) and SEQ ID NO: 56 (light chain CDR1 of AT32) and SEQ ID NO: 74 (light chain CDR2 of AT32) and SEQ ID NO: 92 (light chain CDR3 of AT32); and
        SEQ ID NO:3 (heavy chain CDR1 of AT33) and SEQ ID NO: 21 (heavy chain CDR2 of AT33) and SEQ ID NO: 39 (heavy chain CDR3 of AT33) and SEQ ID NO: 57 (light chain CDR1 of AT33) and SEQ ID NO: 75 (light chain CDR2 of AT33) and SEQ ID NO: 93 (light chain CDR3 of AT33); and
        SEQ ID NO: 5 (heavy chain CDR1 of AT35) and SEQ ID NO: 23 (heavy chain CDR2 of AT35) and SEQ ID NO: 41 (heavy chain CDR3 of AT35) and SEQ ID NO: 59 (light chain CDR1 of AT35) and SEQ ID NO: 77 (light chain CDR2 of AT35) and SEQ ID NO: 95 (light chain CDR3 of AT35); and
        CDR sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical to the sequences of any of these combinations.
    
    


In another preferred embodiment, the heavy and light sequences of one given antibody of interest (or sequences at least 70% identical thereto) are combined. Also provided is, therefore, a human isolated, synthetic or recombinant antibody or functional part thereof, or immunoglobulin or functional equivalent thereof, capable of binding to a G protein of Respiratory Syncytial Virus (RSV), which antibody or functional part or immunoglobulin or functional equivalent is capable of potentiating RSV neutralizing activity of an antibody capable of binding an F protein of RSV, the antibody or functional part or immunoglobulin or functional equivalent having a combination of a heavy and light chain sequence selected from the group consisting of:

    
    
        SEQ ID NO: 109 (heavy chain of AT46) and SEQ ID NO: 127 (light chain of AT46); and
        SEQ ID NO: 110 (heavy chain of AT32) and SEQ ID NO: 128 (light chain of AT32); and
        SEQ ID NO: 111 (heavy chain of AT33) and SEQ ID NO: 129 (light chain of AT33); and
        SEQ ID NO: 113 (heavy chain of AT35) and SEQ ID NO: 131 (light chain of AT35); and
        heavy and light chain sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical to the sequences of any of these combinations.
    
    


The terms “AT46,” “AT32,” “AT33,” and “AT35,” as used herein, encompass all antibodies and functional equivalents with the indicated heavy chain and light chain sequences, for instance, isolated and/or purified or recombinantly produced.
An advantage of a combination of an RSV F-specific antibody and an antibody, according to the disclosure, that is capable of potentiating RSV neutralizing activity of the RSV F-specific antibody is that a lower dosis of the RSV F-specific antibody is needed in order to obtain the same neutralizing capacity. Therefore, less of the RSV F-specific antibody has to be administered to an individual for treatment and/or prevention of an RSV infection or RSV-related disorder. It is favorable to use an amount as low as possible to achieve a desired effect from both a health care of view, it is preferred to administer to a subject as less as possible of any substance, and from an economical point of view, a reduction of the amount of the therapeutic antibody needed generally reduces the cost of the treatment. Alternatively, with a similar amount of RSV F-specific antibody, a more effective treatment and/or prevention of an RSV infection and/or RSV-related disorder is achieved.
Furthermore, an RSV G-specific antibody, according to the disclosure, obviously recognizes a different epitope of RSV as an RSV F-specific antibody. By combining at least one RSV 6-specific antibody, according to the disclosure, with an RSV F-specific antibody, two or more different targets in RSV are recognized during the same therapy. This way, a more potent anti-RSV treatment is obtained. Such a combination will result in more effective treatment and/or prevention of an RSV infection and/or an RSV-related disorder.
Furthermore, in a preferred embodiment, a lower overall antibody dosage is needed, as compared to current treatment with palivizumab. As already mentioned above, a lower amount of antibody capable of binding an F protein of RSV is needed to obtain the same neutralizing capacity. However, an RSV G-specific antibody, according to the disclosure, itself is also capable of counteracting RSV. Thus, in order to obtain a desired activity in counteracting RSV a lower total amount of (RSV G-specific and RSV F-specific) antibodies is needed if an RSV G-specific antibody, according to the disclosure, is combined with an RSV F-specific antibody.
An RSV G-specific antibody, according to the disclosure, capable of potentiating RSV neutralizing activity of an RSV F-specific antibody is thus advantageously combined with such an RSV F-specific antibody. Provided is, thus, a pharmaceutical composition comprising an RSV G-specific antibody, according to the disclosure, and an antibody capable of binding an F protein of RSV, and a pharmaceutically acceptable carrier, diluent and/or excipient. Such a pharmaceutical composition is particularly suitable for use in the treatment and/or prevention of an RSV infection and/or an RSV-related disorder.
In the Examples, isolation of 17 antibodies, according to the disclosure, is described. The CDRs of these antibodies are depicted in table 1. The disclosure provides the insight that the CDRs with a sequence of SEQ ID NOS:1-17, SEQ ID NOS:19-35, SEQ ID NOS:37-53, SEQ ID NOS:55-71, SEQ ID NOS:73-89, and SEQ ID NOS:91-107 provide particularly desired RSV binding characteristics. The disclosure, therefore, provides an isolated, synthetic or recombinant antibody or functional part thereof, or immunoglobulin chain or functional equivalent thereof comprising:

    
    
        a heavy chain CDR1 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO:1-17, and/or
        a heavy chain CDR2 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO:19-35, and/or
        a heavy chain CDR3 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO:37-53, and/or
        a light chain CDR1 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO:55-71, and/or
        a light chain CDR2 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO:73-89, and/or
        a light chain CDR3 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO:91-107.
    
    


Preferably, an RSV G-specific antibody, according to the disclosure, comprises a heavy and/or light chain CDR sequence which is at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90% identical to a sequence selected from the group consisting of SEQ ID NO:1-17, SEQ ID NO:19-35, SEQ ID NO:37-53, SEQ ID NO: 55-71, SEQ ID NO:73-89, and SEQ ID NO:91-107. Most preferably, an RSV G-specific antibody, according to the disclosure, comprises a heavy and/or light chain CDR sequence which is at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably 100%, identical to a sequence selected from the group consisting of SEQ ID NO:1-17, SEQ ID NO:19-35, SEQ ID NO:37-53, SEQ ID NO: 55-71, SEQ ID NO:73-89, and SEQ ID NO:91-107. As described before, the six CDR sequences of one given antibody of interest (or sequences at least 70% identical thereto) are typically combined. An antibody, functional part, immunoglobulin or functional equivalent according to the disclosure thus preferably comprises CDR sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably 100%, identical to the heavy chain CDR1, CDR2 and CDR3 sequences and the light chain CDR1, CDR2 and CDR3 sequences of antibody AT46, AT32, AT33, AT34, AT35, AT36, AT37, AT39, AT40, AT42, AT43, AT44, AT45, AT47, AT49, AT50 or AT51.
Particularly preferred RSV G-specific antibodies, according to the disclosure, are the antibodies AT46, AT32, AT33, AT34, AT35, AT36, AT37, AT39, AT40, AT42, AT43, AT44, AT45, AT47, AT49, AT50 and AT51, which have heavy chain and light chain CDR sequences as depicted in table 1, because these antibodies have been demonstrated to have particularly desired binding characteristics. In a preferred embodiment, an RSV G-specific antibody, according to the disclosure, therefore, comprises both the heavy and light chain CDR sequences of one of the above mentioned RSV G-specific antibodies.
Provided are thus RSV G-specific antibodies, according to the disclosure, which have heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT46, comprising the sequence of SEQ ID NO:1, SEQ ID NO:19, SEQ ID NO:37, SEQ ID NO:55, SEQ ID NO:73 and SEQ ID NO:91, or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT46 does not show competitive binding with any other antibody, described herein, and can thus be advantageously combined with any other RSV G-specific antibody, described herein, and known RSV G-specific antibodies. Antibody AT46 is also preferred because it is capable of binding a conformational epitope of the RSV G protein. Conformational epitopes are generally highly conserved within different RSV strains, as described in more detail herein elsewhere. Thus, antibody AT46 has the advantage that is active against a wide range of RSV strains. Antibody AT46 is furthermore a particularly preferred antibody because it is capable of binding the G protein of both RSV A and B subtypes. Furthermore, antibody AT46 is capable of potentiating the RSV neutralizing activity of several RSV F-specific antibodies, and can thus be advantageously combined with a RSV F-specific antibody, such as palivizumab, AM14, AM16, AM22, AM23 and D25. The characteristics of antibody AT46 are summarized in Tables 4, 5 and 6.
In another embodiment, an RSV G-specific antibody, according to the disclosure, comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT32, comprising the sequence of SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:38, SEQ ID NO:56, SEQ ID NO:74 and SEQ ID NO:92 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT32 is a preferred antibody because it has a particularly high RSV neutralizing capacity, having an IC50 of about 0.02 μg/ml. Antibody AT32 is also preferred because it is capable of binding an epitope of a G protein of RSV, which epitope is RIPNK (amino acids 188-192) of the G protein, and has a high binding affinity, having an affinity constant (KD) of about 0.6 nM for the RSV Ga protein (Table 7a). Thus, antibody AT32 binds to a different epitope as compared to previously disclosed RSV G antibodies. Antibody AT32 can thus be advantageously combined with such known antibodies, with RSV G-specific antibodies, disclosed herein, that are capable of binding to a conformational epitope and with RSV G-specific antibodies, disclosed herein, that are capable of binding the CX3C motif of the RSV G protein. Furthermore, antibody AT32 is capable of potentiating the RSV neutralizing activity of RSV F-specific antibodies, and can thus be advantageously combined with a RSV F-specific antibody, such as palivizumab, AM14, AM16, AM22, AM23 and D25. The characteristics of antibody AT32 are summarized in Tables 4, 5 and 6.
In another embodiment, an RSV G-specific antibody, according to the disclosure, comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT33, comprising the sequence of SEQ ID NO:3, SEQ ID NO:21, SEQ ID NO:39, SEQ ID NO:57, SEQ ID NO:75 and SEQ ID NO:93 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT33 is a preferred antibody because it has a particularly high RSV neutralizing capacity, having an IC50 of about 0.01 μg/ml. Antibody AT33 is also preferred because it is capable of binding an epitope of a G protein of RSV, which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of the G protein. Thus, antibody AT33 binds to a different epitope as compared to previously disclosed RSV G antibodies. Antibody AT33 can thus be advantageously combined with such known antibodies, with RSV G-specific antibodies, disclosed herein, that are capable of binding to a conformational epitope and with RSV G-specific antibodies, disclosed herein, capable of binding the CX3C motif of the RSV G protein. Furthermore, antibody AT33 is capable of potentiating the RSV neutralizing activity of several RSV F-specific antibodies, and can thus be advantageously combined with a RSV F-specific antibody, such as palivizumab, AM14, AM16, AM22, AM23 and D25. The characteristics of antibody AT33 are summarized in Tables 4, 5 and 6.
In another embodiment an RSV G-specific antibody, according to the disclosure, comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT34, comprising the sequence of SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:40, SEQ ID NO:58, SEQ ID NO:76 and SEQ ID NO:94 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT34 is a preferred antibody because it is capable of binding the G protein of both RSV A and B subtype. Antibody AT34 is also preferred because it is capable of binding within or close to the conserved motif and/or the CX3C motif of the RSV G protein. Antibody AT34 can thus be advantageously combined with RSV G-specific antibodies, disclosed herein, that are capable of binding to a conformational epitope and with RSV G-specific antibodies, disclosed herein, that are capable of binding an epitope of a G protein of Respiratory Syncytial Virus, which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of the G protein. The characteristics of antibody AT34 are summarized in Tables 4, 5 and 6.
In another embodiment, an RSV G-specific antibody, according to the disclosure, comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT35, comprising the sequence of SEQ ID NO:5, SEQ ID NO:23, SEQ ID NO:41, SEQ ID NO:59, SEQ ID NO:77 and SEQ ID NO:95 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT35 is a preferred antibody because it has a particularly high RSV neutralizing capacity, having an IC50 of about 0.08 μg/ml. Antibody AT35 is also preferred because it is capable of binding an epitope of a G protein of RSV, which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of the G protein. Thus, antibody AT35 binds to a different epitope as compared to previously disclosed RSV G antibodies. Antibody AT35 can thus be advantageously combined with such known antibodies, with RSV G-specific antibodies, disclosed herein, that are capable of binding to a conformational epitope and with RSV G-specific antibodies, disclosed herein, that are capable of binding the CX3C motif of the RSV G protein. The characteristics of antibody AT35 are summarized in Tables 4, 5 and 6.
In another embodiment, an RSV G-specific antibody, according to the disclosure, comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT36, comprising the sequence of SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:42, SEQ ID NO:60, SEQ ID NO:78 and SEQ ID NO:96 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT36 is a preferred antibody because it is capable of binding an epitope of a G protein of RSV, which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of the G protein. Thus, antibody AT36 binds to a different epitope as compared to previously disclosed RSV G antibodies. Antibody AT36 can thus be advantageously combined with such known antibodies, with RSV G-specific antibodies, disclosed herein, that are capable of binding to a conformational epitope and with RSV G-specific antibodies, disclosed herein, that are capable of binding the CX3C motif of the RSV G protein. The characteristics of antibody AT36 are summarized in Tables 4, 5 and 6.
In another embodiment, an RSV G-specific antibody, according to the disclosure, comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT37, comprising the sequence of SEQ ID NO:7, SEQ ID NO:25, SEQ ID NO:43, SEQ ID NO:61, SEQ ID NO:79 and SEQ ID NO:97 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT37 is a preferred antibody because it is capable of binding an epitope of a G protein of RSV, which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of the G protein. Thus, antibody AT37 binds to a different epitope as compared to previously disclosed RSV G antibodies. Antibody AT37 can thus be advantageously combined with such known antibodies, with RSV G-specific antibodies, disclosed herein, that are capable of binding to a conformational epitope and with RSV G-specific antibodies, disclosed herein, that are capable of binding the CX3C motif of the RSV G protein. The characteristics of antibody AT37 are summarized in Tables 4, 5 and 6.
In another embodiment, an RSV G-specific antibody, according to the disclosure, comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT39, comprising the sequence of SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:44, SEQ ID NO:62, SEQ ID NO:80 and SEQ ID NO:98 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT39 is a preferred antibody because it has a particularly high RSV neutralizing capacity, having an IC50 of about 0.10 μg/ml. Antibody AT39 is also preferred because it is capable of binding an epitope of a G protein of RSV, which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of the G protein. Thus, antibody AT39 binds to a different epitope as compared to previously disclosed RSV G antibodies. Antibody AT39 can thus be advantageously combined with such known antibodies, with RSV G-specific antibodies, disclosed herein, that are capable of binding to a conformational epitope and with RSV G-specific antibodies, disclosed herein, that are capable of binding the CX3C motif of the RSV G protein. The characteristics of antibody AT39 are summarized in Tables 4, 5 and 6.
In another embodiment, an RSV G-specific antibody, according to the disclosure, comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT40, comprising the sequence of SEQ ID NO:9, SEQ ID NO:27, SEQ ID NO:45, SEQ ID NO:63, SEQ ID NO:81 and SEQ ID NO:99 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT40 is a preferred antibody because it is capable of binding the G protein of both RSV A and B subtype and because it has a particularly high RSV neutralizing capacity, having an IC50 of about 0.02 μg/ml. Furthermore, AT40 has a high binding affinity, having an affinity constant (KD) of about 0.2 nM for RSV-Ga and about 0.1 nM for Gb as measured by IBIS-iSPR technology (Table 7a). Antibody AT40 is also preferred because it is capable of binding the epitope FEVFNF (amino acids 165-170) of the RSV G protein. Antibody AT40 can thus be advantageously combined with RSV G-specific antibodies, disclosed herein, that are capable of binding to a conformational epitope and with RSV G-specific antibodies, disclosed herein, that are capable of binding an epitope of a G protein of Respiratory Syncytial Virus, which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of the G protein. The characteristics of antibody AT40 are summarized in Tables 4, 5, 6 and 7.
In another embodiment, an RSV G-specific antibody, according to the disclosure, comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT42, comprising the sequence of SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:46, SEQ ID NO:64, SEQ ID NO:82 and SEQ ID NO:100 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT42 is a preferred antibody because it is capable of binding the G protein of both RSV A and B subtype. Antibody AT42 is also preferred because it is capable of binding a conformational epitope of the RSV G protein, which domain is at least partially within the conserved domain (amino acids 164-172) and/or the CX3C binding domain (CWAIC) because AT42 competes with antibody 131-2G and partially competes with antibody 3D3 (Table 4 and FIG. 5). Conformational epitopes are generally highly conserved within different RSV strains, as described in more detail herein elsewhere. Thus, antibody AT42 has the advantage that is active against a wide range of RSV strains. Furthermore, because it binds a conformational epitope, antibody AT42 can be advantageously combined with RSV G-specific antibodies, disclosed herein, that are capable of binding to the CX3C motif of the RSV G protein and with RSV G-specific antibodies, disclosed herein, that are capable of binding an epitope of a G protein of Respiratory Syncytial Virus, which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of the G protein. Furthermore, AT42 has a high binding affinity, having an affinity constant (KD) of about 1.3 nM for RSV Ga and about 0.3 nM for Gb as measured by IBIS-iSPR technology (Table 7). The characteristics of antibody AT42 are summarized in Tables 4, 5, 6 and 7.
In another embodiment, an RSV G-specific antibody, according to the disclosure, comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT43, comprising the sequence of SEQ ID NO:11, SEQ ID NO:29, SEQ ID NO:47, SEQ ID NO:65, SEQ ID NO:83 and SEQ ID NO:101 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT43 is a preferred antibody because it is capable of binding a conformational epitope of the RSV G protein. Conformational epitopes are generally highly conserved within different RSV strains, as described in more detail herein elsewhere. Thus, antibody AT43 has the advantage that is active against a wide range of RSV strains. Furthermore, because it binds a conformational epitope, antibody AT43 can be advantageously combined with RSV G-specific antibodies, disclosed herein, that are capable of binding to the CX3C motif of the RSV G protein and with RSV G-specific antibodies, disclosed herein, that are capable of binding an epitope of a G protein of Respiratory Syncytial Virus, which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of the G protein. The characteristics of antibody AT43 are summarized in Tables 4, 5 and 6.
In another embodiment, an RSV G-specific antibody, according to the disclosure, comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT44, comprising the sequence of SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:48, SEQ ID NO:66, SEQ ID NO:84 and SEQ ID NO:102 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT44 is a preferred antibody because it is capable of binding the G protein of both RSV A and B subtype and because it has a particularly high RSV neutralizing capacity, having an IC50 of about 0.02 μg/ml. Furthermore, AT44 has a high binding affinity, having an affinity constant (KD) of about 0.1 nM for both RSV Ga and Gb as measured by IBIS-iSPR technology (Table 7a and b). Antibody AT44 is also preferred because it is capable of binding the epitope EVFNF (amino acids 166-170) of the RSV G protein. Antibody AT44 can thus be advantageously combined with RSV G-specific antibodies, disclosed herein, that are capable of binding to a conformational epitope and with RSV G-specific antibodies, disclosed herein, that are capable of binding an epitope of a G protein of Respiratory Syncytial Virus, which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of the G protein. The characteristics of antibody AT44 are summarized in Tables 4, 5, 6 and 7.
In another embodiment, an RSV G-specific antibody, according to the disclosure, comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT45, comprising the sequence of SEQ ID NO:13, SEQ ID NO:31, SEQ ID NO:49, SEQ ID NO:67, SEQ ID NO:85 and SEQ ID NO:103 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT45 is a preferred antibody because it is capable of binding the G protein of both RSV A and B subtype and because it has a particularly high RSV neutralizing capacity, having an IC50 of about 0.11 μg/ml. Antibody AT45 is also preferred because it is capable of binding within or in the proximity of the CX3C motif of the RSV G protein. Antibody AT45 can thus be advantageously combined with RSV G-specific antibodies, disclosed herein, that are capable of binding to a conformational epitope and RSV G-specific antibodies, disclosed herein, that are capable of binding an epitope of a G protein of Respiratory Syncytial Virus, which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of the G protein. The characteristics of antibody AT45 are summarized in Tables 4, 5 and 6.
In another embodiment, an RSV G-specific antibody, according to the disclosure, comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT47, comprising the sequence of SEQ ID NO:14, SEQ ID NO:32, SEQ ID NO:50, SEQ ID NO:68, SEQ ID NO:86 and SEQ ID NO:104 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT47 is a preferred antibody because it is capable of binding a conformational epitope of the RSV G protein. Conformational epitopes are generally highly conserved within different RSV strains, as described in more detail herein elsewhere. Thus, antibody AT47 has the advantage that is active against a wide range of RSV strains. Furthermore, because it binds a conformational epitope, antibody AT47 can be advantageously combined with RSV G-specific antibodies, disclosed herein, that are capable of binding to the CX3C motif of the RSV G protein and with RSV G-specific antibodies, disclosed herein, that are capable of binding an epitope of a G protein of Respiratory Syncytial Virus, which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of the G protein. The characteristics of antibody AT47 are summarized in Tables 4, 5 and 6.
In another embodiment, an RSV G-specific antibody, according to the disclosure, comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT49, comprising the sequence of SEQ ID NO:15, SEQ ID NO:33, SEQ ID NO:51, SEQ ID NO:69, SEQ ID NO:87 and SEQ ID NO:105 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT49 is a preferred antibody because it is capable of binding the G protein of both RSV A and B subtype. Antibody AT49 is also preferred because it is capable of binding within or close to the CX3C motif of the RSV G protein. Antibody AT49 can thus be advantageously combined with RSV G-specific antibodies, disclosed herein, that are capable of binding to a conformational epitope and RSV G-specific antibodies, disclosed herein, that are capable of binding an epitope of a G protein of Respiratory Syncytial Virus, which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of the G protein. The characteristics of antibody AT49 are summarized in Tables 4, 5 and 6.
In another embodiment, an RSV G-specific antibody, according to the disclosure, comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT50, comprising the sequence of SEQ ID NO:16, SEQ ID NO:34, SEQ ID NO:52, SEQ ID NO:70, SEQ ID NO:88 and SEQ ID NO:106 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT50 is a preferred antibody because it is capable of binding an epitope of a G protein of RSV which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of the G protein. Thus, antibody AT50 binds to a different epitope as compared to previously disclosed RSV G antibodies. Antibody AT50 can thus be advantageously combined with such known antibodies, with RSV G-specific antibodies, disclosed herein, that are capable of binding to a conformational epitope and with RSV G-specific antibodies, disclosed herein, that are capable of binding the CX3C motif of the RSV G protein. The characteristics of antibody AT50 are summarized in Tables 4, 5 and 6.
In another embodiment, an RSV G-specific antibody, according to the disclosure, comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT51, comprising the sequence of SEQ ID NO:17, SEQ ID NO:35, SEQ ID NO:53, SEQ ID NO:71, SEQ ID NO:89 and SEQ ID NO:107 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT51 is a preferred antibody because it is capable of binding an epitope of a G protein of RSV, which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of the G protein. Thus, antibody AT51 binds to a different epitope as compared to previously disclosed RSV G antibodies. Antibody AT51 can thus be advantageously combined with such known antibodies, with RSV G-specific antibodies, disclosed herein, that are capable of binding to a conformational epitope and RSV G-specific antibodies, disclosed herein, that are capable of binding the CX3C motif of the RSV G protein. The characteristics of antibody AT51 are summarized in Tables 4, 5 and 6.
Preferably, an RSV G-specific antibody, according to the disclosure, comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences that are at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, identical to the sequences of the same antibody of the disclosure as depicted in table 1.
As is well known by the skilled person, a heavy chain of an antibody is the larger of the two types of chains making up an immunoglobulin molecule. A heavy chain comprises constant domains and a variable domain, which variable domain is involved in antigen binding. A light chain of an antibody is the smaller of the two types of chains making up an immunoglobulin molecule. A light chain comprises a constant domain and a variable domain. The variable domain is, together with the variable domain of the heavy chain, involved in antigen binding.
Complementary-determining regions (CDRs) are the hypervariable regions present in heavy chain variable domains and light chain variable domains. The CDRs of a heavy chain and the connected light chain of an antibody together form the antigen-binding site.
Based on the human RSV G-specific antibodies depicted in table 1, it is possible to produce an immunoglobulin chain or functional equivalent thereof comprising at least one CDR sequence of a human immunoglobulin variable domain depicted in table 1, which is specific for RSV G protein. Further provided, is thus an isolated, recombinant or synthetic immunoglobulin chain or functional equivalent thereof, comprising at least one CDR sequence of a human immunoglobulin variable region depicted in table 1. In a preferred embodiment, a human antibody is provided because the use of a human antibody diminishes the chance of side-effects due to an immunological reaction in a human individual. Optionally, the at least one CDR sequence is optimized, preferably in order to improve binding efficacy or stability. This is, for instance, done by mutagenesis experiments where after the stability and/or binding efficacy of the resulting compounds are preferably tested and an improved RSV G-specific antibody is selected.
A skilled person is well capable of generating variants comprising at least one altered CDR sequence, according to the disclosure. For instance, conservative amino acid substitution is applied. It is also possible to alter at least one CDR sequence depicted in table 1 in order to generate a variant antibody, or a functional part thereof, with at least one altered property as compared to the original antibody. Preferably, an antibody or functional part is provided comprising a CDR sequence, which is at least 70% identical to a CDR sequence as depicted in table 1, so that the favorable binding characteristics of an RSV G-specific antibody, according to the disclosure, are at least in part maintained or even improved. A CDR sequence, as depicted in table 1, is preferably altered such that the resulting antibody or functional part comprises at least one improved property, such as, for instance, an improved binding affinity, selectivity and/or stability, as compared to the original antibody. Variant antibodies or functional parts thereof comprising an amino acid sequence, which is at least 70% identical to a CDR sequence as depicted in table 1, are, therefore, also within the scope of the present disclosure. Various methods are available in the art for altering an amino acid sequence. For instance, a heavy chain or light chain sequence with a desired CDR sequence is artificially synthesized. Preferably, a nucleic acid sequence encoding a CDR sequence, according to the disclosure, is mutated, for instance, using random- or site-directed mutagenesis.
Besides optimizing CDR sequences in order to improve binding efficacy or stability, it is often advantageous to optimize at least one sequence in at least one of the framework regions. This is preferably done in order to improve binding efficacy or stability. Framework sequences are, for instance, optimized by mutating a nucleic acid molecule encoding such framework sequence where after the characteristics of the resulting antibody, or functional part, are preferably tested. This way, it is possible to obtain improved antibodies or functional parts. In a preferred embodiment, human germline sequences are used for framework regions in antibodies or functional parts thereof or immunoglobulin chains or functional equivalents according to the disclosure. The use of germline sequences preferably minimizes the risk of immunogenicity of the antibodies or functional parts, immunoglobulin chains or functional equivalents, because these sequences are less likely to contain somatic alterations, which are unique to individuals from which the framework regions are derived, and may cause an immunogenic response when applied to another human individual.
In a preferred embodiment, RSV G-specific antibodies, according to the disclosure, are provided that are capable of binding an epitope of a G protein of RSV, which epitope is a non-linear or conformational epitope. The term “non-linear or conformational epitope” is herein defined as an epitope, which is formed by the amino acid sequence and the three-dimensional shape of an antigen (e.g., folding). The amino acids making up the epitope can be relatively few in number and widely spread along the length of the molecule. Such epitope is brought into the correct conformation via folding of the antigen. In general, antibodies recognizing conformational epitopes afford broader specificity and, therefore, more effective therapeutic application for ameliorating or preventing RSV infection than antibodies able to bind only linear epitopes because conformational epitopes are more conserved. In order to obtain the necessary correct folding of a protein, variation within amino acids which are part of a conformational epitope is limited. Thus, the antibodies capable of binding to a conformational epitope, disclosed herein, have the advantage that they are active against a wider range of RSV strains than antibodies recognizing linear epitopes. An RSV G-specific antibody, according to the disclosure, capable of binding a conformational epitope of a G protein of RSV is particularly suitable for combination with one or more RSV G-specific antibodies, according to the disclosure, capable of binding to another epitope, such as a linear epitope of RSV G protein which epitope is located between amino acids 51-158 and/or between amino acids 189-299 of the G protein or an epitope, which comprises the CX3C motif of the RSV G protein.
Particularly preferred RSV G-specific antibodies, which bind to conformational epitopes, are the antibodies designated AT46 AT42, AT43 and AT47 which have heavy chain sequences of SEQ ID NOS:109, 118, 119 and 122 as depicted in table 1, respectively, and light chain sequences of SEQ ID NOS:127, 136, 137 and 140 as depicted in table 1, respectively. The heavy and light chain CDR sequences of these preferred antibodies are also depicted in table 1, namely SEQ ID NOS:1, 10, 11 and 14 being the heavy chain CDR1 sequences of these antibodies, SEQ ID NOS:19, 28, 29 and 32 being the heavy chain CDR2 sequences of these antibodies, SEQ ID NOS:37, 46, 47 and 50 being the heavy chain CDR3 sequences of these antibodies, SEQ ID NOS:55, 64, 65 and 68 being the light chain CDR1 sequences of these antibodies, SEQ ID NOS:73, 82, 83 and 86 being the light chain CDR2 sequences of these antibodies, and SEQ ID NOS:91, 100, 101 and 104 being the light chain CDR3 sequences of these antibodies.
The disclosure thus provides an isolated, synthetic or recombinant antibody or functional part thereof, or immunoglobulin chain or functional equivalent thereof comprising:

    
    
        a heavy chain CDR1 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 1, 10, 11 and 14, and/or
        a heavy chain CDR2 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 19, 28, 29 and 32, and/or
        a heavy chain CDR3 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 37, 46, 47 and 50, and/or
        a light chain CDR1 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 55, 64, 65 and 68, and/or
        a light chain CDR2 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 73, 82, 83 and 86, and/or
        a light chain CDR3 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 91, 100, 101 and 104.
    
    


Preferably, the antibody or functional part or immunoglobulin chain or functional equivalent comprises heavy chain CDR1, CDR2 and/or CDR3 sequences and/or light chain CDR1, CDR2 and/or CDR3 sequences that are at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% identical to these sequences. As described before, the six CDR sequences of one given antibody of interest (or sequences at least 70% identical thereto) are typically combined. An antibody, functional part, immunoglobulin or functional equivalent, according to the disclosure, thus preferably comprises CDR sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably 100%, identical to the heavy chain CDR1, CDR2 and CDR3 sequences and the light chain CDR1, CDR2 and CDR3 sequences of antibody AT46 AT42, AT43 or AT47.
The terms “AT46,” “AT42,” “AT43,” and “AT47,” as used herein, encompass all antibodies with the indicated heavy chain and light chain sequences, for instance, isolated and/or purified or recombinantly produced.
The disclosure also provides RSV G-specific antibodies, which are capable of binding to or close to the CX3C motif of the RSV G protein. An RSV G-specific antibody, according to the disclosure, capable of binding the CX3C motif of the RSV G protein is particularly suitable for combination with one or more RSV G-specific antibodies, according to the disclosure, capable of binding to another epitope, such as an epitope of RSV G protein, which epitope is located between amino acids 51-158 and/or between amino acids 189-299 of the G protein or an epitope capable of binding a conformational epitope of a G protein of RSV.
Particularly preferred RSV G-specific antibodies, according to the disclosure, which are capable of binding the CX3C motif of the RSV G protein are the antibodies designated AT34, AT40, AT49, AT44 and AT45, which have heavy chain sequences of SEQ ID NOS:112, 117, 123, 120 and 121 as depicted in table 1, respectively, and light chain sequences of SEQ ID NOS:130, 135, 141, 138 and 139 as depicted in table 1, respectively. The heavy and light chain CDR sequences of these preferred antibodies are also depicted in table 1, namely SEQ ID NOS:4, 9, 12, 13 and 15 being the heavy chain CDR1 sequences of these antibodies, SEQ ID NOS:22, 27, 30, 31 and 33 being the heavy chain CDR2 sequences of these antibodies, SEQ ID NOS:40, 45, 48, 49 and 51 being the heavy chain CDR3 sequences of these antibodies, SEQ ID NOS:58, 63, 66, 67 and 69 being the light chain CDR1 sequences of these antibodies, SEQ ID NOS:76, 81, 84, 85 and 87 being the light chain CDR2 sequences of these antibodies, and SEQ ID NOS:94, 99, 102, 103 and 105 being the light chain CDR3 sequences of these antibodies.
The disclosure thus provides an isolated, synthetic or recombinant antibody or functional part thereof, or immunoglobulin chain or functional equivalent thereof comprising:

    
    
        a heavy chain CDR1 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 4, 9, 12, 13 and 15, and/or a heavy chain CDR2 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 22, 27, 30, 31 and 33, and/or a heavy chain CDR3 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 40, 45, 48, 49 and 51, and/or a light chain CDR1 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 58, 63, 66, 67 and 69, and/or a light chain CDR2 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 76, 81, 84, 85 and 87, and/or
        a light chain CDR3 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 94, 99, 102, 103 and 105.
    
    


Preferably, the antibody or functional part or immunoglobulin chain or functional equivalent comprises heavy chain CDR1, CDR2 and/or CDR3 sequences and/or light chain CDR1, CDR2 and/or CDR3 sequences that are at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% identical to these sequences. As described before, the six CDR sequences of one given antibody of interest (or sequences at least 70% identical thereto) are typically combined. An antibody, functional part, immunoglobulin or functional equivalent, according to the disclosure, thus preferably comprises CDR sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably 100%, identical to the heavy chain CDR1, CDR2 and CDR3 sequences and the light chain CDR1, CDR2 and CDR3 sequences of antibody AT34, AT40, AT49, AT44 or AT45.
The terms “AT34,” “AT40,” “AT49,” “AT44,” and “AT45,” as used herein, encompass all antibodies with the heavy chain and light chain sequences, for instance, isolated and/or purified or recombinantly produced.
In a preferred embodiment, at least two RSV G-specific antibodies, according to the disclosure, are combined because with a combination of different antibodies RSV is more effectively counteracted. Particularly, preferred is the combination of at least two RSV G-specific antibodies, according to the disclosure, which bind to different epitopes of the G protein. By combining at least two RSV G-specific antibodies, which bind to different epitopes on the RSV G protein, two or more different epitopes of RSV G protein are recognized during the same therapy. This way, a more potent anti-RSV response is obtained. With a stronger response to RSV, such combination will result in more effective treatment and/or prevention of an RSV infection and/or an RSV-related disorder.
The disclosure, therefore, provides a composition comprising a combination of at least two RSV G-specific antibodies, according to the disclosure. In a preferred embodiment, a composition, according to the disclosure, comprises at least two RSV G-specific antibodies selected from at least two of the following groups:

    
    
        1) an RSV G-specific antibody, according to the disclosure, capable of binding an epitope of a G protein of RSV, which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of the G protein. Preferred antibodies are AT35, AT37, AT39, AT43, AT51, AT47, AT32, AT33, AT36 and AT50, which have heavy chain sequences of SEQ ID NOS:113, 115, 116, 119, 125, 122, 110, 111, 114 and 124 as depicted in table 1, respectively, and light chain sequences of SEQ ID NOS:131, 133, 134, 137, 143, 140, 128, 129, 132 and 142 as depicted in table 1, respectively;
        2) an RSV G-specific antibody, according to the disclosure, capable of binding a conformational epitope of an RSV G protein. Preferred antibodies are AT46, AT42, AT43 and AT47, which have heavy chain sequences of SEQ ID NOS:109, 118, 119 and 122 as depicted in table 1, respectively, and light chain sequences of SEQ ID NOS:127, 136, 137 and 140 as depicted in table 1, respectively; and
        3) an RSV G-specific antibody, according to the disclosure, capable of binding the CX3C motif of the RSV G protein. Preferred antibodies are AT34, AT40, AT49, AT44 and AT45, which have heavy chain sequences of SEQ ID NOS:112, 117, 123, 120 and 121 as depicted in table 1, respectively, and light chain sequences of SEQ ID NOS:130, 135, 141, 138 and 139 as depicted in table 1, respectively.
    
    


A particularly preferred RSV G-specific antibody, according to the disclosure, is AT46, which has heavy and light chain sequences SEQ ID NO:109 and SEQ ID NO:127 as depicted in table 1. This antibody does not show competitive binding with any other antibody described herein. AT46 can thus be advantageously combined with any other RSV G-specific antibody, described herein, including other antibodies, which are capable of binding a conformational epitope, i.e., AT42, AT43 and AT47, which have heavy and light chain sequences as depicted in table 1. Thus, any combination of two RSV G-specific antibodies, according to the disclosure, which comprises at least AT46 is a combination of two antibodies binding to different epitopes of the RSV G protein. Antibody AT46 can thus be advantageously used in combination with any other RSV G-specific antibody, according to the disclosure, and any known RSV G-specific antibody. Antibody AT46 is furthermore a particularly preferred antibody, according to the disclosure, because it is capable of binding the G protein of both RSV A and B subtypes and has a high binding affinity. Furthermore, antibody AT46 is capable of potentiating the RSV neutralizing activity of several RSV F-specific antibodies. The characteristics of antibody AT46 are summarized in Tables 4, 5 and 6. Thus, in a preferred embodiment of the disclosure, a composition comprises a combination of AT46 and another RSV G-antibody, according to the disclosure.
Other preferred combinations of two RSV G-specific antibodies are depicted in tables 2 and 3. Therefore, in another preferred embodiment of the disclosure, a composition comprises a combination of two RSV G-antibodies, according to the disclosure, wherein the combination is selected from table 2. More preferably, the combination is selected from table 3. One or more RSV G-specific antibodies, according to the disclosure, that are capable of binding a conformational epitope of RSV G protein are also advantageously combined with RSV G-specific antibodies that are already known, such as antibodies disclosed in US 2010-0285022. One or more RSV G-specific antibodies, according to the disclosure, are capable of binding to an epitope of RSV G protein, which epitope is located between amino acids 51-158 and/or between amino acids 189-299 of the G protein are also advantageously combined with RSV G-specific antibodies that are already known, such as antibodies disclosed in US 2010-0285022.
The disclosure, therefore, also provides a composition comprising an RSV G-specific antibody, according to the disclosure, capable of binding an epitope of a G protein of RSV, which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of the G protein (preferably, antibody AT35, AT37, AT39, AT43, AT51, AT47, AT32, AT33, AT36 or AT50, which have heavy chain sequences of SEQ ID NOS:113, 115, 116, 119, 125, 122, 110, 111, 114 and 124, as depicted in table 1, respectively, and light chain sequences of SEQ ID NOS:131, 133, 134, 137, 143, 140, 128, 129, 132 and 142, as depicted in table 1, respectively), and a known RSV G-specific antibody, which is capable of binding an epitope of a G protein of RSV, which epitope is located between amino acids 164-186 of the G protein. Also provided is a composition comprising an RSV G-specific antibody, according to the disclosure, capable of binding a conformational epitope of an RSV G protein (preferably, antibody AT46, AT42, AT43 or AT47, which have heavy chain sequences of SEQ ID NOS:109, 118, 119 and 122, as depicted in table 1, respectively, and light chain sequences of SEQ ID NOS:127, 136, 137 and 140, as depicted in table 1, respectively), and a known RSV G-specific antibody, which is capable of binding an epitope of a G protein of RSV, which epitope is located between amino acids 164-186 of the G protein.
Particularly preferred, are combinations of three RSV G-specific antibodies, according to the disclosure, which bind to different epitopes of the G protein. By combining at least three of such RSV G-specific antibodies at least three different epitopes of RSV G protein are recognized during the same therapy. This way, often an even stronger immunogenic response to RSV is obtained and a higher antibody specificity against RSV is reached as compared to the use of one antibody or a combination of two antibodies. As indicated above, with a stronger immunogenic response to and/or a higher specificity against RSV, a more effective treatment and/or prevention of an RSV infection and/or an RSV-related disorder can be achieved. A combination of three RSV G-specific antibodies, according to the disclosure, preferably comprises three antibodies that do not compete for the same or overlapping epitopes in the RSV G protein. The disclosure, therefore, provides a composition comprising a combination of three RSV G-specific antibodies, according to the disclosure, wherein the combination is selected from the group consisting of:

    
    
        AT34+AT46+AT42;
        AT40+AT46+AT42;
        AT44+AT46+AT42;
        AT45+AT46+AT42;
        AT49+AT46+AT42;

which antibodies have heavy and light chain sequences as depicted in table 1.

    
    


Other preferred combinations of three RSV G-specific antibodies, according to the disclosure, are AT34, AT33 and AT46; and AT36, AT46 and AT45. These combinations of three RSV G-specific antibodies, according to the disclosure, have been proven to be able to non-competitively bind the RSV G protein using IBIS-iSPR technology (IBIS Technologies BV Hengelo, the Netherlands).
Other preferred combinations of two or three antibodies are:

    
    
        AT42+AT33;
        AT42+AT44;
        AT40+AT46+AT32;
        AT40+AT46+AT33;
        AT44+AT42+AT33;
        AT44+AT46+AT33.
    
    


These combinations of anti-RSV G antibodies were able to neutralize the virus without the addition of complement factors as demonstrated in example 2 and FIG. 3A.
Preferred RSV G-specific antibodies, according to the disclosure, are capable of binding the G protein of both RSV subtype A and RSV subtype B because such antibodies can be used for counteracting both RSV subtypes. However, RSV G-specific antibodies, according to the disclosure, capable of binding the G protein of RSV subtype A only are also particularly useful. For instance, RSV G-specific antibodies, according to the disclosure, that only bind the G protein of RSV subtype A, bind to a different epitope in the G protein of RSV than RSV G-specific antibodies, described herein, that bind to both subtype A and B RSV. Therefore, they are particularly suitable to be used in combination with RSV G-specific antibodies that bind to both subtype A and B RSV, as described above. Furthermore, RSV G-specific antibodies, according to the disclosure, that only bind the G protein of RSV subtype A are particularly suitable for diagnosing RSV subtype A.
Preferred RSV G-specific antibodies, according to the disclosure, have a high affinity for the RSV G protein. Measurement of the affinity constant and specificity of binding between antigen and antibody is preferred in determining the efficacy of prophylactic, therapeutic, diagnostic and research methods using anti-RSV G antibodies of the disclosure. “Binding affinity” generally refers to the strength of the sum total of the noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity can generally be represented by the equilibrium dissociation constant (KD), which is calculated as the ka to kd ratio (see, e.g., Chen, Y., et al., (1999) J. Mol Biol 293:865-881, and Table 7 and FIG. 7). Affinity can be measured by common methods known in the art, such as, for instance, a surface plasmon resonance (SPR) assay such as BiaCore or IBIS-iSPR instrument at IBIS Technologies BV (Hengelo, the Netherlands) or solution phase assays, such as Kinexa. Preferably, an RSV G-specific antibody, according to the disclosure, has an affinity constant (KD) as measured by IBIS-iSPR technology of at most 10 nM, more preferably at most 5 nM, more preferably at most 2 nM, more preferably at most 1 nM, more preferably at most 0.5 nM, more preferably at most 0.3 nM, more preferably at most 0.1 nM.
Other preferred RSV G-specific antibodies, according to the disclosure, have a high RSV neutralizing activity in the presence of complement. RSV neutralizing activity is, for instance, determined in vitro in the presence of complement, for instance, rabbit serum complement. Rabbit serum complement is a mixture of complement factors prepared from the serum of rabbits and is commercially available from, for instance, GTi Diagnostics or Calbiochem. An in vitro neutralization assay in the presence of complement is, for instance, performed as described in the Examples. Preferably, an RSV G-specific antibody, according to the disclosure, is capable of neutralizing RSV in vitro in the presence of complement with an IC50<500 ng/ml, more preferably with an IC50<400 ng/ml, more preferably with an IC50<350 ng/ml, more preferably with an IC50>300 ng/ml, more preferably with an IC50<250 ng/ml, more preferably with an IC50<200 ng/ml, more preferably with an IC50<150 ng/ml, most preferably with IC50<125 ng/ml. Further provided is, therefore, an RSV G-specific antibody, according to the disclosure, which has an RSV neutralizing capacity in vitro in the presence of complement with an IC50<500 ng/ml, more preferably with an IC50<400 ng/ml, more preferably with an IC50<350 ng/ml, more preferably with an IC50>300 ng/ml, more preferably with an IC50<250 ng/ml, more preferably with an IC50<200 ng/ml, more preferably with an IC50<150 ng/ml, most preferably with IC50<125 ng/ml. In one embodiment, an RSV G-specific antibody, according to the disclosure, has RSV neutralizing capacity in vitro in the presence of complement with an IC50<100 ng/ml, such as <80 ng/ml, or <25 ng/ml.
In a preferred embodiment, an RSV G-specific antibody, according to the disclosure, comprises a heavy chain sequence and/or light chain sequence, or a sequence which has at least 70% sequence identity thereto, as depicted in table 1. Also provided is, therefore, an antibody or functional part, or immunoglobulin chain or functional equivalent, having a heavy chain sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO:109-125 and/or having a light chain sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO:127-143.
Preferably, an RSV G-specific antibody, according to the disclosure, comprises a heavy chain sequence which is at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90% identical to a sequence selected from the group consisting of SEQ ID NO:109-125 and/or a light chain which is at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90% identical to a sequence selected from the group consisting of SEQ ID NO:127-143. Most preferably, an RSV G-specific antibody, according to the disclosure, comprises a heavy chain sequence which is at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% identical to a sequence selected from the group consisting of SEQ ID NO:109-125 and/or a light chain sequence which is at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% identical to a sequence selected from the group consisting of SEQ ID NO:127-143. The higher the identity, the more closely an antibody resembles an antibody depicted in table 1.
An antibody or functional part or immunoglobulin chain or functional equivalent, according to the disclosure, preferably comprises a heavy chain as well as a light chain, which resemble the heavy and the light chain of the same antibody depicted in table 1. Thus, in a preferred embodiment an RSV G-specific antibody, according to the disclosure, comprises a heavy chain sequence of a given antibody, preferably antibody AT46, comprising the sequence of SEQ ID NO:109 and a light chain sequence of the same antibody, preferably AT46, comprising the sequence of SEQ ID NO:127, or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, identical thereto.
In another embodiment, an RSV G-specific antibody, according to the disclosure, or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT32, comprising the sequence of SEQ ID NO:110 and the light chain sequence of antibody AT32, comprising the sequence of SEQ ID NO:128.
In another embodiment, an RSV G-specific antibody, according to the disclosure, or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT33, comprising the sequence of SEQ ID NO:111 and a light chain sequence comprising the sequence of SEQ ID NO:129.
In another embodiment, an RSV G-specific antibody, according to the disclosure, or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT34, comprising the sequence of SEQ ID NO:112 and a light chain sequence of antibody AT34, comprising the sequence of SEQ ID NO:130.
In another embodiment, an RSV G-specific antibody, according to the disclosure, or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT35, comprising the sequence of SEQ ID NO:113 and a light chain sequence of antibody AT35, comprising the sequence of SEQ ID NO:131.
In another embodiment, an RSV G-specific antibody, according to the disclosure, or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT36, comprising the sequence of SEQ ID NO:114 and a light chain sequence of antibody AT36, comprising the sequence of SEQ ID NO:132.
In another embodiment, an RSV G-specific antibody, according to the disclosure, or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT37, comprising the sequence of SEQ ID NO:115 and a light chain sequence of antibody AT37, comprising the sequence of SEQ ID NO:133.
In another embodiment, an RSV G-specific antibody, according to the disclosure, or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT39, comprising the sequence of SEQ ID NO:116 and a light chain sequence of antibody AT39, comprising the sequence of SEQ ID NO:134.
In another embodiment, an RSV G-specific antibody, according to the disclosure, or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT40, comprising the sequence of SEQ ID NO:117 and a light chain sequence of antibody AT40, comprising the sequence of SEQ ID NO:135.
In another embodiment, an RSV G-specific antibody, according to the disclosure, or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT42, comprising the sequence of SEQ ID NO:118 and a light chain sequence of antibody AT42, comprising the sequence of SEQ ID NO:136.
In another embodiment, an RSV G-specific antibody, according to the disclosure, or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT43, comprising the sequence of SEQ ID NO:119 and a light chain sequence of antibody AT43, comprising the sequence of SEQ ID NO:137.
In another embodiment, an RSV G-specific antibody, according to the disclosure, or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT44, comprising the sequence of SEQ ID NO:120 and a light chain sequence of antibody AT44, comprising the sequence of SEQ ID NO:138.
In another embodiment, an RSV G-specific antibody, according to the disclosure, or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT45, comprising the sequence of SEQ ID NO:121 and a light chain sequence of antibody AT45, comprising the sequence of SEQ ID NO:139.
In another embodiment, an RSV G-specific antibody, according to the disclosure, or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT47, comprising the sequence of SEQ ID NO:122 and a light chain sequence of antibody AT47, comprising the sequence of SEQ ID NO:140.
In another embodiment, an RSV G-specific antibody, according to the disclosure, or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT49, comprising the sequence of SEQ ID NO:123 and a light chain sequence of antibody AT49, comprising the sequence of SEQ ID NO:141.
In another embodiment, an RSV G-specific antibody, according to the disclosure, or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT50, comprising the sequence of SEQ ID NO:124 and a light chain sequence of antibody AT50, comprising the sequence of SEQ ID NO:142.
In another embodiment, an RSV G-specific antibody, according to the disclosure, or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT51, comprising the sequence of SEQ ID NO:125 and a light chain sequence of antibody AT51, comprising the sequence of SEQ ID NO:143.
Preferably, an RSV G-specific antibody, according to the disclosure, or functional part thereof comprises sequences that are at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, identical to the heavy and light chain sequences of antibody AT46, AT32, AT33, AT34, AT35, AT36, AT37, AT39, AT40, AT42, AT43, AT44, AT45, AT47, AT49, AT50 or AT51 as depicted in table 1.
The disclosure further provides an isolated, synthetic or recombinant nucleic acid sequence with a length of at least 15 nucleotides, or a functional equivalent thereof, encoding at least one CDR sequence of an antibody or functional part, or immunoglobulin chain or functional equivalent, according to the disclosure. Preferably, a nucleic acid, according to the disclosure, has a length of at least 30 nucleotides, more preferably at least 50 nucleotides, more preferably at least 75 nucleotides. A nucleic acid, according to the disclosure, is, for instance, isolated from a B-cell which is capable of producing an RSV G-specific antibody, according to the disclosure. In a preferred embodiment, a nucleic acid encoding an RSV G-specific antibody, according to the disclosure, is provided.
As used herein, “an isolated, synthetic or recombinant nucleic acid sequence with a length of at least 15 nucleotides, or a functional equivalent thereof, encoding at least one CDR sequence of an antibody or functional part thereof, or immunoglobulin chain or functional equivalent thereof, according to the disclosure,” is herein also referred to as “a nucleic acid sequence or functional equivalent thereof, according to the disclosure.”
As used herein, a nucleic acid molecule or nucleic acid sequence of the disclosure, preferably comprises a chain of nucleotides, more preferably DNA and/or RNA. In other embodiments, a nucleic acid molecule or nucleic acid sequence of the disclosure comprises other kinds of nucleic acid structures such as, for instance, a DNA/RNA helix, peptide nucleic acid (PNA), locked nucleic acid (LNA) and/or a ribozyme. Such other nucleic acid structures are referred to as functional equivalents of a nucleic acid sequence. The term “functional equivalent of a nucleic acid sequence” also encompasses a chain comprising non-natural nucleotides, modified nucleotides and/or non-nucleotide building blocks, which exhibit the same function as natural nucleotides.
Nucleic acid sequences encoding preferred heavy chain and light chain CDRs of antibodies AT46, AT32, AT33, AT34, AT35, AT36, AT37, AT39, AT40, AT42, AT43, AT44, AT45, AT47, AT49, AT50 and AT51 are depicted in table 1. Nucleic acid sequences encoding a heavy or light chain CDR of a RSV G-specific antibody, according to the disclosure, which differ from the CDR nucleic acid sequences depicted in table 1 but have nucleic acid codons encoding for the same amino acids of the heavy or light chain CDR are also encompassed by the disclosure. Nucleic acid sequences encoding a heavy or light chain CDR of a RSV G-specific antibody depicted in table 1 which has been altered, for instance, through conservative amino acid substitution, whereby an amino acid residue is substituted by another residue with generally similar properties (size, hydrophobicity, etc.), are also encompassed by the disclosure, as long as the resulting CDR has at least 70% sequence identity with a CDR depicted in table 1.
A preferred nucleic acid sequence, according to the disclosure, comprises:

    
    
        a heavy chain CDR1 encoding sequence which has at least 70% sequence identity to a sequence which is selected from the group consisting of SEQ ID NO:145-161, and/or
        a heavy chain CDR2 encoding sequence which has at least 70% sequence identity to a sequence which is selected from the group consisting of SEQ ID NO:163-179, and/or
        a heavy chain CDR3 encoding sequence which has at least 70% sequence identity to a sequence which is selected from the group consisting of SEQ ID NO:181-197, and/or
        a light chain CDR1 encoding sequence which has at least 70% sequence identity to a sequence which is selected from the group consisting of SEQ ID NO:199-215, and/or
        a light chain CDR2 encoding sequence which has at least 70% sequence identity to a sequence which is selected from the group consisting of SEQ ID NO:217-233, and/or
        a light chain CDR3 encoding sequence which has at least 70% sequence identity to a sequence which is selected from the group consisting of SEQ ID NO:235-251.
    
    


A nucleic acid sequence, according to the disclosure, preferably comprises a sequence which has at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity to the sequence. Preferably, the nucleic acid sequence comprises at least one CDR encoding sequence. Further provided is a nucleic acid sequence or functional equivalent thereof comprising a sequence which has at least 70% sequence identity, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:145-161, SEQ ID NO:163-179, SEQ ID NO: 199-215, SEQ ID NO: 199-215, SEQ ID NO: 217-233, and SEQ ID NO: 235-251, the nucleic acid sequence or functional equivalent having at least 15 nucleotides. As described before, the six CDR sequences of one given antibody of interest (or sequences at least 70% identical thereto) are typically combined. A preferred nucleic acid sequence, according to the disclosure, therefore, comprises CDR encoding sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably 100%, identical to the heavy chain CDR1, CDR2 and CDR3 encoding sequences and the light chain CDR1, CDR2 and CDR3 encoding sequences of antibody AT46, AT32, AT33, AT34, AT35, AT36, AT37, AT39, AT40, AT42, AT43, AT44, AT45, AT47, AT49, AT50 or AT51.
A nucleic acid sequence or functional equivalent thereof, according to the present disclosure, preferably encodes a region which has at least 70% sequence identity to a heavy chain and/or a light chain as depicted in table 1. Thus, a preferred nucleic acid sequence or a functional equivalent comprises a sequence which has at least 70% sequence identity to a sequence selected from the group consisting of SEQ ID NO:253-269 and/or a sequence which has at least 70% sequence identity to a sequence selected from the group consisting of SEQ ID NO:271-287. More preferably, a nucleic acid sequence or a functional equivalent, according to the disclosure, comprises a heavy chain encoding sequence as well as a light chain encoding sequence, which resemble the heavy and the light chain encoding sequences of the same antibody depicted in table 1. Thus, in a preferred embodiment, a nucleic acid or functional equivalent, according to the disclosure, comprises a heavy chain encoding sequence of antibody AT46, comprising the sequence of SEQ ID NO:253 and a light chain encoding sequence of antibody AT46, comprising the sequence of SEQ ID NO:271 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, most preferably at least 99% identical thereto.
In another embodiment, a nucleic acid or functional equivalent, according to the disclosure, comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT32, comprising the sequence of SEQ ID NO:254 and the light chain encoding sequence of antibody AT32, comprising the sequence of SEQ ID NO:272.
In another embodiment, a nucleic acid or functional equivalent, according to the disclosure, comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT33, comprising the sequence of SEQ ID NO:255 and a light chain encoding sequence comprising the sequence of SEQ ID NO:273.
In another embodiment, a nucleic acid or functional equivalent, according to the disclosure, comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT34, comprising the sequence of SEQ ID NO:256 and a light chain encoding sequence of antibody AT34, comprising the sequence of SEQ ID NO:274.
In another embodiment, a nucleic acid or functional equivalent, according to the disclosure, comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT35, comprising the sequence of SEQ ID NO:257 and a light chain encoding sequence of antibody AT35, comprising the sequence of SEQ ID NO:275.
In another embodiment, a nucleic acid or functional equivalent, according to the disclosure, comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT36, comprising the sequence of SEQ ID NO:258 and a light chain encoding sequence of antibody AT36, comprising the sequence of SEQ ID NO:276.
In another embodiment, a nucleic acid or functional equivalent, according to the disclosure, comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT37, comprising the sequence of SEQ ID NO:259 and a light chain encoding sequence of antibody AT37, comprising the sequence of SEQ ID NO:277.
In another embodiment, a nucleic acid or functional equivalent, according to the disclosure, comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT39, comprising the sequence of SEQ ID NO:260 and a light chain encoding sequence of antibody AT39, comprising the sequence of SEQ ID NO:278.
In another embodiment, a nucleic acid or functional equivalent, according to the disclosure, comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT40, comprising the sequence of SEQ ID NO:261 and a light chain encoding sequence of antibody AT40, comprising the sequence of SEQ ID NO:279.
In another embodiment, a nucleic acid or functional equivalent, according to the disclosure, comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT42, comprising the sequence of SEQ ID NO:262 and a light chain encoding sequence of antibody AT42, comprising the sequence of SEQ ID NO:280.
In another embodiment, a nucleic acid or functional equivalent, according to the disclosure, comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT43, comprising the sequence of SEQ ID NO:263 and a light chain encoding sequence of antibody AT43, comprising the sequence of SEQ ID NO:281.
In another embodiment, a nucleic acid or functional equivalent, according to the disclosure, comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT44, comprising the sequence of SEQ ID NO:264 and a light chain encoding sequence of antibody AT44, comprising the sequence of SEQ ID NO:282.
In another embodiment, a nucleic acid or functional equivalent, according to the disclosure, comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT45, comprising the sequence of SEQ ID NO:265 and a light chain encoding sequence of antibody AT45, comprising the sequence of SEQ ID NO:283.
In another embodiment, a nucleic acid or functional equivalent, according to the disclosure, comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT47, comprising the sequence of SEQ ID NO:266 and a light chain encoding sequence of antibody AT47, comprising the sequence of SEQ ID NO:284.
In another embodiment, a nucleic acid or functional equivalent, according to the disclosure, comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT49, comprising the sequence of SEQ ID NO:267 and a light chain encoding sequence of antibody AT49, comprising the sequence of SEQ ID NO:285.
In another embodiment, a nucleic acid or functional equivalent, according to the disclosure, comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT50, comprising the sequence of SEQ ID NO:268 and a light chain encoding sequence of antibody AT50, comprising the sequence of SEQ ID NO:286.
In another embodiment, a nucleic acid or functional equivalent, according to the disclosure, comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT51, comprising the sequence of SEQ ID NO:269 and a light chain encoding sequence of antibody AT51, comprising the sequence of SEQ ID NO:287.
The term “% sequence identity” is defined herein as the percentage of residues in a candidate amino acid of nucleic acid sequence that is identical with the residues in a reference sequence after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for the alignment are well known in the art.
Further provided is a vector comprising a nucleic acid sequence or functional equivalent, according to the disclosure. As used herein “a vector comprising a nucleic acid sequence or functional equivalent, according to the disclosure,” is also referred to as “a vector, according to the disclosure.” Methods for constructing a vector with a nucleic acid sequence or functional equivalent, according to the disclosure, are well known in the art. Non-limiting examples of vectors suitable for generating a vector of the disclosure, are retroviral and lentiviral vectors. Such vector is suitable for a variety of applications. For instance, a vector of the disclosure comprising a therapeutically beneficial nucleic acid sequence is suitable for prophylactic or therapeutic applications. Administration of such vector to an individual, preferably a human, in need thereof results in expression of the prophylactic or therapeutic nucleic acid sequence in vivo. The vector can also be used in applications involving in vitro expression of a nucleic acid sequence of interest, for instance, for (commercial) production of antibodies or functional equivalents, according to the disclosure. Also provided is, therefore, an isolated or recombinant cell comprising a nucleic acid sequence or functional equivalent a vector, according to the disclosure.
A nucleic acid sequence or vector, according to the present disclosure, is particularly useful for generating antibodies or functional parts, or immunoglobulin chains or functional equivalents, which are specific for RSV G protein. This is, for instance, done by introducing such nucleic acid sequence or vector into a cell so that the cell's nucleic acid translation machinery will produce the encoded antibodies or functional parts, or immunoglobulin chains or functional equivalents. In one embodiment, a nucleic acid sequence or vector encoding a heavy and/or light chain, according to the disclosure, is expressed in so called producer cells, such as, for instance, cells of a Chinese hamster ovary (CHO), NSO (a mouse myeloma) or 293(T) cell line, some of which are adapted to commercial antibody production. Proliferation of the producer cells results in a producer cell line capable of producing RSV G-specific antibodies, according to the disclosure. Preferably, the producer cell line is suitable for producing antibodies for use in humans. Hence, the producer cell line is preferably free of pathogenic agents such as pathogenic micro-organisms. Most preferably, RSV G-specific antibodies consisting of human sequences are generated using at least one nucleic acid sequence or vector, according to the disclosure.
An isolated or recombinant antibody producing cell capable of producing an RSV G-specific antibody, according to the disclosure, is, therefore, also provided. An antibody producing cell is defined herein as a cell, which is capable of producing and/or secreting antibodies or functional equivalents thereof, and/or which is capable of developing into a cell, which is capable of producing and/or secreting antibodies or functional equivalents thereof. An antibody producing cell, according to the disclosure, is preferably a producer cell, which is adapted to commercial antibody production. Preferably, the producer cell is suitable for producing antibodies for use in humans. A method for producing an RSV G-specific antibody, according to the disclosure, is also provided, the method comprising providing a cell, preferably an antibody producing cell, with a nucleic acid sequence or functional equivalent, or a vector, according to the disclosure, and allowing the cell to translate the nucleic acid sequence or functional equivalent, or vector, thereby producing RSV G-specific antibodies, according to the disclosure. A method, according to the disclosure, preferably further comprises a step of harvesting, purifying and/or isolating RSV G-specific antibodies, according to the disclosure. Obtained RSV G-specific antibodies, according to the disclosure, are preferably used in human therapy, optionally after additional purifying, isolation or processing steps.
In one embodiment, an RSV G-specific antibody, according to the disclosure, is coupled to another moiety to form an antibody-drug conjugate. An RSV G-specific antibody, according to the disclosure, is, for instance, coupled to an antiviral agent, such as acyclovir, penciclovar, lamivudine, ribavirin, zanamivir, laninamivir, peramivir, idoxuridine, amantadine, remantidine, maxamine or thymalfasin. The term “antiviral agent,” as used herein, refers to any substance that reduces or blocks the function, or growth, of a virus and/or causes destruction of a virus. In another embodiment, a moiety that is coupled to an RSV G-specific antibody, according to the disclosure, is an antimicrobial peptide. The term “antimicrobial peptide,” as used herein, refers to small amphipathic peptides of variable length (typically 6 to 100 aminoacids), sequence and structure with activity against microorganisms such as, for instance, bacteria, protozoa, yeast, fungi and/or viruses. Antimicrobial peptides usually act through relatively non-specific mechanisms resulting in membranolytic activity but several antimicrobial peptides can also stimulate the innate immune response. In a preferred embodiment, the antimicrobial peptide has anti-viral activity. Non-limiting examples of suitable antimicrobial peptides are magainins, PGLa, cathelicidins (such as LL-37 and cathelicidin-related antimicrobial peptide (CRAMP)), alamethicin, mellitin and cecropin, hydramacin-1, pexiganan, MSI-78, MSI-843, MSI-594, polyphemusin, human antimicrobial peptide, defensins, protegrins and indolicidin. In yet another embodiment, a moiety that is coupled to an RSV G-specific antibody, according to the disclosure, is an immunomodulatory molecule such as an CD3 antibody. Such CD3 antibody is capable of binding T cells and, if coupled to an RSV G-specific antibody, according to the disclosure, targeting T cells to RSV infected cells.
The other moiety, for example, a cytotoxic agent, is preferably coupled to an RSV G-specific antibody, according to the disclosure, via a linker such as an acid-labile hydrazone linker, via a peptide linker like citruline-valine, through a thioether linkage, or by sortase catalized transamidation, which is described in detail in WO 2010/087994.
Sortase catalized transamidation involves engineering of a sortase recognition site (LPETGG) on the heavy chain of an antibody, preferably on the C-terminal part of the heavy chain, and on the moiety to be coupled to the antibody. The antibody and the moiety further typically contain a GGGGS sequence and a tag for purification purposes, such as a HIS tag. Subsequently, sortase mediated transamidation is performed followed by click chemistry linkage. In a sortase catalized transaminidation, “click chemistry linkage” typically involves chemical coupling of, for instance, an alkyne-containing reagent and, for instance, an azide-containing reagent, which are added by sortase through addition of glycines to the sortase motif on the heavy chain of the antibody and to a sortase motif on the moiety (such as a protein, peptide or antibody) to be coupled to the antibody. In one embodiment, the disclosure, therefore, provides an RSV G-specific antibody, according to the disclosure, wherein a sortase recognition site (LPETGG) is engineered on the heavy chain of the antibody, preferably on the C-terminal part of the heavy chain, the antibody further containing a GGGGS sequence and a purification tag, such as a HIS tag.
In another embodiment, an RSV G-specific antibody, according to the disclosure, is coupled to another moiety via a thioether linkage. In such case, one or more cysteines are preferably incorporated into an RSV G-specific antibody, according to the disclosure. Cysteines contain a thiol group and, therefore, incorporation of one or more cysteines into, or replacement of one or more amino acids by one or more cysteines of an RSV G-specific antibody, according to the disclosure, enable coupling of the RSV G-specific antibody to another moiety. The one or more cysteines are preferably introduced into an RSV G-specific antibody, according to the disclosure, at a position where it does not influence folding of the antibody, and does not alter antigen binding or effector function. The disclosure, therefore, also provides an RSV G-specific antibody, according to the disclosure, wherein at least one amino acid other than cysteine has been replaced by a cysteine.
As described herein before, an RSV G-specific antibody, according to the disclosure, preferably AT46, AT32, AT33 or AT35, and an RSV F-specific antibody, such as palivizumab, AM14, AM16, AM23, AM22 or D25 can be advantageously used in combination. Furthermore, it is also advantageous to combine an RSV G-specific antibody, according to the disclosure, with another RSV G-specific antibody, according to the disclosure, recognizing a different epitope or with a known RSV G-specific antibody recognizing a different epitope. In another embodiment, however, the disclosure provides an RSV bispecific antibody with specificity for both an RSV G protein and an RSV F protein, or with specificity to different epitopes within an RSV G protein. An “RSV bispecific antibody,” as used herein, is defined as an antibody capable of simultaneously binding two different epitopes, which epitopes may be located within the same antigen, i.e., the RSV G protein, or located within different antigens, i.e., the RSV G and F protein, and is also referred to as “an RSV bispecific antibody, according to the disclosure.” The term “RSV bispecific antibody” also encompasses functional parts of such RSV bispecific antibodies, which has retained its capability of binding a least two different epitopes simultaneously, such as bispecific single chain variable fragments (scFv), bispecific Fab fragments and a bispecific F(ab′)2 fragment. Also provided is a pharmaceutical composition comprising an RSV bispecific antibody, according to the disclosure.
In one embodiment, a bispecific antibody, according to the disclosure, comprises two non-identical heavy chain-light chain combinations, thus having two antigen-binding regions, which recognize two different epitopes within the RSV G protein or which recognize one epitope in an RSV G protein and one epitope within an RSV F protein. For instance, in one embodiment, an RSV bispecific antibody comprises a heavy and light chain of an RSV G-specific antibody, according to the disclosure, as depicted in table 1, and a heavy and light chain of another RSV G-specific antibody, according to the disclosure, as depicted in table 1. In another embodiment, an RSV bispecific antibody comprises a heavy and light chain of an RSV G-specific antibody, according to the disclosure, as depicted in table 1, and a heavy and light chain of an RSV F-specific antibody. Bispecific single chain variable fragments (scFv), bispecific Fab fragments and a bispecific F(ab′)2 fragment comprise, for instance, a scFv or Fab fragment of an RSV G-specific antibody, according to the disclosure, and a scFv or Fab fragment of another RSV G-specific antibody, according to the disclosure. Alternatively, bispecific single chain variable fragments (scFv), bispecific Fab fragments and a bispecific F(ab′)2 fragment comprise a scFv or Fab fragment of an RSV G-specific antibody and a scFv or Fab fragment of an RSV F-specific antibody. In a preferred embodiment, an RSV bispecific antibody, according to the disclosure, comprises a heavy and light chain of antibody AT46, AT32, AT33 or AT35 as depicted in Table 1, or a scFv or Fab fragment thereof, and a heavy and light chain of an RSV F-specific antibody such as palivizumab, AM14, AM16, AM23, D25 (WO 2008/147196), or AM22 (WO 2011/043643) or a scFv or Fab fragment thereof. In another preferred embodiment, an RSV bispecific antibody, according to the disclosure, comprises two heavy and light chains of two different RSV G-specific antibodies, according to the disclosure, as depicted in Table 1, or a scFv or Fab fragment thereof, whereby the different RSV G-specific antibodies preferably form a combination depicted in Table 2 or 3.
In another embodiment, an RSV G-specific antibody, according to the disclosure, is coupled to an RSV F-specific antibody or another RSV G-specific antibody by sortase catalized transamidation, which is described herein before and in detail in WO 2010/087994. For this purpose, sortase catalized transamidation involves engineering of a sortase recognition site (LPETGG) on heavy chain of both antibodies to be coupled, preferably on the C-terminal part of the heavy chain. The antibodies further typically contain a GGGGS sequence and a purification tag, such as a HIS tag. Thus, if an RSV G-specific antibody, according to the disclosure, and an RSV F-specific antibody are coupled, both the RSV G-specific and the RSV F-specific antibodies are engineered, as described herein before and in detail in WO 2010/087994. If two RSV G-specific antibodies recognizing different epitopes in the G protein are coupled, both the RSV G-specific antibodies are engineered, as described herein before and in detail in WO 2010/087994. Subsequently, sortase mediated transamidation is preferably performed followed by click chemistry linkage to couple both antibodies via their heavy chains. As herein explained before, “click chemistry linkage” involves chemical coupling of, for instance, an alkyne-containing reagent and, for instance, an azide-containing reagent which are added by sortase through addition of glycines to the sortase motif on the heavy chain of a first antibody and to the heavy chain of a second antibody to be coupled to the first antibody. In a preferred embodiment, antibody AT46, AT32, AT33 or AT35, as depicted in Table 1, is coupled by sortase catalized transamidation to an RSV F-specific antibody, such as palivizumab, AM14, AM16, AM23, D25, or AM22. In another preferred embodiment, two RSV G-specific antibodies are coupled to each other by sortase catalized transamidation, whereby the RSV G-specific antibodies preferably form a combination depicted in Table 2 or 3.
RSV G-specific antibodies, according to the disclosure, are capable of counteracting Respiratory Syncytial Virus. RSV G-specific antibodies, according to the disclosure, are, therefore, particularly suitable for use as a medicine or prophylactic agent. Preferably, RSV G-specific antibodies, according to the disclosure, are used which consist of human sequences, in order to reduce the chance of adverse side effects when human individuals are treated. Such human sequences can be isolated from a human or synthetically or recombinantly produced based on the sequence of human antibodies. Provided is, therefore, an RSV G-specific antibody, according to the disclosure, or a composition comprising a combination of at least two RSV G-specific antibodies, according to the disclosure, for use as a medicament and/or prophylactic agent. Also provided is a nucleic acid sequence or functional equivalent thereof, according to the disclosure, or a vector, according to the disclosure, comprising such nucleic acid or functional equivalent for use as a medicament and/or prophylactic agent. When a nucleic acid or functional equivalent, according to the disclosure, is administered, it will be translated in situ by the host's machinery into an RSV G-specific antibody, according to the disclosure. Produced RSV G-specific antibodies, according to the disclosure, are capable of preventing and/or counteracting an RSV infection or RSV related disorder. RSV G-specific antibodies, according to the disclosure, are particularly suitable for use as a medicament because they are capable of counteracting RSV after an individual has been infected. On the contrary, palivizumab, the only anti-RSV antibody currently registered, is only useful for prophylactic treatment of premature infants and is thus not able to treat an established RSV infection. In a particularly preferred embodiment, the antibody comprises antibody AT46, or a functional part thereof. Provided is, thus, antibody AT46, comprising a heavy chain sequence of SEQ ID NO:109 and a light chain sequence of SEQ ID NO:127, for use as a medicament and/or prophylactic agent.
An RSV G-specific antibody, according to the disclosure, or a nucleic acid sequence or functional equivalent thereof, according to the disclosure, or a composition comprising a combination of at least two RSV G-specific antibodies, according to the disclosure, or an RSV bispecific antibody or a cell, according to the disclosure, is preferably used for at least in part treating and/or preventing an RSV infection and/or an RSV related disorder. As used herein, “at least in part treating an RSV infection” includes counteracting an RSV infection, alleviating symptoms resulting from an RSV infection and/or counteracting inflammation resulting from an RSV infection. Also provided is, therefore, an RSV G-specific antibody, according to the disclosure, or a nucleic acid sequence or functional equivalent thereof, according to the disclosure, or a composition comprising a combination of at least two RSV G-specific antibodies, according to the disclosure, or a vector, according to the disclosure, or a cell, according to the disclosure, or a RSV bispecific antibody, according to the disclosure, for use in a method of at least in part treating and/or preventing an RSV infection and/or an RSV related disorder. Examples of such RSV related disorders are bronchiolitis, pneumonia and tracheobronchitis resulting from an RSV infection. Further provided is a use of an RSV G-specific antibody, according to the disclosure, or a composition, according to the disclosure, or a vector, according to the disclosure, or a cell, according to the disclosure, or an RSV bispecific antibody, according to the disclosure, for the preparation of a medicament and/or prophylactic agent for at least in part treating and/or preventing an RSV infection and/or an RSV related disorder.
The disclosure further provides a method for at least in part treating and/or preventing an RSV infection and/or an RSV related disorder comprising administering to an individual, preferably a human, in need thereof, a therapeutically effective amount of an RSV G-specific antibody, according to the disclosure, and/or a nucleic acid sequence or functional equivalent thereof, according to the disclosure, and/or a composition comprising a combination of at least two RSV G-specific antibodies, according to the disclosure, and/or a vector, according to the disclosure, and/or a pharmaceutical composition, according to the disclosure, and/or a cell, according to the disclosure. In order to at least in part treat or prevent a disorder related to RSV, an RSV G-specific antibody, a nucleic acid sequence or functional equivalent thereof, an RSV bispecific antibody, a composition comprising a combination of at least two RSV G-specific antibodies, a vector, a pharmaceutical composition and/or a cell, according to the disclosure, is preferably administered to an individual before an RSV infection has taken place. Alternatively, an RSV G-specific antibody, a nucleic acid sequence or functional equivalent thereof, an RSV bispecific antibody, a composition comprising a combination of at least two RSV G-specific antibodies, a vector, a pharmaceutical composition and/or a cell, according to the disclosure, is administered when an individual is already infected. In that case, an RSV infection is counteracted, symptoms resulting from an RSV infection are alleviated and/or inflammation resulting from an RSV infection is counteracted. The antibody, nucleic acid sequence, functional equivalent, composition, vector, pharmaceutical composition and/or cell is preferably administered to individuals with an increased risk of complications, such as hospitalized individuals, for instance, infants, individuals with compromised immunity and/or elderly people. An RSV G-specific antibody, a nucleic acid sequence or functional equivalent thereof, a composition comprising a combination of at least two RSV G-specific antibodies, a vector, a pharmaceutical composition and/or a cell, according to the disclosure, is preferably administered via one or more injections. Typical doses of administration of an RSV G-specific antibody, according to the disclosure, or combinations of at least two thereof, or of an RSV bispecific antibody are between 0.1 and 10 mg per kg body weight. For prophylactic or therapeutic application RSV G-specific antibodies, according to the disclosure, or RSV bispecific antibodies, according to the disclosure, are preferably combined with a pharmaceutically acceptable carrier, diluent and/or excipient.
The disclosure further provides a pharmaceutical composition comprising an RSV G-specific antibody, according to the disclosure, or a composition comprising a combination of at least two RSV G-specific antibodies, according to the disclosure, and a pharmaceutical acceptable carrier, diluent and/or excipient. Also provided, is a pharmaceutical composition comprising an RSV bispecific antibody, according to the disclosure, and a pharmaceutical composition comprising an RSV G-specific antibody, according to the disclosure, coupled to an antiviral agent, antimicrobial peptide or immunomodulatory molecule, as described herein. Further provided, is a pharmaceutical composition comprising a nucleic acid sequence or functional equivalent, according to the disclosure, or a vector or a cell, according to the disclosure, comprising such nucleic acid or functional equivalent, and a pharmaceutical acceptable carrier, diluent and/or excipient. Examples of suitable carriers, for instance, comprise keyhole limpet haemocyanin (KLH), serum albumin (e.g., BSA or RSA) and ovalbumin. In one preferred embodiment, the suitable carrier comprises a solution, like, for example, saline. A pharmaceutical composition, according to the disclosure, is preferably suitable for human use. In one embodiment, the pharmaceutical composition further comprises at least one other RSV specific antibody, preferably an RSV F protein specific antibody such as palivizumab, D25, AM14, AM16, AM22 and/or AM23.
An RSV G-specific antibody, according to the present disclosure, is also particularly suitable for diagnostic uses. For instance, if an individual, preferably a human, is suspected of suffering from an RSV infection, a sample, such as a saliva, sputum, blood, or tissue sample, can be obtained from the individual. Subsequently, the sample can be tested for the presence of G protein of RSV, using an RSV G-specific antibody, according to the disclosure. Preferably, the sample is mixed with an RSV G-specific antibody, according to the disclosure, which will specifically bind to a G protein of RSV. The presence of G proteins of RSV in a sample is indicative for the presence of an RSV infection. G proteins of RSV and/or RSV comprising a G protein bound to an RSV G-protein, according to the disclosure, can be isolated from the sample and/or detected using any method known in the art, for example, but not limited to, isolation using magnetic beads, streptavidin-coated beads, or isolation through the use of secondary antibodies immobilized on a column. Alternatively, or additionally, an RSV G-specific antibody, according to the disclosure, is labeled in order to be able to detect the antibody, for instance, but not limited to, fluorescently labeled, or radioactively labeled. Alternatively, an RSV G-specific antibody, according to the disclosure, is detected using a labeled secondary antibody which is directed against the antibody. If binding of the antibody is detected, G protein of RSV is present, which is indicative for the presence of an RSV infection. The disclosure thus provides an RSV G-specific antibody, according to the disclosure, or a composition comprising a combination of at least two RSV G-specific antibodies, according to the disclosure, for use in diagnosis of an RSV infection.
The disclosure thus further provides a method for determining whether an RSV G protein is present in a sample comprising:

    
    
        contacting the sample with an RSV G-specific antibody, according to the disclosure, or a composition comprising a combination of at least two RSV G-specific antibodies, according to the disclosure,
        allowing the antibody or an antibody component of the composition to bind the RSV G protein, if present, and
        determining whether RSV G protein is bound to the antibody, or to an antibody component of the composition, thereby determining whether an RSV G protein is present.
    
    


In a preferred embodiment, it is determined whether an individual is suffering from an RSV infection. Provided is, therefore, a method for determining whether an individual is suffering from an RSV infection comprising:

    
    
        contacting a sample from the individual with an RSV G-specific antibody, according to the disclosure, or a composition comprising a combination of at least two RSV G-specific antibodies, according to the disclosure,
        allowing the antibody, or an antibody component of the composition to bind the RSV, if present, and
        determining whether RSV is bound to the antibody, or to an antibody component of the composition, thereby determining whether the individual is suffering from an RSV infection. Preferably, the individual is a human.
    
    


The disclosure is further explained in the following examples. These examples do not limit the scope of the disclosure, but merely serve to clarify the disclosure.







TABLE 1







Preferred RSV G-specific antibodies according to the disclosure.










SEQ ID





NO
Antibody
Identity
Sequence













1
AT46
Heavy chain CDR1
SRYVMS


 


2
AT32
Heavy chain CDR1
ELSIH


 


3
AT33
Heavy chain CDR1
SLAIS


 


4
AT34
Heavy chain CDR1
HYGMH


 


5
AT35
Heavy chain CDR1
TYWVS


 


6
AT36
Heavy chain CDR1
YNFIDHSVS


 


7
AT37
Heavy chain CDR1
SGGYSWN


 


8
AT39
Heavy chain CDR1
TYAVH


 


9
AT40
Heavy chain CDR1
DRHALH


 


10
AT42
Heavy chain CDR1
SNVYYWG


 


11
AT43
Heavy chain CDR1
NYGVS


 


12
AT44
Heavy chain CDR1
SGHYWA


 


13
AT45
Heavy chain CDR1
GHAIS


 


14
AT47
Heavy chain CDR1
NYGIC


 


15
AT49
Heavy chain CDR1
SLALN


 


16
AT50
Heavy chain CDR1
NYGIS


 


17
AT51
Heavy chain CDR1
KYGIN


 


18
AM22
Heavy chain CDR1
KLSIH


 


19
AT46
Heavy chain CDR2
SITGSGATTYYADSVKGRFTIS


 


20
AT32
Heavy chain CDR2
GFEPEDGEYIYPQKSQG


 


21
AT33
Heavy chain CDR2
GIIPKFNRRDYAQKFQG


 


22
AT34
Heavy chain CDR2
VISYDGDKKYYADSVKG


 


23
AT35
Heavy chain CDR2
NINQDGSEKSYVDSVEG


 


24
AT36
Heavy chain CDR2
WISPYNHRTVYAEKFQG


 


25
AT37
Heavy chain CDR2
YIYQNDITYYNPSLMS


 


26
AT39
Heavy chain CDR2
WINPDNGDTKYSQRFQGRVVIT


 


27
AT40
Heavy chain CDR2
ILSYDGTTDYYADSVKG


 


28
AT42
Heavy chain CDR2
SIFHSGITHYTPSLNS


 


29
AT43
Heavy chain CDR2
WISTYNGNTWYSQKFQA


 


30
AT44
Heavy chain CDR2
GIHHSGSTYTNPPLKS


 


31
AT45
Heavy chain CDR2
GIIPGLGTTRYARKFQD


 


32
AT47
Heavy chain CDR2
WISGYNGNTYYAQNFQG


 


33
AT49
Heavy chain CDR2
GIIPLFGTQNYAQKFQG


 


34
AT50
Heavy chain CDR2
WISAYNGNTYYRQELQG


 


35
AT51
Heavy chain CDR2
WISAYNGNTYYAQKFQG


 


36
AM22
Heavy chain CDR2
GYEGEVDEIFYAQKFQ


 


37
AT46
Heavy chain CDR3
CGRAGQIFDD


 


38
AT32
Heavy chain CDR3
EARYCDNSRCSPNFDH


 


39
AT33
Heavy chain CDR3
DAEWAAGSDYFFDY


 


40
AT34
Heavy chain CDR3
QGAKGGHELSFYCALDV


 


41
AT35
Heavy chain CDR3
EVFVTQVEPAQWGF


 


42
AT36
Heavy chain CDR3
DRVQQGEGNFFDH


 


43
AT37
Heavy chain CDR3
GAYGSGTYYSADALDI


 


44
AT39
Heavy chain CDR3
GRIFDI


 


45
AT40
Heavy chain CDR3
GRALDDFADYGGYYFDY


 


46
AT42
Heavy chain CDR3
HWAGLYFDS


 


47
AT43
Heavy chain CDR3
HGSGNYYGEANYFDH


 


48
AT44
Heavy chain CDR3
DLYDLSTGPFWFDP


 


49
AT45
Heavy chain CDR3
VAGGYFDSATRG


 


50
AT47
Heavy chain CDR3
GFHYHSADQRIFDP


 


51
AT49
Heavy chain CDR3
FLWFGDQTSDDGFDV


 


52
AT50
Heavy chain CDR3
GGAQEMVRIHYYYYGMDV


 


53
AT51
Heavy chain CDR3
PATSYDDLRSGYLNYCDY


 


54
AM22
Heavy chain CDR3
LGVTVTEAGLGIDDY


 


55
AT46
Light chain CDR1
TLSSGHRNYAIA


 


56
AT32
Light chain CDR1
KSSQSVLYDSNNKNYLA


 


57
AT33
Light chain CDR1
SADAFSDQYAY


 


58
AT34
Light chain CDR1
RASQGIGSWLA


 


59
AT35
Light chain CDR1
RASQSIDNYLN


 


60
AT36
Light chain CDR1
KSSQSLLHSSNNKIYLA


 


61
AT37
Light chain CDR1
RASQSVSASNLA


 


62
AT39
Light chain CDR1
QASQDISNFLN


 


63
AT40
Light chain CDR1
RASQGISTWLA


 


64
AT42
Light chain CDR1
RASQTVSSSHLA


 


65
AT43
Light chain CDR1
RASESVSRNYLA


 


66
AT44
Light chain CDR1
RASQSVSTKVV


 


67
AT45
Light chain CDR1
RSSQSLLHSNGYNYLD


 


68
AT47
Light chain CDR1
RASESISTWLA


 


69
AT49
Light chain CDR1
RSSQSLLHGNGYKYLH


 


70
AT50
Light chain CDR1
RASQVISSYLA


 


71
AT51
Light chain CDR1
RASQGITSYLA


 


72
AM22
Light chain CDR1
RASQIVSRNHLA


 


73
AT46
Light chain CDR2
TNGSHYPGD


 


74
AT32
Light chain CDR2
WASTRES


 


75
AT33
Light chain CDR2
KDTERPS


 


76
AT34
Light chain CDR2
NASGLES


 


77
AT35
Light chain CDR2
LASTLQS


 


78
AT36
Light chain CDR2
WASTRES


 


79
AT37
Light chain CDR2
GASRTAT


 


80
AT39
Light chain CDR2
DASKLQT


 


81
AT40
Light chain CDR2
SASRLQS


 


82
AT42
Light chain CDR2
GSSSRAT


 


83
AT43
Light chain CDR2
GASSRAI


 


84
AT44
Light chain CDR2
GASTRAT


 


85
AT45
Light chain CDR2
GSNRAP


 


86
AT47
Light chain CDR2
KASSLES


 


87
AT49
Light chain CDR2
LGSNRAS


 


88
AT50
Light chain CDR2
GASTLQT


 


89
AT51
Light chain CDR2
AASTLQS


 


90
AM22
Light chain CDR2
GASSRAT


 


91
AT46
Light chain CDR3
QTWGAGI


 


92
AT32
Light chain CDR3
QQYYDPL


 


93
AT33
Light chain CDR3
QSTDTSGPL


 


94
AT34
Light chain CDR3
QQYNSHT


 


95
AT35
Light chain CDR3
QQSHSSP


 


96
AT36
Light chain CDR3
QQYYTTHP


 


97
AT37
Light chain CDR3
QQYGSSP


 


98
AT39
Light chain CDR3
QKFDNLL


 


99
AT40
Light chain CDR3
QQANTFP


 


100
AT42
Light chain CDR3
QYYGDSP


 


101
AT43
Light chain CDR3
QQYTIFP


 


102
AT44
Light chain CDR3
QQYNKWP


 


103
AT45
Light chain CDR3
MQALQTP


 


104
AT47
Light chain CDR3
QQYKSYP


 


105
AT49
Light chain CDR3
MQALQSP


 


106
AT50
Light chain CDR3
QQLNTYP


 


107
AT51
Light chain CDR3
QQFHTYP


 


108
AM22
Light chain CDR3
LSSDSSI


 


109
AT46
Heavy chain
EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYVMSWVRQAPGRGLEWVSSITGSGA





TTYYADSVKGRFTISRDNSKNTVYLQMNRLRAEDTAIYYCANCGRAGQIFDDWGQ





GTLVTVSS


 


110
AT32
Heavy chain
QVQLVQSGAEMKKPGASVKVSCQVAGYTLTELSIHWVRQTPGNGLEWMGGFEPE





DGEYIYPQKSQGRVTMTEDTSTGTAYMELRSLRSDDTAVYYCAAEARYCDNSRCS





PNFDHWGQGTLVAVSS


 


111
AT33
Heavy chain
QVQLVQSGAEVKKPGSSVKVSCKASGDSFNSLAISWVRQAPGQGLEWMGGIIPKFN





RRDYAQKFQGRVTITADDSASTAYIELSSLTSDDTALYYCARDAEWAAGSDYFFDY





WGQGTLVIVSS


 


112
AT34
Heavy chain
QVQLMESGGGVVQPGKSLRLSCAASGFTFSHYGMHWVRQAPGKGLEWVAVISYD





GDKKYYADSVKGRFTISRDNSKNTLHLHMNSLRHEDTAVYFCASQGAKGGHELSF





YCALDVWGQGTTVAVSS


 


113
AT35
Heavy chain
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYWVSWVRQTPGKGLEWVARFTISNI





NQDGSEKSYVDSVEGRDNAKNSLYLQMNSLRADDTAVYYCAREVFVTQVEPAQW





GFWGQGTPVIVSS


 


 


114
AT36
Heavy chain
QVQVVQSGAEVKKPGASVKVSCKTSGYNFIDHSVSWVRQAPGQGLEWMGWISPY





NHRTVYAEKFQGRVTMTTDTSTRTVSMELRRLTSDDTAVYFCARDRVQQGEGNFF





DHWGQGTPVTVTSA


 


115
AT37
Heavy chain
QLQLQESGSRLVKPSQTLSLTCGVSGGSISSGGYSWNWIRQPPGKGLEWVGYIYQN





DITYYNPSLMSRVTISADTSKNQFSLKLSSVTAADTAVYYCARGAYGSGTYYSADA





LDIWGQGTMVTVSS


 


116
AT39
Heavy chain
QVQLVQSGPEVKKPGASVRLSCTASGNTFRTYAVHWVRQASGQRLEWMGWINPD





NGDTKYSQRFQGRVVITRDTSARIIYLDLSSLTSEDTAVFYCFSGRIFDIWGQGTTITV





SS


 


117
AT40
Heavy chain
QVQLVESGGGVVQPGMSHRLSCAASTLIFDRHALHWVRQAPGAGLEWVAILSYDG





TTDYYADSVKGRFTVSRDTSKNTVFLQMNGLRPQDTAVYYCARGRALDDFADYG





GYYFDYWGQGILVTVSS


 


118
AT42
Heavy chain
QVQLQESGPGLVQPSETLSLTCTVSGDSITSNVYYWGWIRQPPGKGLEWIGSIFHSGI





THYTPSLNSRVTISVDTSKNQFSLRLSSATAADTAVYYCARHWAGLYFDSWGQGAL





VAVSS


 


119
AT43
Heavy chain
QVQVVQSGPEVKKPGASVRVSCKASGYTFTNYGVSWVRQAPGQGLEWMGWISTY





NGNTWYSQKFQARVTMTTDTSTSTAYMEVRSLRSDDTAIYYCACHGSGNYYGEAN





YFDHWGQGTLVTVSS


 


120
AT44
Heavy chain
QVQLQASGPGLVKPSETLSLTCNVSGYSVSSGHYWAWVRQSPGKGLEWIGGIHHSG





STYTNPPLKSRVSISIDTSKNQFSLRLTSVTAADTAVYFCARDLYDLSTGPFWFDPW





GQGTLVTVSS


 


121
AT45
Heavy chain
QVHLVQSGAEVKKPGSSVKVSCKASGGTFNGHAISWIRQAPGQGLEWKGGIIPGLG





TTRYARKFQDRVTITADESTRTAYMELSSLRSEDTAVYYCARVAGGYFDSATRGW





GQGTLVTVSS


 


 


122
AT47
Heavy chain
QVQLVQSGGEVKKPGASVKVSCKASGYTFTNYGICWVRQAPGQGLEWMGWISGY





NGNTYYAQNFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGFHYHSADQRI





FDPWGQGTLVTVSS


 


 


123
AT49
Heavy chain
QVLLVQSGAEIKKPGSSVKISCKASGGTFSSLALNWVRQAPGQGLQWMGGIIPLFGT





QNYAQKFQGRVTITADESTSTAYMELSGLRPEDTAVYYCALFLWFGDQTSDDGFD





VWGQGTVVTVSS


 


124
AT50
Heavy chain
QVQLVQSGTEVKKPGASVKVSCKASGYTFSNYGISWVRQAPGQGLEWMGWISAY





NGNTYYRQELQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGGAQEMVRIHY





YYYGMDVWGQGTTVTVSS


 


125
AT51
Heavy chain
QVQLVQSGAEVKKPGASMTVSCKASGYTFSKYGINWVRQAPGQGLEWLGWISAY





NGNTYYAQKFQGRVTMTTDTATSTAYMDVRNLRSDDTAMYYCARPATSYDDLRS





GYLNYCDYWGQGTLVTVSS


 


126
AM22
Heavy chain
QVQLVQSGAEVKKPGATVKVSCKISGHTLIKLSIHWVRQAPGKGLEWMGGYEGEV





DEIFYAQKFQHRLTVIADTATDTVYMELGRLTSDDTAVYFCGTLGVTVTEAGLGID





DYWGQGTLVTVSS


 


127
AT46
Light chain
QPVLTQSPSASASLGASVKLTCTLSSGHRNYAIAWHQQRPEKGPRYLMKIYTNGSH





YPGDGTPDRFSGSSSGAERYLTISSLQSEDEADYYCQTWGAGIWVFGGGTKLTVLG





QPK


 


128
AT32
Light chain
DIVMTQSPDSLAVSLGERATFSCKSSQSVLYDSNNKNYLAWYQQRPGQPPKLLIYW





ASTRESGVPDRFSGSGSGTDFTLTISSLQPEDVAVYYCQQYYDPLITFGQGTRLEIKR





TV


 


129
AT33
Light chain
SYELTQPPSVSVSPGQTARITCSADAFSDQYAYWYQQKPGQAPVLVIYKDTERPSGI





PERISGSSSGTTATLSISGVQAEDEADYYCQSTDTSGPLFGGGTKLTLLGQPK


 


130
AT34
Light chain
DIQMTQSPSTLSASVGDRVTITCRASQGIGSWLAWYQQKPGKAPKLLIYNASGLESG





VPSGFSGSGSGTEFTLTISSLQPDDSATYYCQQYNSHTWTFGQGTKVEFKRTV


 


131
AT35
Light chain
AIQMTQSPSSLSASVGDRVTISCRASQSIDNYLNWYQQKPGKAPKLLLFLASTLQSG





VPSRFTGSGSGTDFTLTISSLQPEDFATYYCQQSHSSPYSFGQGTKLEIKRTV


 


132
AT36
Light chain
DIVMTQSPDSLAVSLGERATINCKSSQSLLHSSNNKIYLAWYQQKPGQPPKLLLYW





ASTRESGVPDRFTGSGSGTDFTLTINSLQAEDVAVYYCQQYYTTHPTFGQGTRLEIK





RTV


 


133
AT37
Light chain
KIVLTQSPGTLSLSPGERATLSCRASQSVSASNLAWYQQKPGQAPRLLIYGIPDRFSG





SGSGTDFTLSISRLEPEDFAVYYCGASRTATQQYGSSPLTFGGGTKVEIKRTV


 


134
AT39
Light chain
DIQMTQSPSSLSASVGDRVTITCQASQDISNFLNWYQQKPGQAPKLLIYDASKLQTG





VPSRFSGSGSETDFTFTISSLQPEDVATYYCQKFDNLLLTFGGGTKVELKRTV


 


135
AT40
Light chain
DIQMTQSPSSVSASVGDKVTITCRASQGISTWLAWYQQKPGKAPALLIYSASRLQSG





VPSRFSGSGSGTDFTLTISSLQPEDYATYYCQQANTFPFTFGPGTKVDIKRTV


 


136
AT42
Light chain
EIVLTQSPGTLSLSPGERATLSCRASQTVSSSHLAWYQQKPGQAPRLLIHGSSSRATG





IPERFSGSGSGPDFTLTISRLKPEDFAVYYCQYYGDSPGSFGEGTKVEIKRTV


 


137
AT43
Light chain
DIVLTQSPGTLSLSPGEGATLSCRASESVSRNYLAWYQQKPGQAPRLLIYGASSRAIG





IPDRFSGSGSGTDFTLTISRLEPEDFAVYCCQQYTIFPLTFGGGTKVEIKRTV


 


138
AT44
Light chain
EIVMTQSPATLSVSPGERVTLSCRASQSVSTKVVWYQQKFGQAPRLLIYGASTRATG





IPVRFSGSGSGTEFTLTISSLQSEDLAVYFCQQYNKWPMYTFGQGTKLEIKRTV


 


139
AT45
Light chain
DIVMTQSPLSLPVTPGESASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSN





RAPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPTFGQGTKVEIKRTV


 


140
AT47
Light chain
DIQMTQSPSTLSASVGDRVTITCRASESISTWLAWYQQKPGKAPNLLIYKASSLESG





VPSRFSGSGSGTEFTLAISSLQPDDFATYYCQQYKSYPYTFGQGTKLELKRTV


 


141
AT49
Light chain
DIVMTQSPLSLTVTPGEPASISCRSSQSLLHGNGYKYLHWYLQKPGQSPQLLIYLGSN





RASGVPARFSGSGSDTDFTLKISTVETEDVGVYYCMQALQSPTFGQGTKVEIKRTV


 


142
AT50
Light chain
DIQLTQSPSFLSASVGDRVTITCRASQVISSYLAWYQQTPGRAPKLLIYGASTLQTGV





PSRFSGSGSGTEFTLTISSLQPEDFATYFCQQLNTYPLTFGPGTKVEIKRTV


 


143
AT51
Light chain
DIQLTQSPSFLSASVGDRVTITCRASQGITSYLAWYQQKPGRAPKLLIYAASTLQSGV





ASRFSGSGSGTEFTLTISSLQPEDFATYYCQQFHTYPLTFGGGTKVEIKRTV


 


144
AM22
Light chain
EIVLTQSPGTLSLSPGERATLSCRASQIVSRNHLAWYQQKPGQAPRLLIFGASSRATG





IPVRFSGSGSGTDFTLTINGLAPEDFAVYYCLSSDSSIFTFGPGTKVDFK


 


145
AT46
Heavy chain CDR1
agt aga tat gtc atg agt


 


146
AT32
Heavy chain CDR1
gaa tta tee ata cac


 


147
AT33
Heavy chain CDR1
agt ctt gcc atc agt


 


148
AT34
Heavy chain CDR1
cat tat ggc atg cac


 


149
AT35
Heavy chain CDR1
acc tat tgg gtg agc


 


150
AT36
Heavy chain CDR1
tac aac ttt atc gac cat agt gtc agc


 


151
AT37
Heavy chain CDR1
agt ggt ggt tac tee tgg aac


 


152
AT39
Heavy chain CDR1
acc tat gct gta cat


 


153
AT40
Heavy chain CDR1
gat aga cat gct ctc cac


 


154
AT42
Heavy chain CDR1
agt aat gtt tac tac tgg ggc


 


155
AT43
Heavy chain CDR1
aac tat ggt gtc agc


 


156
AT44
Heavy chain CDR1
agc ggt cac tac tgg gcc


 


157
AT45
Heavy chain CDR1
ggc cat gct atc agc


 


158
AT47
Heavy chain CDR1
aac tac ggt atc tgt


 


159
AT49
Heavy chain CDR1
agc ctt gct ctc aat


 


160
AT50
Heavy chain CDR1
aac tat ggt atc agt


 


161
AT51
Heavy chain CDR1
aag tat ggc atc aac


 


162
AM22
Heavy chain CDR1
aaa tta tcc att cac


 


163
AT46
Heavy chain CDR2
agc att act gga agt ggt gct acg aca tac tat gca gac tcc





gtg aag ggc cgc ttc acc atc tcc


 


164
AT32
Heavy chain CDR2
ggt ttt gag cct gag gat ggt gag tac atc tac cca cag aaa





tcc cag ggc


 


165
AT33
Heavy chain CDR2
ggg atc atc cct aag ttc aat aga aga gac tac gca cag aag





ttt cag ggc


 


166
AT34
Heavy chain CDR2
gtc ata tcc tat gat ggc gat aaa aaa tat tat gca gac tca





gtg aag ggc


 


167
AT35
Heavy chain CDR2
aac att aac caa gat gga agt gag aag tcc tat gtg gac tct





gtg gag ggc cga ttc acc atc tcc


 


168
AT36
Heavy chain CDR2
tgg atc agc cct tac aac cac aga aca gta tat gca gag aag





ttc cag ggc


 


169
AT37
Heavy chain CDR2
tac atc tat cag aat gac atc acc tac tac aac ccg tcc ctc





atg agt


 


170
AT39
Heavy chain CDR2
tgg atc aac cct gac aat ggt gac aca aaa tat tca cag agg





ttc cag ggt aga gtc gtc att acc


 


171
AT40
Heavy chain CDR2
att ctc tct tat gat ggg acc aca gac tac tac gca gac tcc





gtg aag ggc


 


172
AT42
Heavy chain CDR2
agt atc ttt cat agt ggg atc acc cac tat acc ccg tcc ctc





aat agt


 


173
AT43
Heavy chain CDR2
tgg atc agc act tac aat ggt aac aca tgg tat tca cag aag





ttc cag gcc


 


174
AT44
Heavy chain CDR2
ggt atc cat cat agt ggg agt acc tac acc aat ccg ccc ctc





aag agc


 


175
AT45
Heavy chain CDR2
ggg atc atc cct ggc ctt ggt aca aca agg tac gca cgg aag





ttc cag gac


 


176
AT47
Heavy chain CDR2
tgg atc agc ggt tac aat ggt aac aca tac tat gca cag aac





ttc cag ggc


 


177
AT49
Heavy chain CDR2
ggg atc atc cct ctc ttt ggc act caa aac tac gca cag aag





ttc cag ggc


 


178
AT50
Heavy chain CDR2
tgg atc agc gct tac aat ggt aac aca tac tat aga cag gag





ctc cag ggc


 


179
AT51
Heavy chain CDR2
tgg atc agc gca tac aat ggc aac aca tac tat gca cag aag





ttc cag ggc


 


180
AM22
Heavy chain CDR2
ggt tat gag ggt gag gtc gat gag att ttc tac gca cag aag





ttc cag cac


 


181
AT46
Heavy chain CDR3
tgt ggt agg gcg ggc caa att ttt gac gac


 


182
AT32
Heavy chain CDR3
gag gca aga tat tgt gat aac agc aga tgt tcc cct aac ttt





gac cac


 


183
AT33
Heavy chain CDR3
gac gcc gag tgg gca gct ggc tcg gat tac ttc ttt gac tac


 


184
AT34
Heavy chain CDR3
cag ggg gca aag ggc ggt cac gaa ctt tct ttc tac tgt gct





ttg gac gtc


 


185
AT35
Heavy chain CDR3
gaa gtc ttc gtg act cag gtg gag ccc gcg cag tgg ggc ttc


 


186
AT36
Heavy chain CDR3
gat cga gta caa cag ggc gag gga aac ttc ttt gac cac


 


187
AT37
Heavy chain CDR3
ggg gcc tat ggt tcg gga act tat tat tcc gct gat gct ctt





gat ata


 


188
AT39
Heavy chain CDR3
ggg aga att ttt gat ata


 


189
AT40
Heavy chain CDR3
gga agg gcc cta gat gac ttc gct gac tac ggg gga tac tac





ttt gac tac


 


190
AT42
Heavy chain CDR3
cat tgg gct ggc ctc tac ttt gac tct


 


191
AT43
Heavy chain CDR3
cac ggg agt ggc aat tac tac ggc gaa gcg aac tac ttt gac





cac


 


192
AT44
Heavy chain CDR3
gat ctg tac gat ctt tcg acg ggg cct ttt tgg ttc gac ccc


 


193
AT45
Heavy chain CDR3
gtg gcc ggg gga tac ttc gat agt gct act cga ggc


 


194
AT47
Heavy chain CDR3
ggg ttt cac tat cat agt gct gat cag aga ata ttc gac ccc


 


195
AT49
Heavy chain CDR3
ttt ctt tgg ttc ggg gac caa acg agt gat gat ggt ttt gat





gtc


 


 


196
AT50
Heavy chain CDR3
ggg ggt gcc caa gag atg gtt aga ata cac tac tac tac tac





gga atg gac gtc


 


197
AT51
Heavy chain CDR3
ccc gca acc tca tat gac gat ctt cgg agt ggt tat ttg aac





tac tgt gac tac


 


198
AM22
Heavy chain CDR3
cta ggt gtg aca gtg act gag gct gga ctg ggg atc gat gac





tac


 


199
AT46
Light chain CDR1
act ctg agc agt ggg cac agg aac tac gcc atc gca


 


200
AT32
Light chain CDR1
aag tcc agc cag agt gtt tta tac gac tcc aac aat aag aac





tac tta gct


 


201
AT33
Light chain CDR1
tct gca gat gca ttt tca gac caa tat gct tat


 


202
AT34
Light chain CDR1
cgg gcc agt cag ggt att ggt agt tgg ttg gcc


 


203
AT35
Light chain CDR1
cgg gca agt cag agc att gac aac tat tta aat


 


204
AT36
Light chain CDR1
aag tcc agc cag agt ctt tta cac agc tcc aac aat aag atc





tac tta gct


 


205
AT37
Light chain CDR1
agg gcc agt cag agt gtt agc gcc agc aac tta gcc


 


206
AT39
Light chain CDR1
cag gcg agt cag gac att agc aac ttt tta aat


 


207
AT40
Light chain CDR1
cgg gcg agt cag ggt att agt acc tgg tta gcc


 


208
AT42
Light chain CDR1
agg gcc agt cag act gta agc agc agc cac tta gcc


 


209
AT43
Light chain CDR1
agg gcc agt gag agt gtt agc cgc aac tac tta gcc


 


210
AT44
Light chain CDR1
agg gcc agt cag agt gtc agc acc aag gta gtc


 


211
AT45
Light chain CDR1
agg tct agt cag agc ctc ctg cat agt aat gga tac aac tat





ttg gat


 


212
AT47
Light chain CDR1
cgg gcc agt gag agt att agt acc tgg ttg gcc


 


213
AT49
Light chain CDR1
agg tct agt cag agc ctc ctg cat ggt aat gga tac aaa tat





ctg cac


 


214
AT50
Light chain CDR1
cgg gca agc cag gtc att agc agt tat tta gcc


 


215
AT51
Light chain CDR1
cgg gca agt cag ggc att acc agt tat tta gcc


 


216
AM22
Light chain CDR1
agg gcc agt cag att gtt agc agg aac cac tta gcc


 


217
AT46
Light chain CDR2
act aat ggc agc cac tac ccg ggg gac


 


218
AT32
Light chain CDR2
tgg gcg tct acc cgg gaa tcc


 


219
AT33
Light chain CDR2
aaa gac act gag agg ccc tca


 


220
AT34
Light chain CDR2
aac gcg tct ggc tta gaa agt


 


221
AT35
Light chain CDR2
ctt gcg tcc act ttg caa agt


 


222
AT36
Light chain CDR2
tgg gca tct acc cgg gag tcc


 


223
AT37
Light chain CDR2
ggt gca tcc agg acg gcc act


 


224
AT39
Light chain CDR2
gat gcg tcc aaa ttg caa aca


 


225
AT40
Light chain CDR2
tct gca tcc aga ttg cag agt


 


226
AT42
Light chain CDR2
ggt tca tct agc agg gcc aca


 


227
AT43
Light chain CDR2
ggt gca tcc agc agg gcc att


 


228
AT44
Light chain CDR2
ggt gca tcc acc agg gcc act


 


229
AT45
Light chain CDR2
ggt tct aat cgg gcc ccc


 


230
AT47
Light chain CDR2
aag gcg tct agt tta gaa agt


 


231
AT49
Light chain CDR2
ttg ggt tct aat cgg gcc tcc


 


232
AT50
Light chain CDR2
ggt gca tcc acg tta caa act


 


233
AT51
Light chain CDR2
gct gca tcc act ttg caa agt


 


 


234
AM22
Light chain CDR2
ggt gcg tcc agt cgg gcc act


 


235
AT46
Light chain CDR3
cag acc tgg ggc gct ggc att


 


236
AT32
Light chain CDR3
caa caa tat tat gat cct ctc


 


237
AT33
Light chain CDR3
caa tca aca gac acc agt ggt cct tta


 


238
AT34
Light chain CDR3
caa caa tac aat agt cac acg


 


239
AT35
Light chain CDR3
caa cag agc cac tct tcc ccc


 


240
AT36
Light chain CDR3
cag caa tat tat act act cat ccc


 


241
AT37
Light chain CDR3
caa cag tat ggt agc tca ccg


 


242
AT39
Light chain CDR3
caa aag ttt gat aat ctc ctt


 


243
AT40
Light chain CDR3
caa cag gct aac act ttc ccc


 


244
AT42
Light chain CDR3
cag tac tat ggt gac tca ccc


 


245
AT43
Light chain CDR3
tgt cag cag tat act atc ttc cct


 


246
AT44
Light chain CDR3
cag cag tat aat aag tgg ccc


 


247
AT45
Light chain CDR3
atg caa gct cta caa act cct


 


248
AT47
Light chain CDR3
caa cag tat aaa agt tac ccg


 


249
AT49
Light chain CDR3
atg caa gct cta caa agt ccg


 


250
AT50
Light chain CDR3
caa cag ctt aat act tac ccc


 


251
AT51
Light chain CDR3
caa cag ttt cat act tac ccg


 


252
AM22
Light chain CDR3
ctg tcc tct gat tcc tcc ata


 


253
AT46
Heavy chain
gag gtg cag ctg gtg gag tct ggg gga ggc ttg gta cag cct ggg





ggg tcc ctg cga ctc tcc tgt gca gcc tct gga ttc acc ttt agt





aga tat gtc atg agt tgg gtc cgc cag gct cca ggg agg ggc ctg





gag tgg gtc tca agc att act gga agt ggt gct acg aca tac tat





gca gac tcc gtg aag ggc cgc ttc acc atc tcc aga gac aat tcc





aag aac acg gtg tat ctg caa atg aac agg ctg aga gcc gag gac





acg gcc ata tat tac tgt gcg aat tgt ggt agg gcg ggc caa att





ttt gac gac tgg ggc cag gga acc ctg gtc acc gtc tcc tca


 


254
AT32
Heavy chain
cag gtc cag ctg gta caa tct ggg gct gag atg aag aag cct ggg





gcc tca gtg aag gtc tcc tgc cag gtt gcc gga tac acc ctc act





gaa tta tcc ata cac tgg gtg cga cag act cct gga aac ggg ctt





gag tgg atg gga ggt ttt gag cct gag gat ggt gag tac atc tac





cca cag aaa tcc cag ggc aga gtc acc atg acc gag gac aca tct





aca ggc aca gcc tac atg gaa ctg agg agc ctg aga tct gac gac





acg gcc gtg tat tac tgt gca gcc gag gca aga tat tgt gat aac





agc aga tgt tcc cct aac ttt gac ac tgg ggc cag gga acc ctg





gtc gcc gtc tcc tca


 


255
AT33
Heavy chain
cag gtg cag ttg gtg cag tct ggg gct gag gtg aag aag cct ggg





tcc tcg gtg aag gtc tcc tgc aag gct tct gga gac tcc ttc aac





agt ctt gcc atc agt tgg gtg cga cag gcc cct gga caa gga ctc





gag tgg atg gga ggg atc atc cct aag ttc aat aga aga gac tac





gca cag aag ttt cag ggc aga gtc acg att acc gcg gac gac tcc





gcg agc aca gcc tac ata gag ttg agc agc ctg aca tct gac gac





aca gcc ctg tat tac tgt gcg aga gac gcc gag tgg gca gct ggc





tcg gat tac ttc ttt gac tac tgg ggc cag gga acc ctg gtc atc





gtc tcc tca


 


256
AT34
Heavy chain
cag gtg caa ttg atg gag tct ggg gga ggc gtg gtc cag cct ggg





aag tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttc agt





cat tat ggc atg cac tgg gtc cgc cag gct cca ggc aag ggg ctg





gag tgg gtg gca gtc ata tcc tat gat ggc gat aaa aaa tat tat





gca gac tca gtg aag ggc cga ttc acc atc tcc aga gac aat tcc





aag aac acg ctg cat ctc cac atg aat agc ctg aga cat gag gac





acg gct gtc tat ttc tgt gcc tcc cag ggg gca aag ggc ggt cac





gaa ctt tct ttc tac tgt gct ttg gac gtc tgg ggc caa ggg acc





acg gtc gcc gtc tcc tca


 


257
AT35
Heavy chain
gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtc cag ccg ggg





ggg tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttt agt





acc tat tgg gtg agc tgg gtc cgc cag act cca ggg aag gga ctg





gag tgg gtg gcc aac att aac caa gat gga agt gag aag tcc tat





gtg gac tct gtg gag ggc cga ttc acc atc tcc aga gac aac gct





aag aac tcg ctg tat ctg caa atg aac agc ctg aga gcc gac gac





acg gct gta tat tat tgt gcg aga gaa gtc ttc gtg act cag gtg





gag ccc gcg cag tgg ggc ttc tgg ggc cag gga acc ccg gtc atc





gtc tcc tcc


 


258
AT36
Heavy chain
cag gtt cag gtg gtg cag tct gga gcc gag gtg aag aag cct ggg





gcc tca gtc aag gtc tct tgc aag act tct ggt tac aac ttt atc





gac cat agt gtc agc tgg gtg cga cag gcc ccc ggc caa ggg ctt





gag tgg atg gga tgg atc agc cct tac aac cac aga aca gta tat





gca gag aag ttc cag ggc aga gtc acc atg acc aca gac aca tcg





acg agg aca gtc tcc atg gag ttg agg agg ctg aca tct gac gac





acg gcc gtc tac ttc tgt gcg cga gat cga gta caa cag ggc gag





gga aac ttc ttt gac cac tgg ggc cag gga acc ccg gtc acc gtc





acc tca gcc


 


259
AT37
Heavy chain
cag ctg cag ctg cag gag tcc ggc tcc aga ctg gtg aag cct tca





cag acc ctg tcc ctc acc tgc ggt gtc tct ggt ggc tcc atc agc





agt ggt ggt tac tcc tgg aac tgg atc cgg cag cca cca ggg aag





ggc ctg gag tgg gtt ggg tac atc tat cag aat gac atc acc tac





tac aac ccg tcc ctc atg agt cga gtc acc ata tca gca gac acg





tcc aag aac cag ttc tcc ctg aag ttg agc tct gtg acc gcc gcg





gac acg gcc gtg tat tac tgt gcc cga ggg gcc tat ggt tcg gga





act tat tat tcc gct gat gct ctt gat ata tgg ggc caa ggg aca





atg gtc acc gtc tct tca


 


260
AT39
Heavy chain
cag gtc cag ctt gtg cag tct ggg cct gag gtg aag aag cct ggg





gcc tca gtg agg ctt tcc tgt acg gcc tct gga aac acc ttc cgt





acc tat gct gta cat tgg gtg cgc cag gcc tcc gga caa aga ctt





gag tgg atg gga tgg atc aac cct gac aat ggt gac aca aaa tat





tca cag agg ttc cag ggt aga gtc gtc att acc agg gac aca tcc





gcg agg ata atc tac ttg gac ctg agc agc ctg aca tct gaa gac





acg gct gtg ttc tat tgt ttc agc ggg aga att ttt gat ata tgg





ggc caa ggg aca acg atc acc gtc tct tca


 


261
AT40
Heavy chain
cag gtg cag ctg gtg gag tcc ggg gga ggc gtg gtc cag cct ggg





atg tcc cac aga ctc tcc tgt gca gcc tct aca ttg atc ttc gat





aga cat gct ctc cac tgg gtc cgc cag gct cca ggc gcg ggc ctg





gag tgg gtg gcg att ctc tct tat gat ggg acc aca gac tac tac





gca gac tcc gtg aag ggc cga ttc acc gtc tcc aga gac acc tcc





aag aac aca gtg ttt cta caa atg aac ggc ctg aga cct caa gac





acg gct gtt tat tac tgt gcg aga gga agg gcc cta gat gac ttc





gct gac tac ggg gga tac tac ttt gac tac tgg ggc cag gga atc





ctg gtc acc gtc tcc tca


 


262
AT42
Heavy chain
cag gtg cag ctg cag gag tcc ggc cca gga ctg gtg cag cct tcg





gag acc ctg tcc ctc act tgc act gtt tct ggt gac tcc atc acc





agt aat gtt tac tac tgg ggc tgg atc cgc cag ccc cca ggg aag





ggg ctg gag tgg att ggg agt atc ttt cat agt ggg atc acc cac





tat acc ccg tcc ctc aat agt cga gtc acc ata tcc gtc gac acg





tcc aag aac cag ttc tcc ctg aga ctg agt tct gcg acc gcc gca





gac acg gct gta tat tat tgt gcg agg cat tgg gct ggc ctc tac





ttt gac tct tgg ggc cag gga gcc ctg gtc gcc gtc tcc tca


 


263
AT43
Heavy chain
cag gtt cag gtg gtg cag tct gga cct gag gtg aag aag cct ggg





gcc tca gtg agg gtc tcc tgc aag gct tct ggt tac acc ttt acc





aac tat ggt gtc agc tgg gtg cga cag gcc cct gga caa ggg ctt





gag tgg atg gga tgg atc agc act tac aat ggt aac aca tgg tat





tca cag aag ttc cag gcc aga gtc acc atg acc aca gac act tcc





acg agc aca gcc tac atg gag gtg agg agc ctg aga tct gac gac





acg gcc ata tat tac tgt gcg tgc cac ggg agt ggc aat tac tac





ggc gaa gcg aac tac ttt gac cac tgg ggc cag gga acc ctg gtc





acc gtc tcc tcc


 


264
AT44
Heavy chain
cag gtg cag ctg cag gcg tcg ggc cca gga ctg gtg aag cct tcg





gag acc ctg tcc ctc acc tgt aat gtc tct ggc tac tcc gtc agt





agc ggt cac tac tgg gcc tgg gtc cgg cag tcc cca ggg aag ggg





ctg gag tgg att ggg ggt atc cat cat agt ggg agt acc tac acc





aat ccg ccc ctc aag agc cga gtc tcc ata tca ata gac acg tcc





aag aac cag ttc tct ttg agg ttg acc tct gtg acc gcc gca gac





acg gcc gtg tat ttc tgt gcg aga gat ctg tac gat ctt tcg acg





ggg cct ttt tgg ttc gac ccc tgg ggc cag gga acc ctg gtc acc





gtc tcc tca


 


265
AT45
Heavy chain
cag gtg cac ctg gtg cag tct ggg gct gag gtg aag aag cct ggg





tcc tcg gtg aag gtc tcc tgc aag gct tct gga ggc acc ttc aac





ggc cat gct atc agc tgg ata cga cag gcc cct gga caa gga ctt





gag tgg aag gga ggg atc atc cct ggc ctt ggt aca aca agg tac





gca cgg aag ttc cag gac aga gtc acg att acc gcg gac gaa tcc





acg agg aca gcc tac atg gag ctg agc agc ctg aga tct gag gac





acg gcc gtc tat tac tgt gcg aga gtg gcc ggg gga tac ttc gat





agt gct act cga ggc tgg ggc cag gga acc ctg gtc acc gtc tcc





tca


 


266
AT47
Heavy chain
cag gtt cag ctg gtg cag tct gga ggt gag gtg aag aag cct ggg





gcc tca gtg aag gtc tcc tgt aag gct tct ggt tac acc ttt acc





aac tac ggt atc tgt tgg gtg cga cag gcc cct gga caa ggg ctt





gaa tgg atg gga tgg atc agc ggt tac aat ggt aac aca tac tat





gca cag aac ttc cag ggc aga gtc acc atg acc aca gac aca tcc





acg agc aca gcc tac atg gag ctg agg agc ctg aga tct gac gac





acg gcc gta tat tac tgt gcg aga ggg ttt cac tat cat agt gct





gat cag aga ata ttc gac ccc tgg ggc cag gga acc ctg gtc acc





gtc tcc tca


 


267
AT49
Heavy chain
cag gtg ctt ctg gtg cag tct ggg gct gag ata aag aag cct ggg





tcc tcg gtg aaa atc tcc tgc aag gcc tct gga ggg acc ttc agc





agc ctt gct ctc aat tgg gtg cga cag gcc cct gga cag ggg ctt





cag tgg atg gga ggg atc atc cct ctc ttt ggc act caa aac tac





gca cag aag ttc cag ggc aga gtc acc att acc gcg gac gaa tcc





acg agc aca gcc tac atg gag ctg agc ggc ctg cga ccc gag gac





acg gcc gtc tat tac tgt gcc cta ttt ctt tgg ttc ggg gac caa





acg agt gat gat ggt ttt gat gtc tgg ggc caa ggg aca gtg gtc





acc gtg tct tca


 


268
AT50
Heavy chain
cag gtt cag ctg gtg cag tct gga act gag gtg aag aag cct ggg





gcc tca gtg aag gtc tcc tgc aag gct tct ggt tac acc ttt agc





aac tat ggt atc agt tgg gtg cga cag gcc cct gga caa ggg ctt





gag tgg atg gga tgg atc agc gct tac aat ggt aac aca tac tat





aga cag gag ctc cag ggc aga gtc acc atg acc aca gac aca tcc





acg agc aca gcc tac atg gag ctg agg agc ctg aga tct gac gac





acg gcc gtg tat tac tgt gcg aga ggg ggt gcc caa gag atg gtt





aga ata cac tac tac tac tac gga atg gac gtc tgg ggc caa ggg





acc acg gtc acc gtc tcc tca


 


269
AT51
Heavy chain
cag gtt cag ctg gtg cag tct gga gct gag gtg aag aag cct ggg





gcc tca atg acg gtc tcc tgc aag gcc tct ggt tac acc ttt tcc





aag tat ggc atc aac tgg gtg cga cag gcc cct gga caa ggg ctt





gag tgg ctg ggt tgg atc agc gca tac aat ggc aac aca tac tat





gca cag aag ttc cag ggc aga gtc acc atg acc aca gac aca gcc





acg agc aca gcc tac atg gac gtg agg aac ctg aga tct gac gac





acg gcc atg tat tac tgt gcg agg ccc gca acc tca tat gac gat





ctt cgg agt ggt tat ttg aac tac tgt gac tac tgg ggc cag gga





acc ctg gtc acc gtc tcc tca


 


270
AM22
Heavy chain
cag gtc cag ctg gta cag tct ggg gct gag gtg aag aag ccc ggg





gcc aca gtg aaa gtc tcc tgc aag att tcc gga cac acc ctc att





aaa tta tcc att cac tgg gtg cga cag gct cct gga aag ggg ctt





gag tgg atg gga ggt tat gag ggt gag gtc gat gag att ttc tac





gca cag aag ttc cag cac aga ctc acc gtg atc gcc gac aca gcg





aca gac aca gtc tac atg gaa ctg ggc agg ctc acc tct gac gac





acg gcc gtc tat ttc tgt gga aca cta ggt gtg aca gtg act gag





gct gga ctg ggg atc gat gac tac tgg ggc cag gga acc ctg gtc





acc gtc tcc tca


 


271
AT46
Light chain
cag cct gtg ctg act caa tcg ccc tct gcc tct gcc tcc ctg gga





gcc tcg gtc aag ctc acc tgc act ctg agc agt ggg cac agg aac





tac gcc atc gca tgg cat cag cag cga cca gag aag ggc cct cgt





tac ttg atg aag att tat act aat ggc agc cac tac ccg ggg gac





ggg acc cct gat cgc ttc tca ggc tcc agc tct ggg gct gag cgc





tac ctc acc atc tcc agc ctc caa tct gag gat gag gct gac tat





tac tgt cag acc tgg ggc gct ggc att tgg gtt ttc ggc gga ggg





acc aag ctg acc gtc cta ggt cag ccc aag


 


272
AT32
Light chain
gac atc gtg atg acc cag tct cca gac tcc ctg gct gtg tct ctg





ggc gag agg gcc acc ttc agc tgc aag tcc agc cag agt gtt tta





tac gac tcc aac aat aag aac tac tta gct tgg tac cag cag aga





cca gga cag cct cct aag ttg ctc att tac tgg gcg tct acc cgg





gaa tcc ggg gtc cct gac cga ttc agt ggc agc ggg tct ggg aca





gat ttc act ctc acc atc agc agt ctg cag cct gaa gat gtg gca





gtt tat tac tgt caa caa tat tat gat cct ctc atc acc ttc ggc





caa ggg aca cga ctg gag att aaa cga act gtg


 


273
AT33
Light chain
tcc tat gag ctg act cag cca ccc tcg gtg tca gtg tcc cca gga





cag acg gcc agg atc acc tgc tct gca gat gca ttt tca gac caa





tat gct tat tgg tac cag cag aag cca ggc cag gcc cct gtg ttg





gtg ata tat aaa gac act gag agg ccc tca ggg atc cct gag cga





atc tct ggc tcc agc tca ggg aca aca gcc acg ttg agc atc agt





gga gtc cag gca gaa gac gag gct gac tat tac tgt caa tca aca





gac acc agt ggt cct tta ttc ggc gga ggg acg aag ctg acc ctc





cta ggt cag ccc aag


 


274
AT34
Light chain
gac atc cag atg acc cag tct cct tcc acc ctg tct gca tct gtg





gga gac aga gtc acc atc act tgt cgg gcc agt cag ggt att ggt





agt tgg ttg gcc tgg tat cag cag aaa cca ggg aaa gcc cca aaa





ctc ctg atc tat aac gcg tct ggc tta gaa agt ggc gtc cca tca





ggg ttc agc ggc agt gga tct ggg aca gag ttc act ctc acc atc





agc agc ctg cag cct gat gat tct gcg acg tat tac tgc caa caa





tac aat agt cac acg tgg aca ttc ggc caa ggg acc aag gtg gaa





ttc aag cga act gtg


 


275
AT35
Light chain
gcc atc cag atg acc cag tct cca tcc tcc ctg tct gca tct gta





gga gac aga gtc acc atc tct tgc cgg gca agt cag agc att gac





aac tat tta aat tgg tat cag cag aaa ccg ggg aaa gcc cct aaa





ctc ctg ctc ttt ctt gcg tcc act ttg caa agt ggt gtc cct tca





agg ttc act ggc agt gga tct ggg aca gat ttc act ctc acc atc





agc agt ctt caa cct gaa gat ttt gcg act tac tac tgt caa cag





agc cac tct tcc ccc tac agt ttt ggc cag ggg acc aag ctt gag





atc aaa cga act gtg


 


276
AT36
Light chain
gac atc gtg atg acc cag tct cca gac tct ctg gct gtg tct ctg





ggc gag agg gcc acc atc aac tgc aag tcc agc cag agt ctt tta





cac agc tcc aac aat aag atc tac tta gct tgg tac cag cag aaa





cca gga cag cct cct aag tta ctc ctt tac tgg gca tct acc cgg





gag tcc ggg gtc cct gac cgc ttc act ggc agc ggg tct ggg aca





gat ttc act ctc acc atc aac agc ctg cag gct gag gat gtg gct





gtt tat tac tgt cag caa tat tat act act cat ccc act ttt ggc





cag ggg acc agg ctg gag atc aaa cga act gtg


 


277
AT37
Light chain
aaa att gtg ttg acg cag tct cca ggc acc ctg tct ttg tct cca





ggg gaa aga gcc acc ctc tcc tgc agg gcc agt cag agt gtt agc





gcc agc aac tta gcc tgg tac cag cag aaa cct ggc cag gct ccc





agg ctc ctc atc tat ggt gca tcc agg acg gcc act ggc atc cca





gac agg ttc agt ggc agt ggg tct ggg aca gac ttc act ctc tcc





atc agc aga ctg gag cct gaa gat ttt gca gtg tat tac tgt caa





cag tat ggt agc tca ccg ctc act ttc ggc gga ggg acc aag gtg





gag atc aaa cga act gtg


 


278
AT39
Light chain
gac atc cag atg acc cag tct cca tcc tcc ctg tca gca tct gtg





gga gac aga gtc acc atc act tgc cag gcg agt cag gac att agc





aac ttt tta aat tgg tat cag cag aaa ccg ggc caa gcc cct aaa





ctc ctg atc tat gat gcg tcc aaa ttg caa aca ggg gtc ccg tca





agg ttc agt gga agt ggt tct gag aca gac ttt act ttc acc atc





agc agc ctg cag cct gaa gat gtt gca aca tat tac tgt caa aag





ttt gat aat ctc ctt ctc act ttc ggc gga ggg acc aag gtg gag





ctc aag cga act gtg


 


279
AT40
Light chain
gac atc cag atg acc cag tct cca tct tcc gta tct gcg tct gtg





gga gac aaa gtc acc atc acc tgt cgg gcg agt cag ggt att agt





acc tgg tta gcc tgg tat cag cag aaa cct ggg aaa gct cct gcc





ctc ctg ata tat tct gca tcc aga ttg cag agt ggg gtc ccc tca





agg ttt agc ggc agt gga tct ggg aca gat ttc act ctc acc atc





agc agc ctg cag cct gaa gat tat gca acc tat tat tgt caa cag





gct aac act ttc ccc ttc act ttc ggc cct ggg acc aaa gtg gac





atc aaa cga act gtg


 


280
AT42
Light chain
gaa atc gtg ttg acg cag tct cca ggc acc ctg tct ctg tct cca





ggg gaa aga gcc acc ctc tcc tgc agg gcc agt cag act gta agc





agc agc cac tta gcc tgg tac cag cag aaa cct ggc cag gct ccc





agg ctc ctc atc cat ggt tca tct agc agg gcc aca ggc atc cca





gag agg ttc agt ggc agt ggg tct ggg cca gac ttc act ctc acc





atc tcc aga ctg aag cct gaa gat ttt gct gtg tat tac tgt cag





tac tat ggt gac tca ccc ggc tct ttc ggc gaa ggg acc aag gtg





gag atc aaa cga act gtg


 


281
AT43
Light chain
gac att gtg ttg acg cag tct cca ggc acc ctg tct ttg tct cca





ggg gaa gga gcc acc ctc tcc tgc agg gcc agt gag agt gtt agc





cgc aac tac tta gcc tgg tac cag caa aaa cct ggc cag gct ccc





agg ctc ctc atc tat ggt gca tcc agc agg gcc att ggc atc cca





gac agg ttc agt ggc agt ggg tct ggg aca gac ttc act ctc acc





atc agc aga ctg gag cct gaa gat ttt gca gta tac tgc tgt cag





cag tat act atc ttc cct ctc act ttc ggc gga ggg acc aag gtg





gag atc aaa cga act gtg


 


282
AT44
Light chain
gaa atc gtg atg acg cag tca cca gcc acc ctg tct gtg tct cca





ggg gag aga gtc acc ctc tcc tgt agg gcc agt cag agt gtc agc





acc aag gta gtc tgg tac cag cag aaa ttt ggc cag gct ccc agg





ctc ctc atc tat ggt gca tcc acc agg gcc act ggt atc cca gtc





agg ttc agt ggc agt ggg tct ggg aca gag ttc act ctc acc atc





agc agc ctg cag tct gaa gat ctt gca gtt tat ttc tgt cag cag





tat aat aag tgg ccc atg tac act ttt ggc cag ggg acc aag ttg





gaa atc aaa cga act gtg


 


283
AT45
Light chain
gat att gtg atg act cag tct cca ctc tcc ctg ccc gtc acc cct





gga gag tcg gcc tcc atc tcc tgc agg tct agt cag agc ctc ctg





cat agt aat gga tac aac tat ttg gat tgg tac ctg cag aag cca





ggg cag tct cca cag ctc ctg atc tat ttg ggt tct aat cgg gcc





ccc ggg gtc cct gac agg ttt agt ggc agt gga tca ggc aca gat





ttt aca ctg aaa atc agc aga gtg gag gct gag gat gtt ggg gtt





tat tac tgc atg caa gct cta caa act cct acg ttc ggc caa ggg





acc aag gtg gaa atc aaa cga act gtg


 


284
AT47
Light chain
gac atc cag atg acc cag tct cct tcc acc ctg tct gca tct gta





gga gac aga gtc acc atc act tgc cgg gcc agt gag agt att agt





acc tgg ttg gcc tgg tat cag cag aaa cca ggg aaa gcc cct aac





ctc ctg atc tat aag gcg tct agt tta gaa agt ggg gtc cca tca





agg ttc agc ggc agt gga tct ggg aca gaa ttc act ctc gcc atc





agc agc ctg cag cct gat gat ttt gca act tat tac tgc caa cag





tat aaa agt tac ccg tac act ttt ggc cag ggg acc aag ctg gag





ctg aaa cga act gtg


 


285
AT49
Light chain
gat att gtg atg act cag tca ccg ctc tcc ctg acc gtc acc ccg





gga gag ccg gcc tcc atc tca tgc agg tct agt cag agc ctc ctg





cat ggt aat gga tac aaa tat ctg cac tgg tac ctg cag aag cca





ggg cag tct cca cag ctc ctg atc tat ttg ggt tct aat cgg gcc





tcc ggg gtc cct gcc agg ttc agt ggc agt gga tca gac aca gat





ttt act ctg aaa atc agc acc gtg gag act gag gat gtt ggg gtt





tat tac tgc atg caa gct cta caa agt ccg acg ttc ggc caa ggg





act aag gtg gaa atc aaa cga act gtg


 


286
AT50
Light chain
gac atc cag ttg acc cag tct cca tcc ttc ctg tct gca tct gta





gga gac aga gtc acc atc act tgc cgg gca agc cag gtc att agc





agt tat tta gcc tgg tat cag caa aca cca ggg aga gcc cct aag





ctc ctg atc tat ggt gca tcc acg tta caa act ggg gtc cca tca





agg ttc agc ggc agt gga tct ggg aca gaa ttc act ctc aca atc





agc agc ctg cag cct gaa gat ttc gca act tat ttc tgt caa cag





ctt aat act tac ccc ctc act ttc ggc cct ggg acc aaa gtg gag





atc aaa cga act gtg


 


287
AT51
Light chain
gac atc cag ttg acc cag tct cca tcc ttc ctg tct gca tct gta





gga gac aga gtc acc atc act tgc cgg gca agt cag ggc att acc





agt tat tta gcc tgg tat cag caa aaa cca ggg aga gcc cct aag





ctc ctg atc tat gct gca tcc act ttg caa agt ggg gtc gca tca





agg ttc agc ggc agt gga tct ggg aca gaa ttc act ctc aca atc





agc agc ctg cag cct gaa gat ttt gca act tat tac tgt caa cag





ttt cat act tac ccg ctc act ttc ggc gga ggg acc aag gtg gag





atc aaa cga act gtg


 


288
AM22
Light chain
gaa att gtg ttg aca cag tct cca ggc acc ctg tct ttg tct cca





gga gaa aga gcc acc ctc tcc tgc agg gcc agt cag att gtt agc





agg aac cac tta gcc tgg tac cag caa aaa cct ggc cag gct ccc





agg ctc ctc atc ttt ggt gcg tcc agt cgg gcc act ggc atc cca





gtc cgg ttc agt ggc agt ggg tct ggg aca gac ttc act ctc acc





atc aac gga ctg gcg cct gaa gat ttt gca gtt tac tac tgt ctg





tcc tct gat tcc tcc ata ttc aca ttc ggc cct ggg acc aag gtg





gat ttc aaa
















TABLE 2





Preferred combinations of RSV G-specific antibodies, according to the disclosure



















AT43 + AT49
AT43 + AT40





AT51 + AT34
AT51 + AT40


AT47 + AT44
AT47 + AT34
AT47 + AT49
AT35 + AT45
AT35 + AT44


AT35 + AT34
AT35 + AT49
AT35 + AT40


AT37 + AT45
AT37 + AT34
AT37 + AT49
AT37 + AT40


AT39 + AT45
AT39 + AT44
AT39 + AT34
AT39 + AT49
AT39 + AT40


AT32 + AT45
AT32 + AT44
AT32 + AT34
AT32 + AT49
AT32 + AT40


AT32 + AT31


AT33 + AT45
AT33 + AT44
AT33 + AT34
AT33 + AT49
AT33 + AT40


AT33 + AT42
AT33 + AT38
AT33 + AT50
AT33 + AT36
AT33 + AT46


AT42 + AT44


AT46 + AT38
AT46 + AT45
AT46 + AT44
AT46 + AT34
AT46 + AT49


AT46 + AT40


AT36 + AT45
AT36 + AT44
AT36 + AT34
AT36 + AT49


AT50 + AT44
AT50 + AT40


AT31 + AT44
AT31 + AT34
















TABLE 3





Particularly preferred combinations of RSV G-specific


antibodies, according to the disclosure



















AT43 + AT49






AT51 + AT34


AT47 + AT34


AT35 + AT45
AT35 + AT44
AT35 + AT34


AT37 + AT45
AT37 + AT34


AT39 + AT45
AT39 + AT34
AT39 + AT49
AT39 + AT40


AT32 + AT45
AT32 + AT44
AT32 + AT34
AT32 + AT40


AT32 + AT31


AT33 + AT45
AT33 + AT44
AT33 + AT34
AT33 + AT49
AT33 + AT40


AT33 + AT42
AT33 + AT38
AT33 + AT50
AT33 + AT46


AT46 + AT45
AT46 + AT44
AT46 + AT34


AT46 + AT40


AT36 + AT45
AT36 + AT34
AT36 + AT49


AT50 + AT44


AT31 + AT34
















TABLE 4







Summary of preferred RSV G-specific antibodies, according to the disclosure.














competition w/ antibodies binding the 






conserved RSV G domain
















CDC virus IC50
















AT No.
epitope
(ng/ml)
131-2G
3D3
synergy w/D25
















RSV A+
46
non-linear
325
No
No
Yes


RSV B+
42
non-linear
ND
Yes
partially
No



40

18
Yes
Yes
No



44

20
Yes
Yes
No



45

108
Yes
partially
No



49

ND
Yes
Yes
No



34

>1000
Yes
Yes
No


RSV A+
32-33-35

20-10-79
No
No
Yes



36-37-39

98 (AT39)
No
No
No



43-47-50-51
non-linear (43-47)
No
yes (47)
partially (51)
No
















TABLE 5







Binding of B cell supernatants containing anti-


RSV G IgG to RSV infected cells expressing native


viral proteins, detected with anti-huIgG-PE.














RSV X
RSV 2007-2



clone name
RSV A2
subtype A
subtype B







AT46
+
+
+



AT42
+
+
+



AT40
+
+
+



AT44
+
+
+



AT45
+
+
+



AT49
+
+
+



AT34
+
+
+



AT32
+
+
neg



AT33
+
+
neg



AT35
+
+
neg



AT36
+
+
neg



AT37
+
+
neg



AT39
+
+
neg



AT43
+
+
neg



AT47
+
+
neg



AT50
+
+
neg



AT51
+
+
neg



palivizumab
+
+
+



rD25
+
+
+



ctrl anti IgG-PE
neg
neg
neg

















TABLE 6







Summary of antibody binding to RSV infected


cell lysates by ELISA and Western Blot.











antibody clone
ELISA
WB







AT46
−
−



AT42
−
−



AT40
+
+



AT44
+
+



AT45
+
+



AT49
+
+



AT34
+
+



AT32
+
+



AT33
+
+



AT35
+
+



AT36
+
+



AT37
+
+



AT39
+
+



AT43
+/−
+/−



AT47
+/−
+/−



AT50
+
+



AT51
+
+

















TABLE 7a







ka, kd and KD of antibodies AT40, AT44, AT32, AT42


and AT49 to RSV Ga. ka is indicated in 104 sec−1*M−1,


kd in 10−4 sec−1, KD in nM. Constants were fitted in


Scrubber2, using a global fit to all SPR curves.












Antibody:
ka:
kd:
KD (RSV A2 (G)):







AT32
75 (±21)
4.3 (±0.3)
0.6 (±0.1)



AT40
35 (±2)
0.6 (±0.1)
0.2 (±0.01)



AT42
64 (±11)
7.6 (±1.7)
1.3 (±0.4)



AT44
35 (±4)
0.3 (±0.1)
0.1 (±0.02)



AT49
22 (±3)
0.3 (±0.04)
0.1 (±0.01)

















TABLE 7b







ka, kd and KD of antibodies AT40,


AT44, AT42 and AT49 to RSV Gb.












Antibody:
ka:
kd:
KD (RSV G (B1)):







AT40
34 (±10)
0.3 (±0.15)
0.1 (±0.07)



AT42
40 (±14)
1.0 (±0.11)
0.3 (±0.1)



AT44
34 (±16)
0.4 (±0.17)
0.1 (±0.07)



AT49
12 (±6)
0.5 (±0.02)
0.5 (±0.2)













BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Amino acid sequence of RSV G protein of two subtypes A (Ga) A2 and Long strain and of three subtypes B (Gb) viruses from the USA (the B1 strain), Turkey and Uruguay (SEQ ID NOS 289-293, respectively). * indicate conserved/identical amino acid residues.
FIG. 2. Screening B cell supernatants for specificity to the RSV F and G protein. RSV A2 infected, PKH2 Green Fluorescent labeled HEp2 cells were mixed with PFA fixed RSV G expressing VERO cells and incubated with 20 cell/well B cell culture supernatant. IgG antibodies that bound the HEp2 or VERO cells were detected with a mouse anti-human IgG-PE. Antibodies present in the B cell culture supernatant that bound the RSV infected HEp2 cells (population 1) but not the G expressing VERO cells (population 2) presumably recognize the RSV F protein. Antibodies recognizing both cell lines most likely recognize the RSV G protein.
FIG. 3. Neutralization of RSV A2 by anti-RSV G protein specific human monoclonal antibodies in the absence and presence of complement. (A) Before RSV A2 was administered to HEp2 cells in 96-well plates, the virus was co-incubated with purified, recombinant AT32, AT33, AT40, AT42, AT44, AT46 and AT47 at 27 μg/ml. When combinations of 3 mAbs were tested the final concentration was 27 μg/ml, thus 9 μg/ml of each antibody. (B) Monoclonal B cell supernatant with anti-RSV G specific antibodies were first incubated with RSV virus for 1 hour at 37° C. before 10% rabbit serum complement was added for another hour in the presence of HEp2 cells. Cells were washed and cultured for 2 more days in normal culture medium.
FIG. 4. Enhanced neutralization of RSV A2 virus by combinations of anti-RSV G and F protein specific antibodies. (A) Increasing amounts of RSV F specific antibodies and a fixed amount (500 ng/ml) of anti-RSV G antibody were co-incubated with 25 PFU of RSV A2 virus for 1 hour at 37° C. Subsequently, the virus antibody mixture was added to 20,000 HEp2 cells in solution in 96-well flat-bottom culture plates. After two days, the number of infected foci was determined. Antibody virus combinations were tested at least three times; FIG. 4B shows the average increase compared to the F antibody alone.
FIG. 5. 3D3 antibody-binding competition for the conserved domain on the RSV G protein. To analyze if the anti-RSV G antibodies bind within the conserved region of the RSV G protein we performed antibody competition assays. The antibodies were compared to 3D3, which binds the epitope HFEVFNFVP (aa 164-172, FIG. 1, US patent application US 2010-0285022 and Collarini et al. J Immunol (2009) 183: 6338-6345). 3D3 was directly labeled with ALEXA Fluor 647 (Molecular Probes) and antibody competition was determined by incubation of RSV-infected HEp2 cells with an increasing dose of the non-labeled antibody before the labeled antibody was added at a standard concentration. In addition, the assay was also performed by simultaneously incubation of the labeled and non-labeled antibodies, in general no differences between the two methods was detected. Shown in FIG. 5 is the average binding of ALEXA Fluor 647 labeled 3D3 antibody relative to the control of three separate experiments.
FIG. 6. Western Blots showing binding of RSV G-specific antibodies to RSV A2 supernatant (denatured), detected with anti-human IgG IR-dye.
FIG. 7. (A) Example of recombinant full-length RSV Ga protein binding to human anti-RSV G antibodies captured on a SPR anti-human IgG chip. (B) Graph summarizes the binding of RSV G antibodies to biotinilated 12-mer peptides that were coupled to streptavidin coated on an IBIS SPR chip. The peptide library spans the amino acid sequence 149 to 199 of the RSV A2 strain. (C) Amino acid epitope recognize by the antibodies 3D3, AT40, AT44, 131-2G and AT32.



DETAILED DESCRIPTION
Examples
Example 1: Generation of Human Monoclonal Antibodies Against the RSV G Protein by Transduction of Human Peripheral Blood Memory, IgG+ B Cells by BCL6 and Bcl-xL
Materials and Methods
B Cell Isolation
B cells were obtained from PBMCs from 40 to 50 ml Peripheral blood of three healthy adult volunteers by density gradient separation using Lymphoprep (Axis-Shield PoC, Oslo, Norway) and CD22 MACS microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany). IgG memory B cells were isolated as CD19+CD3−CD27+IgM−IgA− population by FACSAria (Becton Dickinson, San Jose, Calif., USA).
The following mAbs against the human molecules CD3 (SK7), CD19 (SJ25C1), CD27 (O323; eBioscience), IgA (F(ab)2; DAKO Glostrup Denmark), IgD (IA6-2), IgG (G18-145), IgM (G20-127) (BD), Ig-kappa (F(ab)2; DAKO, G20-193), and Ig-lambda (F(ab)2; JDC12, DAKO) were directly labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), phycoerythrin cyanine 5, (PE-Cy5), allophycocyanin (APC), phycoerythrin-indotricarbocyanine (PE-Cy7) or allophycocyanin-indotricarbocyanine (APC-Cy7) and were purchased from BD-Pharmingen (San Diego, Calif.) unless otherwise indicated. Stained cells were analyzed on an LSRII or FACSCanto (BD) and flow cytometry data were processed using FlowJo software (Tree Star, Ashland, Oreg., USA).
Retroviral Transduction
Use of the BCL6 and Bcl-xL retroviral construct has been described previously (Kwakkenbos et al. Generation of stable monoclonal antibody-producing B cell receptor-positive human memory B cells by genetic programming. Nature Medicine (2010) vol. 16 (1) pp. 123-8). Briefly, cDNAs encoding human BCL6, Bcl-xL and EGFP were cloned into the LZRS retroviral vector and retrovirus was generated by transfection Phoenix packaging cells (Shvarts et al. A senescence rescue screen identifies BCL6 as an inhibitor of anti-proliferative p19(ARF)-p53 signaling. Genes Dev (2002) 16:681-686). After enrichment (by ficoll density gradient and high speed cell sorting (FACSAria, BD)) and activation of human peripheral memory B cells on CD40L-L cells in the presence of rmIL-21, the cells were transduced. (Diehl et al. STAT3-mediated up-regulation of BLIMP1 is coordinated with BCL6 down-regulation to control human plasma cell differentiation. J Immunol (2008) 180(7):4805-15). Transduced cells express EGFP and can be sorted to enrich for cells that besides EGFP will express BCL6 and Bcl-xL.
B Cell Culture and Screening of Anti-RSV G Protein Specific B Cells
After 4 days from transduction, GFP positive cells were sorted by FACSAria, plated at 20 cells per well in ten 96-well flat-bottom tissue culture-treated plates per donor. After 14 days in culture, B cells and supernatants were harvested. B cells were frozen and supernatants were tested for binding capacity to RSV A2 virus infected HEp-2 cell. In brief, HEp-2 cell culture monolayers were infected with RSV A2 virus at a MOI of 2-3. The infected HEp-2 cells were harvested 48 hours after infection. Cells were stained with PKH2 Green Fluorescent Cell Linker Kit (Sigma-Aldrich, St. Louis, Mo., USA). In addition, B cell supernatants were screened simultaneously on paraformaldehyde (PFA) fixed RSV G protein transduced VERO cells (kindly provided by Myra Widjojoatmodjo, NVI, Bilthoven, The Netherlands) (FIG. 2). A mixture of 2.5E4 PKH2 stained RSV A2 virus infected HEp-2 cell and 2.5E4 RSV G protein expressing VERO cells were incubated for 1 hour at 4° C. with 100 μl of supernatant. Cells were washed once with IMDM supplemented with 1% FBS. IgGs binding to the target cells were detected with PE labeled anti-human IgG (SouthernBiotech, Birmingham, Ala., USA).
Double positive cells were plated at 1 cell per well in 96-well flat-bottom tissue culture-treated plates by FACSAria to obtain single clones. B cells were maintained in standard culture medium containing IMDM (Invitrogen), 8% FBS (HyClone) and penicillin/streptomycin (Roche) and were co-cultured on irradiated (50Gy) mouse L cell fibroblasts stably expressing CD40L (CD40L-L cells, 10E5 cells/ml) and recombinant mouse IL-21 (25 ng/ml, R&D systems, Minneapolis, Minn., USA). After 14 days in culture, supernatants were collected to test binding capacity to A2 virus infected HEp-2 cell by FACS. Table 4 shows an overview and some characteristics of the final 17 B cell clones of which recombinant antibodies were generated. Table 5 shows binding of the antibodies to Hep2 cells infected with RSV A2, RSV X (both subtype A viruses) and RSV 2007-2 (an RSV subtype B virus).
Cloning of Anti-RSV G Monoclonal Antibodies
Total RNA was isolated from approximately 5E5 monoclonal B cells with TRIzol® (Invitrogen). cDNA was generated and subjected to PCR to produce heavy and light chain fragments using 1U AmpliTaq Gold DNA polymerase (Applied Biosystems Inc. Foster City, Calif., USA). PCR products were run on agarose gels, purified and cloned into the pCR2.1 TA cloning vector according to manufacturers' recommendations (Invitrogen). Sequence analysis was performed using BigDye Terminator chemistry (Applied Biosystems Inc.) and Vector-NTI software (Invitrogen). To rule out reverse transcriptase and/or DNA polymerase induced mutations, several independent cDNA conversions and PCR reactions were performed and individually cloned and sequence analyzed.
IgG ELISA
Plates were coated with either anti-human IgG Fc-fragment (Jackson ImmunoResearch Laboratories, Bar Harbor, Me., USA) at 10 μg/ml in PBS for 1 hour at 37° C. or o/n at 4° C. and washed in ELISA wash buffer (PBS, 0.5% TWEEN®-20). 4% Protifar (Nutricia, Zoetermeer, The Netherlands) in PBS was used as blocking agent, before serial dilution of cell culture supernatants and enzyme-conjugated detection Abs were added (dilutions 1:2500 for HRP-conjugated anti-IgG (Jackson ImmunoResearch Laboratories, Inc.). TMB substrate/stop solution (Biosource, Carlsbad, Calif., USA) was used for development of the ELISAs.
Example 2: Functional Testing of 17 Unique, Fully Human Anti-RSV G Protein Specific Antibodies RSV Culture and Neutralization Assay
An RSV A2 virus stock was obtained from supernatant of 3 day infected HEp2 cells maintained in standard culture medium. Supernatants were centrifuged and filtered (0.22 μM filter, Millipore). Subsequently aliquots were snap-frozen, stored in liquid nitrogen and virus titer was determined by standard TCID50 and PFU assay on adherent HEp2 cells. For neutralization assays 10E4 HEp2 cells were seeded in flat-bottom 96-well plates (Costar, Schiphol-Rijk, Netherlands) in standard culture medium. The next day 100TCID50 of RSV A2 and B cell culture supernatant were pre-incubated in the absence or presence of 10% rabbit complement serum (Sigma-Aldrich) before being added in triplicate to HEp2 cells for 1 hour at 37° C. After two days, cells were fixed with 80% acetone and stained with polyclonal anti-RSV-HRP (Biodesign, Kennebunk, Me., USA). 3-Amino-9-ethylcarbazole (AEC) was added for detection and visualization of RSV plaques by light microscopy (plaques were counted). In addition, RSV infected cells could also be stained with polyclonal goat anti-RSV directly labeled with-Alexa Fluor 647 (Molecular Probes). Fluorescent signal was detected with and analyzed by the automated fluorescent microscoop (Operetta, Perkin Elmer). Palivizumab (Medlmmune, Gaithersburg, Md., USA) and D25 (WO 2008/147196) were used as positive control for RSV neutralization.
Results
RSV A2 neutralization experiments with antibodies derived from monoclonal B cell cultures did not result in neutralization in the absence of rabbit serum complement. In general antibody IgG concentrations in B cell supernatant vary between 600 and 2000 ng/ml, which could be too low. When we used increased concentrations of recombinant, purified monoclonal antibodies we did found that AT44 and AT47 could reduce virus infection (FIG. 3a, top panels). AT40, AT33 and AT42 did so only partially. This effect was not seen for the other 12 anti-RSV G antibodies (not shown). More interestingly, we found that combinations of anti-RSV G antibodies were able to neutralize the virus up to 50-60% without the addition of complement (FIG. 3a bottom panels).
Besides the direct neutralization we could identify a large group (9 out of 17) of monoclonal antibodies that neutralized RSV when virus and B cell culture supernatant were co-incubated with 10% rabbit serum complement thereby inducing complement dependent cytotoxicity (CDC) (FIG. 3b). IC50 values were between 10 and 325 ng/ml.
Not all antibodies did broadly recognize RSV-A and RSV-B strains. Depicted in Table 5 is the binding of antibodies to HEp2 cells infected with the RSV A2, RSV-X (subtype A) and a RSV-2007-2 strain of the B subtype (also summarized in Table 4).
Example 3: Synergistic Effect of RSV G Protein Specific Antibodies on the Neutralizing Capacity of Anti-RSV F Antibodies
The role of the G protein on the surface of the RS virus is thought to be associated with target cell attachment. But also for other (unknown) process and mechanisms the G protein could be important, for example, the stabilization of the F protein trimer. It has been shown that the two proteins form a complex (Low et al., The RSV F and G glycoproteins interact to form a complex on the surface of infected cells, Biochemical and Biophysical Research Communications (2008) 366(2):308-13) and it has been shown that an anti-RSV G and RSV-F antibody in vivo can reduce virus titers in mice (Haynes et al., Therapeutic Monoclonal Antibody Treatment Targeting Respiratory Syncytial Virus (RSV) G Protein Mediates Viral Clearance and Reduces the Pathogenesis of RSV Infection in BALB/c Mice, J. Infect. Dis. (2009) 200(3):439-47). Without being bound by theory, antibodies directed against RSV G may influence the interaction between F and G and thereby induce 1) destabilization of the F trimer or 2) expose epitopes on the F trimer that become better accessible for anti-F antibodies; in either situation the F trimer may unfold to its post-fusion state and thereby become non-functional.
To study this we incubated RSV with increasing doses of anti-F antibodies e.g. D25, AM14 and palivizumab and with G specific antibodies (increasing concentrations or fixed at 500 ng/ml). As shown in FIG. 4a we observed that recombinant purified AT46 and AT32 did enhance the neutralizing capacity of AM14 and D25 but less of palivizumab. The synergistic effect was mainly seen at lower concentrations of anti-F antibody, the effect was consistent (the data shown is an average of three or more experiments) and the synergistic effect enhanced neutralization of the F antibodies by a factor 2 (FIG. 4b). Thus the G specific antibodies may induce changes in the presentation and/or stability of the F protein making the F protein more susceptible to neutralization by F specific antibodies.
Example 4: Direct Labeling of Purified Antibodies to Determine Antibody-Binding Competition by FACS
The RSV G protein can bind to the CX3C chemokine receptor 1 (CX3CR1) also named fractalkine receptor or G-protein coupled receptor 13 (GPR13). CX3CR1 is expressed on multiple cell lineages (NK cells, monocytes, Th1 CD4+ T cells and CD8+ T cells, mast cells and B cells. The ligand for CX3CR1, CX3CL1 induces adhesion of leukocytes when the chemokine is expressed as a membrane-anchored protein whereas the soluble form of CX3CL1 induces chemotaxis of leukocytes. The RSV G protein contains a conserved epitope (CWAIC residue 182 to 186, FIG. 1) that mimics the CX3CR1 binding epitope of CX3CL1. Antibodies exist that bind RSV G within the larger conserved domain (aa 169 to 191) and thereby (partially) compete with binding to CX3CR1 (Mekseepralard et al. Protection of mice against Human respiratory syncytial virus by wild-type and aglycosyl mouse-human chimaeric IgG antibodies to subgroup-conserved epitopes on the G glycoprotein. J. Gen. Virol. (2006) 87(Pt 5):1267-73). To analyze if the anti-RSV G antibodies disclosed herein bind similar epitopes we performed antibody competition assays. Antibodies disclosed herein were compared to 3D3 from Trellis Bioscience which binds the epitope HFEVFNFVP (aa 164-172, FIG. 1, U.S. patent application US 2010-0285022, and Collarini et al. Potent high-affinity antibodies for treatment and prophylaxis of respiratory syncytial virus derived from B cells of infected patients. J. Immunol. (2009) 183: 6338-6345). 3D3 was directly labeled with ALEXA Fluor 647 (Molecular Probes) and antibody competition was determined by incubation of RSV-infected HEp2 cells with an increasing dose of the non-labeled antibody before the labeled antibody was added at a standard concentration. In addition, the assay was also performed by simultaneous incubation of the labeled and non-labeled antibodies, in general no differences between the two methods were detected. Shown in FIG. 5 is the average binding of ALEXA Fluor 647 labeled 3D3 antibody relative to the control of three separate experiments. Binding of the 3D3 antibody can be out-competed by itself and antibodies that bind a similar or proximal epitope like the mouse antibody 131-2G (epitope HFEVF). Of the antibodies that bind RSV Ga and Gb (left panel), AT40, AT44 and AT34 strongly reduced 3D3 binding, suggesting that they compete for the same or proximal epitopes. AT42, AT45 and AT49 only partially compete with 3D3, which may suggest that they recognize different epitopes but may sterically hinder 3D3 from efficient binding. The antibody AT46 did not interfere with 3D3 binding. The right panel indicates competition of Ga specific antibodies with 3D3. None of the antibodies (AT32, 33, 36, 37, 39, 43, 50 and 51) did interfere with 3D3 binding, which indicates that they all recognize different RSV Ga specific epitopes.
Example 5: Binding of Anti-RSV G Antibodies in ELISA, SPR and WB
Antibodies, especially human antibodies, which contain relatively long variable domains (CDR regions), often recognize non-linear structures within their putative target. These non-linear structures can be disrupted by standard purification methods, which, for example, include denaturing compounds like TWEEN®. Our B cell technology is utmost suitable to screen for antibodies that recognize these non-linear structures since the method allows for functional screening of antibodies. However, this implies that not all antibodies discovered will recognize its putative target in standard binding assays like western blot (WB), surface plasma resonance (SPR) or ELISA. Besides AT46, AT42, AT43 and AT47 all antibodies gave clear signals in the ELISA (Table 6).
For the RSV ELISA 2 ml of 1% TRITON X®-100 in PBS was added to a cell pellet containing RSV infected Hep2 cells. The lysed cells were mixed thoroughly and kept for 5′ at RT before 10 ml of ice cold PBS was added. The mixture was homogenized using a syringe with needle and cleared though a 0.22 μm filter (Millipore) or centrifuged at 5,000 rpm at 4° C. for 5′. Subsequently, the lysate was dialyzed against 1 L PBS overnight at 4° C. After dialyzation, 0.05% NaN3 was added and samples were stored at 4° C. until use, or stored at −80° C. for long-term storage.
ELISA plates were coated with a lysate of RSV infected HEp-2 cells in PBS for 1 hour at 37° C. or o/n at 4° C. and washed in ELISA wash buffer (PBS, 0.5% TWEEN®-20). Plates were blocked by incubation with 4% milk in PBS, before the anti-RSV antibodies or polyclonal goat anti-RSV (Biodesign) in combination with enzyme-conjugated anti-IgG antibodies were added (dilutions 1:2500 for HRP-conjugated anti-IgG (Jackson). TMB substrate/stop solution (Biosource) was used for development of the ELISAs.
To confirm antibody binding to RSV G, western blots were prepared which were loaded with denatured and boiled supernatants of RSV A2 infected HEp2 cells. These supernatants contain relatively high amounts of the secreted form of RSV G. Summarized in Table 6 and shown in FIG. 6 is a western blot on which antibodies that recognize RSV Ga only (AT32, 33, 35, 36, 37, 39, 50 and 51), bind relative strong to the RSV G protein. From the same group of RSV Ga only binding antibodies, AT47 and AT43 only weakly bind RSV G. From the panel of antibodies that recognize RSV Ga and Gb on infected cells only AT40, 44, 45, 49 and 34 recognize RSV Ga by western blot. AT46 and AT42 do not bind.
In addition, we generated surface plasmon resonance (SPR) data with the IBIS MX96 instrument (Krishnamoorthy et al., Electrokinetic label-free screening chip: a marriage of multiplexing and high throughput analysis using surface plasmon resonance imaging, Lab Chip (2010) 10(8):986-90; van Beers et al., Mapping of citrullinated fibrinogen B-cell epitopes in rheumatoid arthritis by imaging surface plasmon resonance, Arthritis Research & Therapy (2010) 12(6):R219; Krishnamoorthy et al., Electrokinetic lab-on-a-biochip for multi-ligand/multi-analyte biosensing, Anal. Chem. (2010) 82(10):4145-50; de Lau et al., Lgr5 homologues associate with Wnt receptors and mediate R-spondin signaling, Nature (2011) 476(7360):293-7). The rate- and affinity constant of the antibodies were determined using a similar method as described in de Lau et al., Nature (2011).
Briefly, pre-activated SPR sensor chips (IBIS Technologies, Hengelo, Netherlands) were coated with an array of anti-human IgG specific spots (goat anti-human IgG, polyclonal, Fc-specific, Jackson ImmunoResearch Laboratories, Bar Harbor, Me., USA) using a continuous flow microspotter (CFM) (Wasatch Microfluidics, Salt Lake City, Utah, USA). After preparing the sensor chip, the chip was placed in the instrument and treated with RSV-G-specific human IgG. Each anti-IgG spot in the array thus captured a decreasing amount of IgG. After measuring a new baseline for each spot, purified RSV-Ga or Gb protein (Sino Biologics, Beijing, China) was injected to determine label-free surface plasmon resonance (Table 7a, 7b and FIG. 7a).
Kinetic parameters were calculated using Sprint 1.6.8.0 (IBIS Technologies, Hengelo, Netherlands) and Scrubber2 software (BioLogic software, Campbell, Australia). Results between experiments are comparable, the antibodies form stable complexes with the RSV G protein and similar ka, kd and KD were generated. In contrast to the WB and ELISA data we do find binding of the AT42 antibody to RSV Ga and RSV Gb protein in the IBIS SPR (Tables 7a and 7b). AT46 did not bind to either the recombinant or denatured form of the protein, probably because AT46 binds to a conformational epitope, which is not present in the recombinant and denatured form of the G protein because their conformation differs from that of the protein expressed on the surface of the RS virus.
Example 6: Determination of the Epitope of Anti-RSV-G Antibodies
In addition, we performed studies to precisely determine the epitopes recognized by several anti-RSV G antibodies, according to the disclosure. Therefore, we generated 40 peptides containing a 5′ biotin molecule plus a spacer followed by 12-successive amino acids, spanning the amino acid domain 149 to 199 of the RSV A2 G protein. This domain contains the conserved region, which is also recognized by the 131-2G and 3D3 antibody (FHFEVFNFV) and the cysteine rich domain forming the fractakine binding epitope (CWAIC). To detect binding to the peptides we obtained streptavidin-coated sensor chips (IBIS Technologies, Hengelo, Netherlands), on which the biotin labeled peptides were spotted using the CFM. Subsequently, the antibodies were run one by one over the chip at four different concentrations, after each run the chip was regenerated. Since the peptides were still present after regeneration, this indicated that the immobilized strepativin-biotin/peptides complexes were very stable.
FIG. 7b shows the maximum response observed in the SPR instrument when antibodies recognized a certain peptide (1 to 40, as depicted below). The height of the signal is influenced by the affinity of the antibody for the peptide, the concentration of the antibody, the amount of peptide immobilized and the conformation/polarity of the peptide on the sensor chips (polarity of the FHFEVFNF is low). Together we can conclude that the epitope recognized 3D3, 131-2G, AT40 and AT44 are in close proximity of each other (FIGS. 7b and 7c). Antibodies AT42, AT46 and AT49 did not recognize any of the captured peptides on the chip (not shown), indicating that the epitope of these antibodies is at least partly located outside the amino acid domain 149 to 199 of the G protein or that these antibodies recognize a conformation not present when the peptides are captured on the chip.
The domain described for the 131-2G antibody; HFEVF (Tripp et al. CX3C chemokine mimicry by respiratory syncytial virus G glycoprotein, Nat. Immunol., 2001) could be confirmed by us. Regarding 3D3 we find that the antibody binds to the residues FHFEVFNF as core residues and FHFEVFNFV as the complete epitope. The epitope published for 3D3 is HFEVFNFVP (Collarini et al., Potent high-affinity antibodies for treatment and prophylaxis of respiratory syncytial virus derived from B cells of infected patients, J. Immunol., 2009), we, however, find one more residue at the beginning, (F163) which is necessary.
AT40 and AT44 both start at residue 165F. AT40's epitope then continues till residue F170, making the epitope consist of FEVFNF. AT44 needs at least residue E166 till F170, making the complete epitope ranging from EVFNF. To our current knowledge these antibody epitopes have never been described before. Antibody AT32, which only binds to RSV subtype A viruses did bind to the more distal epitope RIPNK (position 188 to 192), an epitope located just after the fractalkine binding site.









 1 KQRQNKPPSKPN



 



 2 QRQNKPPSKPNN



 



 3 RQNKPPSKPNND



 



 4 QNKPPSKPNNDF



 



 5 NKPPSKPNNDFH



 



 6 KPPSKPNNDFHF



 



 7 PPSKPNNDFHFE



 



 8 PSKPNNDFHFEV



 



 9 SKPNNDFHFEVF



 



10 KPNNDFHFEVFN



 



11 PNNDFHFEVFNF



 



12 NNDFHFEVFNFV



 



13 NDFHFEVFNFVP



 



14 DFHFEVFNFVPC



 



15 FHFEVFNFVPCS



 



16 HFEVFNFVPCSI



 



17 FEVFNFVPCSIC



 



18 EVFNFVPCSICS



 



19 VFNFVPCSICSN



 



20 FNFVPCSICSNN



 



21 NFVPCSICSNNP



 



22 FVPCSICSNNPT



 



23 VPCSICSNNPTC



 



24 PCSICSNNPTCW



 



25 CSICSNNPTCWA



 



26 SICSNNPTCWAI



 



27 ICSNNPTCWAIC



 



28 CSNNPTCWAICK



 



29 SNNPTCWAICKR



 



30 NNPTCWAICKRI



 



31 NPTCWAICKRIP



 



32 PTCWAICKRIPN



 



33 TCWAICKRIPNK



 



34 CWAICKRIPNKK



 



35 WAICKRIPNKKP



 



36 AICKRIPNKKPG



 



37 ICKRIPNKKPGK



 



38 CKRIPNKKPGKK



 



39 KRIPNKKPGKKT



 



40 RIPNKKPGKKTT

## Claims

1. A composition comprising: a combination of AT44, AT42 and AT33; wherein said AT44, said AT42 and said AT33 are each isolated, synthetic or recombinant antibodies, or antigen binding fragments thereof; said AT44 comprises SEQ ID NO: 2, SEQ ID NO: 20, SEQ ID NO: 38, SEQ ID NO: 56, SEQ ID NO: 74 and SEQ ID NO: 92; said AT42 comprises SEQ ID NO:10, SEQ ID NO: 28, SEQ ID NO: 46, SEQ ID NO: 64, SEQ ID NO: 82 and SEQ ID NO: 100; and said AT33 comprises SEQ ID NO: 3, SEQ ID NO: 21, SEQ ID NO: 39, SEQ ID NO: 57, SEQ ID NO: 75 and SEQ ID NO: 93.