FIELD OF THE INVENTION

The invention relates to medicine. The invention in particular relates to antibodies and antigen-binding fragments that specifically bind to the attachment glycoprotein (G protein) of Respiratory Syncytial Virus (RSV) and that neutralize RSV. The invention also relates to diagnostic, prophylactic and therapeutic methods using anti-RSV antibodies.

BACKGROUND OF THE INVENTION

Human respiratory syncytial virus (RSV) is a negative-sense, single-stranded RNA virus of the family Paramyxoviridae which also includes common respiratory viruses such as those causing measles and mumps. There are two primary RSV subtypes: subtype A and subtype B. RSV replicates in the upper respiratory track and then spreads to the lower airways leading to bronchiolitis or pneumonia. The virus causes inflammation, edema of the airways, increased mucus production, and breakdown of respiratory epithelium.

An estimated 64 million cases of respiratory illness and 160,000 deaths worldwide are attributable to RSV -induced disease. Severe RSV infection occurs most often in children and infants, especially in premature infants. Underlying health problems such as chronic lung disease or congenital heart disease can significantly increase the risk of serious illness. RSV infections also can cause serious illness in the elderly, individuals with chronic pulmonary disease and in immunocompromised adults, such as bone marrow transplant recipients.

Several approaches to the prevention and treatment of RSV infection have been investigated. Intravenous immunoglobulin (RSV-IGIV; RespiGam®) isolated from donors, and the monoclonal antibody palivizumab (SYNAGIS ^® ) have been approved for RSV prophylaxis in high-risk premature infants. A vaccine or commercially available treatment for RSV, however, is not yet available. Only ribavirin, a RNA inhibitor, is approved for treatment of RSV infection. In order to be effective for treatment of RSV infection, high doses, repeated administrations and/or large volumes of antibody products, such as palivizumab, are required due to low effectivity.

RSV has two major surface glycoproteins, F and G. The F protein mediates fusion, allowing entry of the virus into the cell cytoplasm and facilitating the formation of syncytia in vitro. The F protein sequence is well (~ 90%) conserved among RSV strains (Johnson and Collins, J Gen Virol. (1988) 69: 2623-2628). The sole marketed monoclonal antibody palivizumab is directed against the F protein of RSV.

The G protein of RSV is a surface protein that is heavily glycosylated and functions as the attachment protein. In contrast to the F protein, the G protein is quite variable across strains except for a central conserved domain (CCD), comprising amino acid residues 153-184 of the G protein of the RSV A2 strain, or corresponding amino acid residues in other strains. Both the central conserved domain and adjacent regions (residues 145-193) are bounded by rigid and heavy O-glycosylated mucin-like regions. The N-terminal half of the central conserved domain contains a small region that is conserved among more than 700 strains. The C-terminal half contains 4 conserved cysteines that are connected in a 1-4, 2-3 topology and folds into a cystine noose.

Although passive immunization using antibodies directed to the G protein has generally been considered impractical due to the lack of sequence conservation across strains, neutralizing monoclonal antibodies binding to the RSV G protein are known. Anderson, L. J. et al (J. Virol. (1988) 62:4232-4238) describe the neutralization ability of mixtures of F and G murine monoclonal antibodies, one of which binds to the RSV G protein (i.e, 131-2G). The antigenic site of this antibody was later defined by Sullender (Virol. (1995) 209:70-79). This antibody was found to bind both RSV groups A and B, representing the major strains of RSV. In addition, WO 2009/055711 discloses antibodies, such as 3D3 and 3G12, which are immunoreactive with a conserved motif within the G protein of RSV A2 and have neutralizing activity against RSV A and B subtypes. These antibodies have been shown to recognize linear epitopes in the central conserved domain, but have not been tested in the preferred animal model (i.e., cotton rats) for evaluating RSV antibodies and vaccines.

In view of the severity of the respiratory illness caused by RSV, in particular in young children and in the elderly, there is an ongoing need for effective means to prevent and treat RSV infection. SUMMARY OF THE INVENTION

The present invention provides isolated antibodies, and antigen-binding fragments thereof, that bind specifically to the RSV G protein and that are capable of neutralizing RSV. The antibodies and antigen-binding fragments are preferably capable of specifically binding to and neutralizing RSV of both subtype A and B. Preferably, the antibodies are human antibodies. The antibodies bind to epitopes in the central conserved unglycosylated region (also referred to as central conserved domain, CCD) of the RSV G protein.

The antibodies and antigen-binding fragments have high affinity for the G protein and have potent neutralizing ability. The antibodies and antigen-binding fragments of the invention are useful as diagnostic, prophylactic and/or therapeutic agents, both alone and in combination with other diagnostic, prophylactic and/or therapeutic agents.

The invention further provides compositions which comprise one or more antibodies of the invention and/or antigen binding fragments thereof. The invention also provides diagnostic, prophylactic and therapeutic methods that employ the anti- RSV antibodies. Prophylactic and therapeutic methods include administering to human subjects the anti-RSV antibodies and/or antigen-binding fragments thereof for the prevention or treatment of a RSV infection and RSV-mediated diseases or conditions, and/or amelioration of one or more symptoms of a RSV infection.

Combinations of a plurality of different anti-RSV antibodies and/or antigen-binding fragments thereof and/or with other anti-RSV antibodies can be used for combination therapy. Compositions comprising the anti-RSV antibodies and/or antigen-binding fragments thereof in combination with other prophylactic or therapeutic agents are also provided. The invention also provides nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof.

The antibodies of the invention are unique in that the antibodies are more potent against RSV type A and B than any known anti-RSV G antibody, in particular than the known anti-RSV G monoclonal antibody 3D3, at least in an in vitro neutralization assay.

The antibodies of the invention bind to unique epitopes on the RSV G protein. In certain embodiments, the antibodies comprise a heavy chain CDR3 comprising a CXXXXC motif in its amino acid sequence. In certain embodiments, the antibodies and antigen-binding fragments thereof are unique in that they work additively and/or synergistically with anti-RSV F antibodies.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the binding profiles against RSV Ga and RSV Gb protein. IgGs were tested in ELISA assays for their ability to bind to the ectodomain of recombinant RSV Ga and Gb protein. Open circles (dashed lined) denote binding to Ga (RSV A/Long) and closed circles (solid line) denote binding to Gb (RSV B/Bl).

FIG. 2 shows the neutralization profiles against RSV-A and RSV-B strains. IgGs were tested in neutralization assays for their ability to neutralize RSV-A and RSV-B strains. Open circles (dashed line) denote neutralization of RSV-A (RSV A/A2) and closed circles (solid line) denote neutralization of RSV-B (RSV B/18537).

FIG. 3 shows binding of RSV G specific monoclonal antibodies to RSV G peptides (ELISA). Short and long RSV G peptides spanning the central conserved domain (Table 15) were used for binding experiments in an ELISA with varying

concentrations of RSV G specific mAbs: CB003.1 (closed black circles, solid line), CB010.7 (open black circles, dashed line), or no monoclonal antibody (closed light grey circles).

FIG. 4: Minimal epitope mapping by PepScan. The binding activity of RSV G protein specific antibodies to all fully overlapping 5-mer, 8-mer, 10-mer, 14-mer, 18-mer, 25- mer and 32-mer peptides of central region (residues 145-201 of RSV-G type A and type B). The binding activity with a peptide is shown as a vertical line proportional to the PepScan ELISA signal.

FIG. 5: Full substitution analysis of CB003.1 and CB010.7 epitope by PepScan. The binding activity of monoclonal antibodies CB003.1 and CB010.7 at 100 and 30 ng/mL, respectively, with a peptide is shown as a vertical line proportional to the Pepscan ELISA signal. Each group of 20 lines corresponds to the complete replacement set for each amino acid position in the original 14-mer peptide

(FHFEVFNFVPCSIC). Within each group of 20 lines, the substitutions are in alphabetical order based on the one-letter amino acid code

(ACDEFGHIKLMNPQRSTVWY) and the reactivity of the original 14-mer peptide is shown as a grey bar. FIG. 6: Alanine scanning of RSV G protein central region (PepScan). Alanine substitutions at all positions of peptides corresponding to residues 161 - 192 of RSV - G central domain of type A (left panel) and type B (right panel). The alanine at position 180 of type A was substituted with glycine. The reactivity of the original peptide is shown as a grey bar.

FIG. 7 shows binding of the monoclonal antibodies to naturally occurring variants of the RSV G protein central region. Binding of mAbs CB003.1 and CB010.7 with different peptides corresponding to available type A (top panel) and type B (bottom panel) variants. The reactivity of the wild type peptide is shown as a grey bar.

FIG. 8 shows the prophylactic efficacy of anti-RSV G mAbs in cotton rat RSV- A/Long model on lung and nasal turbinate virus load at day 4 post challenge.

FIG. 9 shows the therapeutic efficacy of anti-RSV G mAbs in cotton rat RSV -A/Long model on lung and nasal turbinate virus load at day 4 post challenge.

FIG. 10 shows the therapeutic efficacy of anti-RSV G mAbs in cotton rat RSV- A/Long model on histopathology scores at day 6 post challenge.

DESCRIPTION OF THE INVENTION

Definitions

Definitions of terms as used in the present invention are given below.

The term "included" or "including" as used herein is deemed to be followed by the words "without limitation".

As used herein the term "antibody" refers to immunoglobulin molecules including monoclonal antibodies, such as chimeric, humanized or human monoclonal antibodies. The term "antibody" includes all immunoglobulin classes and subclasses known in the art. Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be divided into the five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgAl, IgA2, IgGl, IgG2, IgG3 and IgG4.

The term antigen-binding fragment refers to antigen-binding and/or variable domain comprising fragments of an immunoglobulin that compete with the intact immunoglobulin for specific binding to the binding partner of the immunoglobulin,

1. e. RSV G protein. Regardless of structure, the antigen-binding fragment binds with the same antigen that is recognized by the intact immunoglobulin. Antigen-binding fragments include, inter alia, Fab, F(ab'), F(ab')2, Fv, dAb, Fd, complementarity determining region (CDR) fragments, single-chain antibodies (scFv), bivalent single- chain antibodies, (single) domain antibodies, diabodies, triabodies, tetrabodies, (poly) peptides that contain at least a fragment of an immunoglobulin that is sufficient to confer specific antigen binding to the (poly) peptide, etc. An antigen-binding fragment may comprise a peptide or polypeptide comprising an amino acid sequence of at least

2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, or 250 contiguous amino acid residues of the amino acid sequence of the antibody. The antigen-binding fragments may be produced synthetically or by enzymatic or chemical cleavage of intact immunoglobulins or they may be genetically engineered by recombinant DNA techniques. The methods of production are well known in the art and are described, for example, in Antibodies: A Laboratory Manual, Edited by: E. Harlow and D, Lane (1988), Cold Spring Harbor Laboratory, Cold Spring Harbor,

New York, which is incorporated herein by reference. An antibody or antigen-binding fragment thereof may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or they may be different.

The term "monoclonal antibody" as used herein refers to antibody molecules of single specificity. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to an antibody displaying a single binding specificity which has variable and constant regions derived from or based on human germline immunoglobulin sequences or derived from completely synthetic sequences. The method of preparing the monoclonal antibody is not relevant for the binding specificity.

The term "functional variant", as used herein, refers to an antibody that comprises a nucleotide and/or amino acid sequence that is altered by one or more nucleotides and/or amino acids compared to the nucleotide and/or amino acid sequences of a reference antibody and that is capable of competing for specific binding to the binding partner, i.e. the RSV, with the reference antibody. In other words, the modifications in the amino acid and/or nucleotide sequence of the reference antibody do not significantly affect or alter the binding characteristics of the antibody encoded by the nucleotide sequence or containing the amino acid sequence, i.e. the antibody is still able to specifically recognize and bind its target. The functional variant may have conservative sequence modifications including nucleotide and amino acid substitutions, additions and deletions. These modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and random PCR-mediated mutagenesis, and may comprise natural as well as non- natural nucleotides and amino acids.

The term "neutralizing" as used herein in relation to the antibodies of the invention refers to antibodies that are capable of preventing or inhibiting infection of a cell by the virus, by neutralizing or inhibiting its biological effect and/or reducing the infectious titer of RSV, regardless of the mechanism by which neutralization is achieved. Neutralization can e.g. be achieved by inhibiting the attachment or adhesion of the virus to the cell surface, or by inhibition of the fusion of viral and cellular membranes following attachment of the virus to the target cell, and the like.

The term "specifically binding", as used herein, in reference to the interaction of an antibody and its binding partner, e.g. an antigen, means that the interaction is dependent upon the presence of a particular structure, e.g. an antigenic determinant or epitope, on the binding partner. In other words, the antibody preferentially binds or recognizes the binding partner even when the binding partner is present in a mixture of other molecules or organisms. The binding may be mediated by covalent or non- covalent interactions or a combination of both. In yet other words, the term

"specifically binding" means that the antibody is specifically immunoreactive with an antigenic determinant or epitope and is not immunoreactive with other antigenic determinants or epitopes. An antibody that (immuno)specifically binds to an antigen may bind to other peptides or polypeptides with lower affinity as determined by, e.g., radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA),

BIACORE, or other assays known in the art. Antibodies or fragments thereof that specifically bind to an antigen may be cross-reactive with related antigens, carrying the same epitope. Preferably, antibodies or fragments thereof that specifically bind to an antigen do not cross-react with other antigens.

Detailed description of the invention

In a first aspect the present invention provides antibodies and antigen-binding fragments capable of specifically binding to the G protein of respiratory syncytial virus (RSV) and that are capable of neutralizing RSV. The antibodies are preferably capable of specifically binding to and neutralizing RSV of both subtype A and B. Preferably, the antibodies are human monoclonal antibodies.

According to the present invention, the antibodies and antigen-binding fragments bind to epitopes in the central conserved domain (CCD) of the RSV G protein. The central conserved domain spans the amino acid sequence comprising the amino acids 153-184 of the G protein of the RSV A2 strain (or corresponding amino acid residues in other strains). In certain embodiments, the antibodies and antigen- binding fragments bind to an epitope comprising one or more amino acid residues within the amino acid sequence comprising amino acid residues 161-169, in particular one or more amino acids within the amino acid sequence comprising the amino acid residues 162-168 of the G protein of the RSV A2 strain (numbering according to RSV strain A2 strain.

Antibodies and antigen-binding fragments thus are provided that bind to an epitope in the G protein that is located at a site that is N-terminal of the cystine noose. According to the invention, it has been shown that despite the fact that at least some of the neutralizing antibodies of the present invention bind to a similar, but not identical linear epitope as e.g. the previously described monoclonal antibody 3D3 (WO2009/055711), the antibodies of the present invention have a higher neutralizing potency, as measured in an in vitro neutralization assay. According to the invention, it has been shown that the antibodies of the invention bind this linear epitope in a unique manner. Thus, according to the present invention it has been shown that these antibodies have different side chain specificity for the 161-169 epitope of RSV type A and B (numbering according to RSV strain A2). This is e.g. reflected by the substitution analysis (see Example 11) which shows that the epitope of the antibodies of the invention has different essential residues, as compared to e.g. 3D3.

The antibodies and antigen-binding fragments of the present invention have been shown to be more potent against RSV type A and B than any of the known anti- RSV G antibodies, in particular more potent than the known anti-RSV G monoclonal antibody 3D3, in an in vitro neutralization assay, in particular an in vitro assay as described in Example 7.

In certain embodiments, the IC50 (effective dilution for 50% neutralization of plaque formation) of the antibodies and antigen-binding fragments for RSV strain A/A2 (ATCC Cat. No. VR-1540) was below 40 ng/ml and/or the IC50 for RSV strains B/18537 (ATCC Cat. No. VR-1589) was below 30 ng/ml.

In an embodiment, the antibody is not an antibody selected from the group consisting of 1F12, 3G12, 1A5, 3D3, 1G1, 2B11, 5D8, 2D10, 3F9, 1D4, 1G8, 6A12, 10C6 (as described in WO 2009/055711).

In certain embodiments, the antibody or antibody fragment of the invention competes for binding to the RSV G protein with an antibody selected from the group consisting of 1F12, 3G12, 1A5, 3D3, 1G1, 2B11, 5D8, 2D10, 3F9, 1D4, 1G8, 6A12, and 10C6 (as described in WO 2009/055711).

In certain embodiments, the antibodies comprise a heavy chain CDR3 comprising a CXXXXC motif in its amino acid sequence.

In certain embodiments, the antibody comprises a heavy chain comprising: a) a heavy chain CDR1 region of SEQ ID NO: 1 , a heavy chain CDR2 region of SEQ ID NO:2, and a heavy chain CDR3 region of SEQ ID NO:3,

b) a heavy chain CDR1 region of SEQ ID NO:4, a heavy chain CDR2 region of SEQ ID NO:5, and a heavy chain CDR3 region of SEQ ID NO:6,

c) a heavy chain CDR1 region of SEQ ID NO:7, a heavy chain CDR2 region of SEQ ID NO:8, and a heavy chain CDR3 region of SEQ ID NO:9, d) a heavy chain CDRl region of SEQ ID NO: 10, a heavy chain CDR2 region of SEQ ID NO : 11 , and a heavy chain CDR3 region of SEQ ID NO : 12,

e) a heavy chain CDRl region of SEQ ID NO: 25, a heavy chain CDR2 region of SEQ ID NO:26, and a heavy chain CDR3 region of SEQ ID NO:27, or

f) a heavy chain CDRl region of SEQ ID NO:31 , a heavy chain CDR2 region of SEQ ID NO:32, and a heavy chain CDR3 region of SEQ ID NO:33.

In certain embodiments, the antibody comprises a light chain comprising: a) a light chain CDRl region of SEQ ID NO: 13, a light chain CDR2 region of SEQ ID NO : 14, and a light chain CDR3 region of SEQ ID NO : 15 ,

b) a light chain CDRl region of SEQ ID NO: 16, a light chain CDR2 region of SEQ ID NO: 17, and a light chain CDR3 region of SEQ ID NO: 18,

c) a light chain CDRl region of SEQ ID NO: 19, a heavy chain CDR2 region of SEQ ID NO:20, and a light chain CDR3 region of SEQ ID NO:21,

d) a light chain CDRl region of SEQ ID NO:22, a light chain CDR2 region of SEQ ID NO:23, and a light chain CDR3 region of SEQ ID NO:24,

e) a light chain CDRl region of SEQ ID NO:28, a light chain CDR2 region of SEQ ID NO:29, and a light chain CDR3 region of SEQ ID NO:30, or

f) a light chain CDRl region of SEQ ID NO:34, a light chain CDR2 region of SEQ ID NO:35, and a light chain CDR3 region of SEQ ID NO:36.

In certain embodiments, the antibody is selected from the group consisting of: a) an antibody comprising a heavy chain CDRl region of SEQ ID NO: 1 , a heavy chain CDR2 region of SEQ ID NO:2, and a heavy chain CDR3 region of SEQ ID NO: 3, a light chain CDRl region of SEQ ID NO: 13, a light chain CDR2 region of SEQ ID NO: 14, and a light chain CDR3 region of SEQ ID NO: 15;

b) a heavy chain CDRl region of SEQ ID NO:4, a heavy chain CDR2 region of SEQ ID NO:5, and a heavy chain CDR3 region of SEQ ID NO:6 and a light chain CDRl region of SEQ ID NO: 16, a light chain CDR2 region of SEQ ID NO:17, and a light chain CDR3 region of SEQ ID NO: 18;

c) an antibody comprising a heavy chain CDRl region of SEQ ID NO:7, a heavy chain CDR2 region of SEQ ID NO: 8, and a heavy chain CDR3 region of SEQ ID

NO:9, a light chain CDRl region of SEQ ID NO:19, a heavy chain CDR2 region of SEQ ID NO:20, and a light chain CDR3 region of SEQ ID NO:21;

d) an antibody comprising a heavy chain CDRl region of SEQ ID NO: 10, a heavy chain CDR2 region of SEQ ID NO: 11, and a heavy chain CDR3 region of SEQ ID NO: 12, a light chain CDR1 region of SEQ ID NO:22, a light chain CDR2 region of SEQ ID NO:23, and a light chain CDR3 region of SEQ ID NO:24;

e) an antibody comprising a heavy chain CDR1 region of SEQ ID NO:25, a heavy chain CDR2 region of SEQ ID NO:26, and a heavy chain CDR3 region of SEQ ID NO:27, a light chain CDR1 region of SEQ ID NO:28, a light chain CDR2 region of SEQ ID NO:29, and a light chain CDR3 region of SEQ ID NO:30; and

f) an antibody comprising a heavy chain CDR1 region of SEQ ID NO: 31 , a heavy chain CDR2 region of SEQ ID NO:32, and a heavy chain CDR3 region of SEQ ID NO:33, a light chain CDR1 region of SEQ ID NO:34, a light chain CDR2 region of SEQ ID NO:35, and a light chain CDR3 region of SEQ ID NO:36.

In certain embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 37, a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 39, a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 41, a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 43, a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 45 or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 47.

In certain embodiments, the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 38, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 40, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 42, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 44, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 46 or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 48.

In certain embodiments, the antibody is selected from the group consisting of: a) an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 37 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 38;

b) an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 39 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 40;

c) an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 41 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 42; d) an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 43 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 44;

e) an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 45 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 46; and

f) an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 47 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 48.

In certain embodiments, antigen-binding fragments of the above described antibodies are provided. The antigen-binding fragments preferably bind to the same epitope.

The antibodies and antigen-binding fragments of the present invention bind to different epitopes as compared to the epitopes of known anti-RSV G proteins, such as e.g. the anti-RSV G antibody 3D3, which also has been shown to bind to an epitope in the central conserved domain of the RSV G protein. With binding to a different epitope it is meant that the antibody binds to different critical amino acid residues as compared to known antibodies, such as 3D3. It has furthermore been shown that the antibodies of the invention are more potent than any of the known RSV G protein binding antibodies, when measured in an in vitro neutralization assay, in particular an in vitro neutralization assay as described in Example 7.

In certain embodiments, the antibodies act synergistically when used in combination with antibodies binding to RVS F protein. As used herein, the term "synergistic" means that the combined effect of the antibodies or antigen-binding fragments when used in combination is greater than their additive effects when used individually. A way of calculating synergy is by means of the combination index. The concept of the combination index (CI) has been described by Chou and Talalay (Adv Enzyme Regul., 22:27-55, 1984).

In certain embodiments, the antibodies and antigen-binding fragments are for use as a medicament, and preferably for use in the diagnostic, therapeutic and/or prophylactic treatment of RSV infection caused by RSV A and/or B subtypes. As used herein, the term "treat" or "treatment" refers to reducing the viral burden in a subject that is already infected with RSV and/or to ameliorating the symptoms of the disease in such a subject. Such symptoms include e.g. bronchiolitis, airway inflammation, congestion in the lungs, and difficulty of breathing. "Prevention" or "prophylaxis" encompasses inhibiting or reducing the spread of RSV or inhibiting or reducing the onset, development or progression of one or more of the symptoms associated with infection with RSV.

The present invention also relates to compositions comprising at least one antibody or antigen-binding fragment of the invention. In certain embodiments, the compositions are pharmaceutical compositions comprising at least one antibody or antigen-binding fragment according to the invention, and at least a pharmaceutically acceptable excipient. By "pharmaceutically acceptable excipient" is meant any inert substance that is combined with an active molecule, such as an antibody, for preparing a convenient dosage form. The "pharmaceutically acceptable excipient" is an excipient that is non-toxic to recipients at the used dosages and concentrations, and is compatible with other ingredients of the formulation comprising the drug, agent or antibody. Pharmaceutically acceptable excipients are widely applied and known in the art.

In yet another embodiment the invention relates to the use of an antibody or antigen-binding fragment of the invention in the preparation of a medicament for the diagnosis, prophylaxis, and/or treatment of RSV infection. The invention also relates to methods of prevention or treatment of RSV infection by administering a therapeutically effective amount of an antibody according to the invention to a subject in need thereof. The term "therapeutically effective amount" refers to an amount of the antibody as defined herein that is effective for preventing, ameliorating and/or treating a condition resulting from infection with RSV. Amelioration as used herein may refer to the reduction of visible or perceptible disease symptoms, viremia, or any other measurable manifestation of RSV infection.

For use in therapy, the antibodies or fragments thereof are formulated into pharmaceutical compositions using suitable excipients and administered according to standard protocols. The pharmaceutical compositions may comprise one or more antibodies or antigen-binding fragments according to the invention. Additional therapeutic agents may be present, including one or more antibodies that are immunoreactive with the F protein of RSV or other therapeutic agents that are effective against RSV or inflammation. Thus, anti -inflammatory agents such as both steroidal and non-steroidal anti-inflammatory compounds may be included in the compositions. In certain embodiments, complete antibodies, i.e. containing the complement- containing Fc region are used.

In certain embodiments, e.g. in order to reduce the inflammatory response in the lungs, only the antigen-binding fragments of the antibodies are used.

Administration of mixtures of immunospecific fragments and entire antibodies is also included within the scope of the invention.

Treatment may be targeted at patient groups that are susceptible to RSV infection. Such patient groups include, but are not limited to e.g., the elderly (e.g. > 50 years old, > 60 years old, and preferably > 65 years old), the young (e.g. < 5 years old, < 1 year old), hospitalized patients, immuno-compromised patients and patients who have been treated with an antiviral compound but have shown an inadequate antiviral response.

Administration of the antibody compositions of the invention is typically by injection, generally intramuscular or intravenous injection. The formulations are prepared in ways generally known in the art for administering antibody compositions. Suitable formulations may be found in standard formularies, such as Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Co., Easton, PA, incorporated herein by reference. The formulations are typically those suitable for parenteral administration including isotonic solutions, which include buffers, antioxidants and the like, as well as emulsions that include delivery vehicles such as liposomes, micelles and nanoparticles.

The desired protocols and formulations are dependent on the judgment of the attending practitioner as well as the specific condition of the subject. Dosage levels will depend on the age, general health and severity of infection, if appropriate, of the subject.

Another aspect of the invention includes functional variants of the antibodies as defined herein. Molecules are considered to be functional variants of an antibody according to the invention, if the variants are capable of competing for specifically binding to RSV or a fragment thereof with the "parental" or "reference" antibodies. In other words, molecules are considered to be functional variants of an antibody according to the invention when the functional variants are still capable of binding to the same or overlapping epitope of RSV or a fragment thereof. Functional variants include, but are not limited to, derivatives that are substantially similar in primary structural sequence, including those that have modifications in the Fc receptor or other regions involved with effector functions, and/or which contain e.g. in vitro or in vivo modifications, chemical and/or biochemical, that are not found in the parental antibody. Such modifications include inter alia acetylation, acylation, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, cross-linking, disulfide bond formation, glycosylation, hydroxylation, methylation, oxidation, PEGylation, proteolytic processing, phosphorylation, and the like.

Alternatively, functional variants can be antibodies as defined in the present invention comprising an amino acid sequence containing substitutions, insertions, deletions or combinations thereof of one or more amino acids compared to the amino acid sequences of the parental antibodies. Furthermore, functional variants can comprise truncations of the amino acid sequence at either or both the amino or carboxyl termini. Functional variants according to the invention may have the same or different, either higher or lower, binding affinities compared to the parental antibody but are still capable of binding to RSV or a fragment thereof. For instance, functional variants according to the invention may have increased or decreased binding affinities for RSV or a fragment thereof compared to the parental antibodies. Functional variants intended to fall within the scope of the present invention have at least about 50% to about 99%, preferably at least about 60% to about 99%, more preferably at least about 70% to about 99%, even more preferably at least about 80% to about 99%, most preferably at least about 90%) to about 99%, in particular at least about 95% to about 99%), and in particular at least about 97% to about 99% amino acid sequence identity and/or homology with the parental antibodies as defined herein. Computer algorithms such as inter alia Gap or Bestflt known to a person skilled in the art can be used to optimally align amino acid sequences to be compared and to define similar or identical amino acid residues. Functional variants can be obtained by altering the parental antibodies or parts thereof by general molecular biology methods known in the art including, but not limited to, error-prone PCR, oligonucleotide-directed mutagenesis, site-directed mutagenesis and heavy and/or light chain shuffling.

The present invention also provides immunoconjugates, i.e. molecules comprising at least one antibody, antigen-binding fragment or functional variant and further comprising at least one tag, such as inter alia a detectable moiety/agent. Also contemplated in the present invention are mixtures of immunoconjugates according to the invention or mixtures of at least one immunoconjugate according to the invention and another molecule, such as a therapeutic agent or another antibody or

immunoconjugate. In a further embodiment, the immunoconjugates of the invention may comprise more than one tag. These tags can be the same or distinct from each other and can be joined/conjugated non-covalently to the antibodies. The tag(s) can also be joined/conjugated directly to the human antibodies through covalent bonding. Alternatively, the tag(s) can be joined/conjugated to the antibodies by means of one or more linking compounds. Techniques for conjugating tags to antibodies are well known to the skilled artisan. The tags of the immunoconjugates of the present invention may be therapeutic agents, but they can also be detectable moieties/agents. Tags suitable in therapy and/or prevention may be toxins or functional parts thereof, antibiotics, enzymes, other antibodies that enhance phagocytosis or immune stimulation. Immunoconjugates comprising a detectable agent can be used diagnostically to, for example, assess if a subject has been infected with RSV or to monitor the development or progression of RSV infection as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. However, they may also be used for other detection and/or analytical and/or diagnostic purposes. Detectable moieties/agents include, but are not limited to, enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, and non-radioactive paramagnetic metal ions. The tags used to label the antibodies for detection and/or analytical and/or diagnostic purposes depend on the specific detection/analysis/diagnosis techniques and/or methods used such as inter alia immunohistochemical staining of (tissue) samples, flow cytometric detection, scanning laser cytometric detection, fluorescent immunoassays, enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), bioassays (e.g., phagocytosis assays), Western blotting applications, etc.

Suitable labels for the detection/analysis/diagnosis techniques and/or methods known in the art are well within the reach of the skilled artisan.

Furthermore, the human antibodies or immunoconjugates of the invention can also be attached to solid supports, which are particularly useful for in vitro immunoassays or purification of RSV or fragments thereof. The antibodies of the present invention can be fused to marker sequences, such as a peptide to facilitate purification. Examples include, but are not limited to, the hexa-histidine tag, the hemagglutinin (HA) tag, the myc tag or the flag tag. Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate. In another aspect the antibodies of the invention may be conjugated/attached to one or more antigens. Preferably, these antigens are antigens which are recognized by the immune system of a subject to which the antibody-antigen conjugate is administered. The antigens may be identical, but may also differ from each other. Conjugation methods for attaching the antigens and antibodies are well known in the art and include, but are not limited to, the use of cross-linking agents.

Next to producing immunoconjugates chemically by conjugating, directly or indirectly, via for instance a linker, the immunoconjugates can be produced as fusion proteins comprising the antibodies of the invention and a suitable tag. Fusion proteins can be produced by methods known in the art such as, e.g. , recombinantly by constructing nucleic acid molecules comprising nucleotide sequences encoding the antibodies in frame with nucleotide sequences encoding the suitable tag(s) and then expressing the nucleic acid molecules.

The present invention furthermore provides nucleic acid molecules encoding an antibody, antigen-binding fragment, or functional variant according to the invention. Such nucleic acid molecules can be used as intermediates for cloning purposes, e.g. in the process of affinity maturation as described above. In a preferred embodiment, the nucleic acid molecules are isolated or purified. The skilled artisan will appreciate that functional variants of these nucleic acid molecules are also intended to be a part of the present invention. Functional variants are nucleic acid sequences that can be directly translated, using the standard genetic code, to provide an amino acid sequence identical to that translated from the parental nucleic acid molecules. Preferably, the nucleic acid molecules encode antibodies comprising the CDR regions as described above. In a further embodiment the nucleic acid molecules encode antibodies comprising two, three, four, five or even all six CDR regions of the antibodies of the invention.

It is another aspect of the invention to provide vectors, i.e. nucleic acid constructs, comprising one or more nucleic acid molecules according to the present invention. Vectors can be derived from plasmids such as inter alia F, Rl, RP1, Col, pBR322, TOL, Ti, etc; cosmids; phages such as lambda, lambdoid, M13, Mu, PI, P22, QP, T-even, T-odd, T2, T4, T7, etc; plant viruses. Vectors can be used for cloning and/or for expression of the antibodies of the invention and might even be used for gene therapy purposes. Vectors comprising one or more nucleic acid molecules according to the invention operably linked to one or more expression- regulating nucleic acid molecules are also covered by the present invention. The choice of the vector is dependent on the recombinant procedures followed and the host used. Introduction of vectors in host cells can be effected by inter alia calcium phosphate transfection, virus infection, DEAE-dextran mediated transfection, lipofectamine transfection or electroporation. Vectors may be autonomously replicating or may replicate together with the chromosome into which they have been integrated. Preferably, the vectors contain one or more selection markers. The choice of the markers may depend on the host cells of choice, although this is not critical to the invention as is well known to persons skilled in the art. They include, but are not limited to, kanamycin, neomycin, puromycin, hygromycin, zeocin, thymidine kinase gene from Herpes simplex virus (HSV-TK), dihydrofolate reductase gene from mouse (dhfr). Vectors comprising one or more nucleic acid molecules encoding the human antibodies as described above operably linked to one or more nucleic acid molecules encoding proteins or peptides that can be used to isolate the human antibodies are also covered by the invention. These proteins or peptides include, but are not limited to, glutathione-S-transferase, maltose binding protein, metal-binding polyhistidine, green fluorescent protein, luciferase and beta-galactosidase.

The invention also provides host cells containing one or more copies of the vectors mentioned above. Host cells include, but are not limited to, cells of mammalian, plant, insect, fungal or bacterial origin. Bacterial cells include, but are not limited to, cells from Gram-positive bacteria or Gram-negative bacteria such as several species of the genera Escherichia, such as E. coli, and Pseudomonas. In the group of fungal cells preferably yeast cells are used. Expression in yeast can be achieved by using yeast strains such as inter alia Pichia pastoris, Saccharomyces cerevisiae and Hansenula polymorpha. Furthermore, insect cells such as cells from Drosophila and Sf9 can be used as host cells. Besides that, the host cells can be plant cells such as inter alia cells from crop plants such as forestry plants, or cells from plants providing food and raw materials such as cereal plants, or medicinal plants, or cells from ornamentals, or cells from flower bulb crops. Transformed (transgenic) plants or plant cells are produced by known methods, for example, Agrobacterium- mediated gene transfer, transformation of leaf discs, protoplast transformation by polyethylene gly col-induced DNA transfer, electroporation, sonication, microinjection or ballistic gene transfer. Additionally, a suitable expression system can be a baculovirus system. Expression systems using mammalian cells, such as Chinese Hamster Ovary (CHO) cells, COS cells, BH cells, NSO cells or Bowes melanoma cells are preferred in the present invention. Mammalian cells provide expressed proteins with posttranslational modifications that are most similar to natural molecules of mammalian origin. Since the present invention deals with molecules that may have to be administered to humans, a completely human expression system would be particularly preferred. Therefore, even more preferably, the host cells are human cells. Examples of human cells are inter alia HeLa, 911, AT1080, A549, 293 and HEK293 cells. In preferred embodiments, the human producer cells comprise at least a functional part of a nucleic acid sequence encoding an adenovirus El region in expressible format. In even more preferred embodiments, said host cells are derived from a human retina and immortalized with nucleic acids comprising adenoviral E 1 sequences, such as 911 cells or the cell line deposited at the European Collection of Cell Cultures (ECACC), CAMR, Salisbury, Wiltshire SP4 OJG, Great Britain on 29 February 1996 under number 96022940 and marketed under the trademark PER.C6 ^® (PER.C6 is a registered trademark of Crucell Holland B.V.). For the purposes of this application "PER.C6 cells" refers to cells deposited under number 96022940 or ancestors, passages up-stream or downstream as well as descendants from ancestors of deposited cells, as well as derivatives of any of the foregoing. Production of recombinant proteins in host cells can be performed according to methods well known in the art. The use of the cells marketed under the trademark PER.C6 ^® as a production platform for proteins of interest has been described in WO 00/63403 the disclosure of which is incorporated herein by reference in its entirety.

The antibodies of the invention can be prepared by various means. A method of producing an antibody according to the invention is an additional part of the invention. The method comprises the steps of a) culturing a host cell according to the invention under conditions conducive to the expression of the antibody, and b) optionally, recovering the expressed antibody. The expressed antibodies can be recovered from the cell free extract, but preferably they are recovered from the culture medium. The above method of producing can also be used to make functional variants of the antibodies and/or immunoconjugates of the present invention. Methods to recover proteins, such as antibodies, from cell free extracts or culture medium are well known to the artisan skilled in the art.

Alternatively, next to the expression in hosts, such as host cells, the antibodies and immunoconjugates of the invention can be produced synthetically by conventional peptide synthesizers or in cell-free translation systems using RNA nucleic acid derived from DNA molecules according to the invention. The antibodies according to the present invention may also be generated by transgenic non-human mammals, such as for instance transgenic mice or rabbits that express human immunoglobulin genes. Preferably, the transgenic non-human mammals have a genome comprising a human heavy chain transgene and a human light chain transgene encoding all or a portion of the human antibodies as described above. The transgenic non-human mammals can be immunized with a purified or enriched preparation of RSV or a fragment thereof. Protocols for immunizing non-human mammals are well established in the art. See Using Antibodies: A Laboratory Manual, Edited by: E. Harlow, D. Lane (1998), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York and Current Protocols in Immunology, Edited by: J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W. Strober (2001), John Wiley & Sons Inc., New York, the disclosures of which are incorporated herein by reference. Immunization protocols often include multiple immunizations, either with or without adjuvants such as Freund's complete adjuvant and Freund's incomplete adjuvant, but may also include naked DNA immunizations. In other embodiments, the human antibodies are produced by B-cells, plasma and/or memory cells derived from the transgenic animals. In yet another embodiment, the human antibodies are produced by hybridomas, which are prepared by fusion of B-cells obtained from the above- described transgenic non-human mammals to immortalized cells. B-cells, plasma cells and hybridomas as obtainable from the above-described transgenic non-human mammals and human antibodies as obtainable from the above-described transgenic non-human mammals, B-cells, plasma and/or memory cells and hybridomas are also a part of the present invention.

The invention further provides kits comprising at least an antibody, an antigen-binding fragment, an immunoconjugate, a functional variant, and/or at least a nucleic acid according to the invention. Optionally, the above-described components of the kits of the invention are packed in suitable containers and labelled for diagnosis, prophylaxis and/or treatment of the indicated conditions. The above- mentioned components may be stored in unit or multi-dose containers as an aqueous, preferably sterile, solution or as a lyophilised, preferably sterile, formulation for reconstitution. The kit may further comprise more containers comprising a pharmaceutically acceptable buffer. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, culture medium for one or more of the suitable hosts and, possibly, even at least one other therapeutic, prophylactic or diagnostic agent. Associated with the kits can be instructions customarily included in commercial packages of therapeutic, prophylactic or diagnostic products, that contain information about for example the indications, usage, dosage, manufacture, administration, contraindications and/or warnings concerning the use of such therapeutic, prophylactic or diagnostic products.

The antibodies according to the present invention can also be advantageously used as a diagnostic agent in an in vitro method for the detection of RSV. The invention thus further provides a method of detecting RSV in a sample, wherein the method comprises the steps of (a) assaying the level of RSV antigen in a sample, e.g. by contacting a sample with a diagnostically effective amount of an antibody (or fragments thereof) or an immunoconjugate according to the invention, and (b) comparing the assayed level of RSV antigen with a control level, whereby an increase in the assayed level of RSV antigen compared to the control level is indicative of RSV infection. The sample may be a biological sample including, but not limited to blood, serum, stool, sputum, nasophargyal aspirates, bronchial lavages, urine, tissue or other biological material from (potentially) infected subjects, or a non-biological sample such as water, drink, etc. The sample may first be manipulated to make it more suitable for the method of detection. Manipulation means inter alia treating the sample suspected to contain and/or containing the virus in such a way that the virus will disintegrate into antigenic components such as proteins, (poly) peptides or other antigenic fragments. Preferably, the antibodies or immunoconjugates of the invention are contacted with the sample under conditions which allow the formation of an immunological complex between the antibody and the virus or antigenic components thereof that may be present in the sample. The formation of an immunological complex, if any, indicating the presence of the virus in the sample, is then detected and measured by suitable means. Such methods include, inter alia, homogeneous and heterogeneous binding immunoassays, such as radio-immunoassays (RIA), ELISA, immunofluorescence, immunohistochemistry, FACS, BIACORE and Western blot analyses. Preferred assay techniques, especially for large-scale clinical screening of patient sera and blood and blood-derived products are ELISA and Western blot techniques. ELISA tests are particularly preferred. The invention is further illustrated in the following examples which are not intended to limit the invention.

EXAMPLES

EXAMPLE 1

Antigen production and labelling

Unlike the fusion protein (RSV F) expressed on the surface of the viral membrane, the attachment protein (RSV G) is highly variable, thus defining the two broad subtypes of RSV (i.e. subtypes A and B). Despite the sequence variability, RSV G contains a central and highly conserved region. In an effort to obtain broadly neutralizing monoclonal antibodies, RSV G corresponding to a representative subgroup A (RSV A/Long) and subgroup B strain (RSV B/Bl) were expressed recombinantly in 293 freestyle cells, purified, and labelled for use in single cell sorting experiments.

Expression of RSV Ga and Gb

Recombinant RSV attachment protein (G protein) corresponding to RSV A/Long (Accession No. P20895) and RSV B/Bl (Accession No. NP_056862), herein referred to RSV Ga and Gb, were expressed from a CMV -based promoter mammalian expression vector (Invitrogen Corp., pcDNA3.1) with both a Myc (EQKLISEEDL) and 6X histidine tag (Table 1). Leader sequence corresponding to human V kappa I signal peptide was introduced at amino terminus to promote secretion. Both RSV Ga and Gb were expressed lacking the transmembrane domain and included amino acids 65-288 and 65-299 of RSV Ga and Gb, respectively.

RSV Ga and Gb were transfected according to manufacturer guidelines.

Recombinantly expressed RSV Ga and Gb proteins were purified using Nickel NTA chromatography. Seventy-two hours after transfection the supernatant was harvested and dialyzed overnight against 20 mM Tris-HCL pH8 and 300 mM NaCl. The following day, the dialysis was repeated with fresh buffer and for an additional 6 hours. The dialyzed supernatant was then supplemented with 5% glycerol and 10 mM imidazole (VWR, Cat. No. EM-5720) and loaded onto a column packed with 2 mL of Ni-NTA agarose beads (Qiagen, Cat. No. 30310). The bound protein was

subsequently washed with 2 column volumes of wash buffer consisting of 20 mM Tris-HCl, pH8, 300 mM NaCl, 5% glycerol, and 20 mM imidazole. The proteins were then eluted with 5 mL of elution buffer containing 20 mM Tris-HCl, pH8, 300 mM NaCl, 5% glycerol, and 50 mM imidazole. Finally, the eluate was dialyzed against four liters of phosphate buffered saline (PBS) at 4°C overnight. The dialyzed protein was then concentrated to 0.5-1.0 mL in a 3 OK MWCO concentrator (Millipore, Amicon Ultracel concentrator) and quantitated by bicinchoninic acid assay (BCA assay; Thermo Fisher, per manufacturer instructions). In addition, the purified proteins were each quality-controlled by SDS-PAGE/Coomassie.

RSV Ga was fluorescently labelled with Alexa Fluor 647 (AF 647) using the Alexa Fluor 647 microscale protein labelling kit (Invitrogen Cat. No. A30009) according to manufacturer's instructions. After purification, the degree of labelling was determined to be 1.2 moles of AF 647 per mole of protein using a NanoDrop UV spectrophotometer (manufacturer). Similarly, the RSV Gb protein was labelled with Alexa Fluor 488 (AF 488) using a microscale protein labelling kit (Invitrogen Cat. No. A30006) according to manufacturer's instructions and after final purification, the degree of labelling was determined using a NanoDrop spectrophotometer to be about 2 moles of AF 488 per mole of protein.

Table 1. Recombinant RSV G protein sequences used

Protein Amino Acid Sequence

(Accession No.)

RSV G A/Long ANHKVTLTTAI I QDATSQIKNTTPTYLTQDPQLGI S FSNLSE ITSQTTTI LASTTPGV

KSNLQPTTVKTKNTTTTQTQPSKPTTKQ QNKPPNKPNNDFHFEVFNFVPCS ICSNNP

(P20895)

TCWAICKRI PNKKPGKKTTTKPTKKPTFKTTKKDLKPQTTKPKEVPTTKPTEEPTINT TKTNITTTLLTNNTTGNPKLTSQMETFHSTSSEGNLSPSQVSTTSEHPSQPS SPPNTT RQQAYVEQ

KLI SEEDLNSAVDHHHHHH ( SEQ I D NO : 4 9 )

RSV G B/B1 ANHKVTLTTVTVQTIKNHTEKNITTYLTQVPPERVS SSKQPTTTSPIHTNSATTSPNT

KSETHHTTAQTKGRTTTSTQTNKPSTKPRLKNPPKKPKDDYHFEVFNFVPCS ICGNNQ

(NP_056862)

LCKS ICKT I PSNKPKKKPTI KPTNKPTTKTTNKRDPKTPAKTTKKETTTNPTKKPTLT TTERDTSTSQSTVLDTTTLEHTIQQQSLHSTTPENTPNSTQTPTASEPSTSNSTQNTQ SHAQAYVE

QKLI SEEDLNSAVDHHHHHH ( SEQ I D NO : 50 ) EXAMPLE 2

Identification of anti-RSV G-specific antibodies

Broadly neutralizing monoclonal antibodies against RSV G protein were recovered from memory B-cells (CD19+CD27+IgG+) isolated from peripheral blood mononuclear cells (PBMCs) obtained through the San Diego Blood Bank. In short, CD22+ enriched B-cells were stained with fluorescently labeled antibodies to memory B cell surface markers and incubated with RSV Ga, Gb (labeled with Alexa Fluor 647 and 488, respectively, as described in Example 1), or the RSV G central conserved domain (CCD) biotin-conjugated peptide (SYM-1706).

CD 19/CD27/IgG/RS VGa/RS VGb or CD 19/CD27/IgG/SYM- 1706 (used in certain sorting experiments). Positive cells were sorted and single cells deposited into individual wells of a 96-well plate using a FACSAria II (BD Biosciences) or MoFlo XDP (Beckman Coulter). Plates were stored at -80°C until processed. On average, approximately 10-25xl0 ⁶ B-cells per donor were surveyed.

EXAMPLE 3

Recovery of heavy and light chain genes from single B-cells specific to RSV Ga and Gb

As described in Example 2, broadly neutralizing monoclonal antibodies against RSV were isolated from memory B-cells (CD 19+CD27+IgG+) with reactivity to RSV Ga and Gb protein or the RSV G central conserved domain (CCD) biotin-conjugated peptide (SYM-1706). Heavy and light chain genes were then recovered by a two-step PCR approach from individual B-cells, cloned, and expressed in vitro as Fab antibodies.

First strand cDNA synthesis

Complementary DNA (cDNA) was generated from individually sorted cells using Invitrogen's Superscript III First Strand Synthesis kit (Superscript III kit, Cat No. 18080-051). IgG heavy and light chain amplification by nested PCR

IgG heavy and light chain variable regions (both kappa and lambda chains) were amplified from freshly prepared cDNA using a two-step, nested PCR approach. Subsequently, heavy and light chain PCR fragments were assembled into a single cassette to facilitate downstream cloning using an overlap extension PCR.

Step I Amplification

For Step I, 2.5 of freshly prepared cDNA generated as mentioned above was used as template to amplify heavy, kappa, and lambda light chains. A pool of primers specifically designed to the leader regions of antibody heavy chain (CB- 5'LVH primers), kappa light chain (CB-5'LVk primers), and lambda light chain (CB- 5' LVlam primers) were used (Table 2-4). A single reverse primer specifically designed to the CHI region, Ck, and CL region of the heavy chain, kappa light chain, and lambda light chain, respectively, were used in the Step I PCR reaction.

Table 2. VH Step I forward primers (5' - 3')

Name Sequence

CB-5'LVHla ATGGACTGGACCTGGAGGTTCCTC (SEQ ID NO: 51] CB-5'LVHlb ATGGACTGGACCTGGAGGATCCTC (SEQ ID NO: 52) CB-5'LVHlc ATGGACTGGACCTGGAGGGTCTTC (SEQ ID NO: 531 CB-5'LVHld ATGGACTGGACCTGGAGCATCC (SEQ ID NO: 54)

GGACATACTTTGTTCCACGCTCCTGC (SEQ ID NO:

CB-5'LVH2

55)

CB-5'LVH3a AGGTGTCCAGTGTCAGGTGCAGC (SEQ ID NO: 56)

CB-5'LVH3b AGGTGTCCAGTGTGAGGTGCAGC (SEQ ID NO: 57)

CB-5'LVH3c AGGTGTCCAGTGTCAGGTACAGC (SEQ ID NO: 58)

CB-5'LVH4 GCAGCTCCCAGATGGGTCCTG (SEQ ID NO: 59)

CB-5'LVH5 TCAACCGCCATCCTCGCCCTC (SEQ ID NO: 60)

GTCTGTCTCCTTCCTCATCTTCCTGC (SEQ ID NO:

CB-5'LVH6

61)

3'CgCHl GGAAGGTGTGCACGCCGCTGGTC (SEQ ID NO: 62) Table 3. Vk Step I forward primers (5' - 3')

Name Sequence

CB-5'LVkla ATGAGGGTCCCCGCTCAGCTC (SEQ ID NO: 63)

CB-5'LVklb ATGAGGGTCCCTGCTCAGCTC (SEQ ID NO: 64)

CB-5'LVklc ATGAGAGTCCTCGCTCAGCTC (SEQ ID NO: 65)

CB-5'LVk2 TGGGGCTGCTAATGCTCTGG (SEQ ID NO: 66)

CB-5'LVk3 CCTCCTGCTACTCTGGCTCCCAG (SEQ ID NO: 67)

TCTCTGTTGCTCTGGATCTCTGGTGC (SEQ ID NO:

CB-5'LVk4 68)

CB-5'LVk5 CTCCTCAGCTTCCTCCTCCTTTGG (SEQ ID NO: 69)

AACTCATTGGGTTTCTGCTGCTCTGG (SEQ ID NO:

CB-5'LVk6 70)

3'Ck-Rev494 GTGCTGTCCTTGCTGTCCTGCTC (SEQ ID NO: 71)

Table 4. VL Step I forward primers (5' - 3')

Name Sequence

CB-5' LVlaml CTCCTCGCTCACTGCACAGG (SEQ ID NO 72)

CB-5' LVlam2 CTCCTCTCTCACTGCACAGG (SEQ ID NO: 73)

CB-5' LVlam3 CTCCTCACTCGGGACACAGG (SEQ ID NO: 74)

CB-5' LVlam4 ATGGCCTGGACCCCTCTCTG (SEQ ID NO: 75)

CB-5' LVlam5 ATGGCATGGATCCCTCTCTTCCTC (SEQ ID NO: 76)

3'Clam-Rev CAAGCCAACAAGGCCACACTAGTG (SEQ ID NO: 77)

Step II Amplification

l) For Step II, 2.5 μΐ, of Step I PCR product generated from the reaction above was used as a template to amplify heavy, kappa, and lambda light chain genes. A pool of forward primers specifically designed to the framework 1 region of antibody heavy chain, kappa light chain, and lambda light chain were used (Table 5-7). A pool of reverse primers specifically designed to the heavy chain junction

(3'SalIJH primers), kappa light chain junction (3'Jk primers), and a 5'region- specific primer corresponding to the lambda light chain (CB-VL primers) were used. Furthermore, Step II forward primers were engineered to introduce an Sill restriction site, while the Step II heavy chain reverse primers were designed to introduce a Sail restriction site Table 5. VH Step II primers (5' - 3')

Name Sequence

GCTCGCAGCATAGCCGGCCATGGCCCAGGTGCAGCTGGTGCAGTC

CB-VHl (SEQ ID NO: 78)

GCTCGCAGCATAGCCGGCCATGGCCCAGGTCCAGCTGGTGCAGTC

CB-VHlb (SEQ ID NO: 79)

GCTCGCAGCATAGCCGGCCATGGCCCAGGTTCAGCTGGTGCAGTC

CB-VHlc (SEQ ID NO: 80)

GCTCGCAGCATAGCCGGCCATGGCCCAGGTCCAGCTTGTGCAGTC

CB-VHld (SEQ ID NO: 81)

GCTCGCAGCATAGCCGGCCATGGCCCAGGTCACCTTGAGGGAGTCTGG

CB-VH2a (SEQ ID NO: 82)

GCTCGCAGCATAGCCGGCCATGGCCCAGGTCACCTTGAAGGAGTCTGG

CB-VH2b (SEQ ID NO: 83)

GCTCGCAGCATAGCCGGCCATGGCCCAGGTGCAGCTGGTGGAGTC

CB-VH3a (SEQ ID NO: 84)

GCTCGCAGCATAGCCGGCCATGGCCGAGGTGCAGCTGTTGGAGTC

CB-VH3b (SEQ ID NO: 85)

GCTCGCAGCATAGCCGGCCATGGCCGAGGTGCAGCTGGTGGAGTC

CB-VH3c (SEQ ID NO: 86)

GCTCGCAGCATAGCCGGCCATGGCCCAGGTACAGCTGGTGGAGTCTG

CB-VH3d (SEQ ID NO: 87)

GCTCGCAGCATAGCCGGCCATGGCCCAGSTGCAGCTGCAGGAG

CB-VH4a (SEQ ID NO: 88)

GCTCGCAGCATAGCCGGCCATGGCCCAGGTGCAGCTACAGCAGTGG

CB-VH4b (SEQ ID NO: 89)

GCTCGCAGCATAGCCGGCCATGGCCGAGGTGCAGCTGGTGCAGTC

CB-VH5 (SEQ ID NO: 90)

GCTCGCAGCATAGCCGGCCATGGCCCAGGTACAGCTGCAGCAGTCAG

CB-VH6 (SEQ ID NO: 91)

GCTCGCAGCATAGCCGGCCATGGCCCAGGTGCAGCTGGTGCAATCTG

CB-VH7 (SEQ ID NO: 92)

3'SalIJH 1/2/4/5 TGCGAAGTCGACGCTGAGGAGACGGTGACCAG (SEQ ID NO: 93) 3'SalIJH3 TGCGAAGTCGACGCTGAAGAGACGGTGACCATTG (SEQ ID NO: 94 )

TGCGAAGTCGACGCTGAGGAGACGGTGACCGTG ( SEQ I D NO : 3'SalIJH6 95 )

Table 6. VK Step II primers (5' - 3')

Name Sequence

CTACCGTGGCCTAGGCGGCCGACATCCAGATGACCCAGTCTCC

CB-VKl (SEQ ID NO: 96 )

CTACCGTGGCCTAGGCGGCCGACATCCAGTTGACCCAGTCTCC

CB-VKlb (SEQ ID NO: 97 )

CTACCGTGGCCTAGGCGGCCGCCATCCAGTTGACCCAGTCTCC

CB-VKlc (SEQ ID NO: 98 )

CTACCGTGGCCTAGGCGGCCGATRTTGTGATGACTCAGTCTCCACTC

CB-VK2a (SEQ ID NO: 99 )

CTACCGTGGCCTAGGCGGCCGAAATTGTGTTGACGCAGTCTCCAG

CB-VK3a (SEQ ID NO: 100 )

CTACCGTGGCCTAGGCGGCCGAAATTGTGTTGACACAGTCTCCAG

CB-VK3b (SEQ ID NO: 101 )

CTACCGTGGCCTAGGCGGCCGAAATAGTGATGACGCAGTCTCCAG

CB-VK3c (SEQ ID NO: 102 )

CTACCGTGGCCTAGGCGGCCGACATCGTGATGACCCAGTCTCC

CB-Vk4 (SEQ ID NO: 103 )

CTACCGTGGCCTAGGCGGCCGAAACGACACTCACGCAGTCTCC

CB-Vk5 (SEQ ID NO: 104 )

CTACCGTGGCCTAGGCGGCCGAAATTGTGCTGACTCAGTCTCCAG

CB-Vk6 (SEQ ID NO: 105 )

GAAGACAGATGGTGCAGCCACAGTTCGTTTGATYTCCACCTTGGTC

3'Jkl/4 Rev Ila-L (SEQ ID NO: 106 )

GAAGACAGATGGTGCAGCCACAGTTCGTTTGATCTCCAGCTTGGTC

3'Jk2 Rev Ilb-L (SEQ ID NO: 107 )

GAAGACAGATGGTGCAGCCACAGTTCGTTTGATATCCACTTTGGTC

3'Jk3 Rev IIc-L (SEQ ID NO: 108 )

GAAGACAGATGGTGCAGCCACAGTTCGTTTAATCTCCAGTCGTGTC

3'Jk5 Rev Ild-L (SEQ ID NO: 109 ) Table 7. VL Step II primers (5' - 3')

Name Sequence

CTACCGTGGCCTAGGCGGCCAATTTTATGCTGACTCAGCCCCACTC CB-VL1 ( SEQ ID NO : 1 10 )

CTACCGTGGCCTAGGCGGCCTCCTATGTGCTGACTCAGCC CB-VL2 ( SEQ ID NO : 1 11 )

CTACCGTGGCCTAGGCGGCCCAGTCTGTGCTGACGCAGCC CB-VL3 ( SEQ ID NO : 1 12 )

CTACCGTGGCCTAGGCGGCCCAGTCTGTCGTGACGCAGCC CB-VL4 ( SEQ ID NO : 1 13 )

CTACCGTGGCCTAGGCGGCCCAGTCTGCCCTGACTCAGCC CB-VL5 ( SEQ ID NO : 1 14 )

CTACCGTGGCCTAGGCGGCCTCTTCTGAGCTGACTCAGGACC CB-VL6 ( SEQ ID NO : 1 15 )

CTACCGTGGCCTAGGCGGCCTCCTATGAGCTGACTCAGCCACC CB-VL7 ( SEQ ID NO : 1 16 )

3'Clam-Step II CTCAGAGGAGGGYGGGAACAGAGTGAC ( SEQ ID NO : 117 )

Step III Amplification: Overlap Extension PCR

For Step III, the heavy and light chain DNA fragments (Step II products) were linked into a single cassette via overlap extension PCR using a: 1) Fab linker (kappa or lambda; Table 8) amplified as outlined below which anneals to the 3 ' end of the light chain Step II fragment and the 5 ' end of the heavy chain Step II fragment and contains either the kappa or lambda constant region, 2) a forward overlap primer with an Sfil restriction site that anneals to the 5' end of the light chain, and 3) a reverse primer with a Sail restriction site that anneals to the 3 ' end of the heavy chain step II fragment (Table 9). This reaction results in a 1200 bp fragment (i.e., cassette) consisting of the light chain-linker-heavy chain. Following amplification, the PCR linker reaction product or the overlap extension PCR reaction product was separated on a 1% agarose gel and gel extracted according to manufacturer's instructions (Qiagen Gel Extraction Kit; Cat. No. 28706). Table 8. Nucleotide Sequence of Kappa and Lambda Linker

Gene Sequence

IGKC CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGCTTAAA TCTGGAACTGCCTCTGTTGTGTGCCTTCTAAATAACTTCTATCCCCGTGAGGCCAAA GTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACA GAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTTACGCTTAGCAAA GCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTCAGC TCGCCCGTCACAAAGAGCTTCAACCGCGGAGAGTGTTAATCTAGAAATAAGGAGGAT ATAATTATGAAATACCTGCTGCCGACCGCAGCCGCTGGTCTGCTGCTGCTCGCAGCA TAGCCGGCCATGGCC ( SEQ ID NO : 118 )

IGLC2 GTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTG

TGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGC AGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAG TACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGC TACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACA GAATGTTCATAATCTAGAAATAAGGAGGATATAATTATGAAATACCTGCTGCCGACC GCAGCCGCTGGTCTGCTGCTGCTCGCAGCATAGCCGGCCATGGCC

( SEQ I D NO : 11 9 )

Table 9. Linker primers (5' - 3')

Name Sequence

CGAACTGTGGCTGCACCATCTGTCTTC

FabLinker-F ( SEQ ID NO : 120 )

GGCCATGGCCGGCTATGCTGCGAGC

FabLinker-R ( SEQ ID NO : 121 )

GTCACTCTGTTCCCRCCCTCCTCTGAG

Lambda-Fab Linker F ( SEQ ID NO : 122 )

Overlap-F CTACCGTGGCCTAGGCGGCC ( SEQ ID NO : 123 )

Overlap-R TGCGAAGTCGACGCTGARGAG ( SEQ I D NO : 124 )

Digestion and Cloning into Bacterial Expression Vector

Following PCR purification (Qiagen) of the overlap extension PCR, the fragment was digested and the digested overlap product was then separated on a 1% agarose gel. The band corresponding to the overlap cassette (-1.1 kb) was purified by gel extraction (Qiagen). Finally, the digested overlap extension product was ligated and cloned into the pCB-Fab bacterial expression vector. All transformations were carried out using DH5a Max Efficiency cells (Invitrogen Corp., Cat. No. 18258-012). Approximately 100 μΐ of recovered cells were plated onto a 100 μg/mL carbenicillin plate supplemented with 20 mM glucose. Plates were incubated overnight at 37° C to allow for colony growth.

EXAMPLE 4

Fab Binding to RSV G and Monoclonal Antibody Rescue

Fab antibodies cloned in Example 3 were expressed in bacteria and again tested for their ability to bind to RSV Ga, RSV Gb, or the RSV G central conserved domain (CCD) peptide (SYM-1706: amino acid sequence: biotin- KQRQNKP PNKPNNDFHFEVFNFVPC S I C SNNPTCWAI CKR; SEQ ID NO: 125).

Bacterial supernatants were added to RSV Ga, Gb, CCD peptide, negative control actin, and anti-human F(ab)2 coated plates and incubated for 2 hours at 37° C (except for the CCD peptide which was incubated on a Streptavidin coated plate and incubated for 2 hours at room temperature). CR9514 (an antibody based on 3D3, i.e. comprising the heavy and light chain variable region of 3D3, as disclosed in WO 2009/055711) was used as positive control against RSV Ga, Gb, CCD peptide, and anti-human F(ab)2 coated plates at a dilution of 0.1 μg/mL in 0.4%

NFDM/PBS/0.05% Tween20. Mouse anti-actin (Sigma, Cat. No. A3853) was used at 1.25 μg/mL as positive control for bovine actin coated plates. Anti-HA HRP (Roche, Cat. No. 12013819001) was used as secondary antibody for bacterial supernatants. Anti-human Fab (Jackson Labs, Cat. No. 109-036-097) was used for CR9514 (comprising the variable regions of 3D3) control wells. Finally, goat anti-mouse HRP (Jackson Labs, Cat. No. 115-035-072) was used for the actin positive control.

Following incubation, plates were washed four times in PBS/0.05% Tween20 and developed with 50 μί 1 :1 v/v TMB:peroxide solution (Pierce, Cat No. 34021) for approximately 5 minutes. The reaction was immediately halted by the addition of 50 μΐ _^ 2N H2SO4 and the absorbance at 450 nm was measured using an ELISA plate reader. Positive binding was indicated by an OD450 greater than 0.5 (0.5-0.9 is moderate binding, >1 is strong binding) and a response that was 3 -fold above background. Based on ELISA results, about six clones on average with reactivity to target antigens were selected. Because each Fab antibody was originally cloned using a pool of framework 1 -specific and junction-specific primers, the potential for cross-priming, especially for highly related primers, was high. For this reason, several bacterial clones representing each overlapped product were selected to sequence. Plasmid miniprep DNA was prepared according to manufacturer guidelines (Qiagen Miniprep kit Cat. No. 27106). Heavy and light chains corresponding to each clone selected were sequenced with the primers highlighted in Table 10. Sequences were analyzed, the closest germline identified, and CDR and framework regions determined. This information was subsequently used to design primers to clone and convert candidate antibodies into IgG.

Table 10. Sequencing Primers for Bacterial Fabs (5' - 3')

Gene Sequence

SeqpCBFab-HCF TGAAATACCTGCTGCCGACC (SEQ ID NO: 126)

Seq-PelB-Rev CAGCAGACCAGCGGCTGC (SEQ ID NO: 127)

EXAMPLE 5

Cloning, Sequencing, and Purification of IgGs

Fab antibodies reactive to RSV Ga, Gb, and CCD peptide identified in the bacterial ELISA outlined in Example 4 were cloned and expressed as IgGs in the human embryonic kidney cells (293 -F cells). IgGs were subsequently purified and quality-controlled by determining concentration, SDS-PAGE, and by size exclusion chromatography.

A. IgG Cloning and Sequencing Information

Fab antibodies identified in the bacterial ELISA (outlined in Example 4) were subsequently converted into IgGs by cloning the variable heavy and light domains

(kappa and lambda) by restriction digest into the pCP9-kappa (SEQ ID NO: 127) and pCP9-lambda (SEQ ID NO: 128) expression vectors. Given the potential for cross- priming (aforementioned in Example 4), the initial amino acids of FR1 and the ending amino acids of the junction region for each bacterial clone selected for conversion into IgG frequently differed to those of its corresponding germline sequence. For this reason, primers specific to each antibody were designed to restore the FR1 and junction regions for both heavy and light chain genes of each bacterial clone selected. Heavy and light chains were amplified using the corresponding bacterial clone (expressed from the pCB-Fab vector in Example 4) and cloned in a sequential manner into the pCP9 expression vectors.

Amplification of the heavy chain resulted in an average sized fragment of 370 bp which was resolved on a 1% agarose gel and gel extracted according to manufacturer's instructions (Qiagen). The heavy chain fragment was then used to attach the HAVT20 leader sequence (5'- ATGGCCTGCCCTGGCTTTCTCTGGGCACTTGTGATCTCCACCTGTCTTGAA TTTTCC ATGGCT-3 ' ; MACPGFLWALVISTCLEFSMA) by overlap extension PCR.

The corresponding overlap HAVT20-heavy chain product was subsequently PCR purified according to manufacturer's instructions (Qiagen). Ligations were carried out sequentially; that is, either the light chain was first digested and ligated or the corresponding heavy chain digested and inserted. Once either the light or heavy chain insertion was sequenced confirmed, a representative bacterial clone was selected, miniprep was prepared and used to clone the second chain (i.e., either light or heavy chain, depending on which was cloned first). For cloning the heavy chain fragment, the pCP9 vector and PCR purified heavy chain overlap product were digested with restriction enzymes BamHI HF (NEB, Cat. No. R3136L) and Xhol (NEB, Cat. No. R0146L). Digested pCP9 vector and heavy chain overlap product were then resolved on a 1% agarose gel and gel extracted (upper -9.5 kB for pCP9 vector). Ligations were carried out at a 1 :3 vector-to-insert ratio and transformed into DH5a Max Efficiency cells (Invitrogen Corp., Cat. No. 18258-012). Upon sequence confirmation, the second chain (e.g., light chain) was cloned. For cloning the light chain fragment, the pCP9 clone containing the corresponding heavy chain and the light chain PCR product were digested with Notl HF (NEB, Cat. No. R3189L) and Xbal (NEB, Cat. No. R0145L. The light chain was then ligated into the pCP9 vector containing the corresponding heavy chain gene and transformed into DH5a Max

Efficiency cells. Several colonies were selected for sequencing and analyzed. Tables 11 and 12 show sequences of the antibody heavy and light chains CDR regions. Table 11. Amino acid sequences of heavy chain variable regions (SEQ ID NO:)

Clone CDR1 CDR2 CDR3

Germline

CB002.1 IGHV4-59 SYFWN (25) YIYGSGSADYNPSLKS (26) SGFCTNDACYRRGSWFDP (27) CB003.1 IGHV1-46 TYYIH (10) I NTGSG VTSYAQKFQG _M YSGSWYPFDY (12)

V SYDGTKKYHADSVKG VGELRSFDWLLADGTAYYYYGMDV

CB010.7 IGHV3-30 THGMH (7)

(8) (9)

CB028.2 IGVH1-18 TYGIT (31) WISG DSD NTN YAQN LQG ALAKWYCSSSSCFCGGGSCYSDY (33)

(32)

CB048.3 IGHV3-30 NHGM H (4) VISYDGNKKYYADSVKG (5) TTFYFDDSNYYEYLDY (6)

CB058.1 IGHV3-23 SYAMS (1) AIRGSVDNTYYADSVKG (2) DPALYCSGETCFSDLTD (3)

Table 12. Amino acid sequences of light chain variable regions (SEQ ID NO:)

VK/VL

Clone CDR1 CDR2 CDR3

Germline

CB002.1 IGKV1-39 RASQSIDNYLN (28) AASSLQS (29) QQSYSTLT (30)

CB003.1 GKV3-20 RASQNINGNYLA (22) EASSRAT (23) QQYGTSPF (24)

CB010.7 IG V4-1 KSSQSVLYSSN N KNYLA (19) WASTREF (20) HQYYSI P (21)

CB028.2 IGKV1-39 RASQG MSNYLN (34) AASTLQS (35) QQSFSTP (36)

CB048.3 IG V1-9 RASQGIRSYLA (16) AASTLQS (17) QQLNTSPP (18)

CB058.1 IGKV1-16 RASQG 1 N N YLA ( 13) AASTLPS (14) QHYI RYP (15)

B. IgG Expression and Purification

To express each IgG, midi-preps of the pCP9 vectors containing both heavy and light chain genes of interest were prepared (Qiagen) and used to transfect 293 -F cells using 293fectin per manufacturer's instructions (Invitrogen, Cat. No. 1-0031).

Following transfection, cells were incubated for 72 hours to allow for sufficient IgG production. Cell media was then harvested and centrifuged to remove the cells.

Purification was effected by column chromatography using a Protein A column (Protein A sepharose beads; Amersham, Cat. No. 17-0963-03). The eluate was then dialyzed against 4 liters of 20 mM Tris-HCl pH7.2, 150 mM NaCl twice. Finally, the dialyzed samples were concentrated down to about 1 mL with a 10 kDa Amicon Ultra column (Millipore).

A series of quality control steps were executed for each IgG to determine concentration and purity, and assess size. IgG concentration was determined initially via NanoDrop readings using a molar extinction coefficient for IgG of 210,000 M-l cm-1. In addition, IgG concentration was confirmed by BCA assay (Thermo Fisher) according to supplier's instructions and by measurements using Protein A sensor tips on the Octet Red384 (ForteBio). As an additional quality control step, SDS-PAGE was performed under non-reducing and reducing conditions (i.e., ±DTT) followed by Bio-Safe Coomassie stain (Biorad) to visualize intact IgG or reduced heavy and light polypeptide chains. Finally, IgGs were quality controlled by size exclusion chromatography a Superdex 200 10/300 GL gel filtration column (Pharmacia).

EXAMPLE 6

IgG binding Assays

IgGs generated and quality controlled as described in Example 5 above, and anti-RSV G antibody CR9514 (comprising the variable regions of 3D3) were tested in ELISA assays for their ability to bind to recombinant RSV Ga and Gb protein.

Briefly, 96 half-well ELISA plates (Costar) were coated with 50 of antigen in IX PBS overnight [RSV Ga: 0.5 μg/mL; RSV Gb: 0.5 μg/mL; bovine actin: 1 μg/mL (Sigma); affinipure goat anti-human F(ab)2: 2 μg/mL (Jackson Immunoresearch). Plates were incubated overnight at 4°C and blocked on the following day with 135 μΐ _^ of 4% non-fat dried milk (NFDM, Biorad) in PBS and incubated for 2 hours at 37° C. mAbs were then diluted in 0.4% NFDM/PBS/0.05% Tween20 starting at 100 ng/mL and titrated down in 5 -fold dilutions, and added to plates for 2 hours at 37° C.

CR9 14 (3D3) mAb was used as positive control against RSV Ga and Gb, and was titrated in a similar manner. Additionally, mouse anti-actin (Sigma, Cat. No. A3853) was used at 1.25 μg/mL as positive control for bovine actin coated plates. After incubation, plates were washed four times with PBS/0.05% Tween20. Secondary antibodies were added each at 1 : 1000 in 0.4% NFDM/PB S/0.05 % Tween20 and incubated for 40 minutes at 37° C. Anti-Fc HRP (Jackson Labs, Cat. No. 109-035- 008) was used as secondary antibody for mAbs. Finally, goat anti-mouse HRP (Jackson Labs, Cat. No. 115-035-072) was used for the actin positive control. Following incubation, plates were washed four times in PBS/0.05% Tween20 and developed with 50 μΐ, 1 :1 v/v TMB:peroxide solution (Pierce, Cat No. 34021) for approximately 5 minutes. The reaction was immediately halted by the addition of 50 μΐ. 2N H2SO4 and the absorbance at 450 nm was measured using an ELISA plate reader. The estimated EC50 values for binding (determined by titrating each IgG) for the antibodies according to the invention ranged between 1.0 and 2.0 ng/ml for RSV strain A/Long and between 0.5 and 2.5 ng/ml for strain B/Bl .

EXAMPLE 7

IgG Neutralization Assays

The anti-PvSV antibodies were analyzed for their ability to bind to and neutralize RSV in solution as assessed by a plaque reduction assay. In this experiment, the virus and the antibodies were pre-incubated in the absence of target cells. The mixture was then added to the cells and virus infection was measured by a standard plaque reduction assay described herein. The anti-RSV antibodies were analyzed for their ability to neutralize several strains of RSV, including RSV A/A2 (ATCC Cat. No. VR-1540), RSV B/18537 (ATCC Cat. No. VR-1580) and RSV A/Long (ATCC Cat. No. VR-26). Antibodies CR9514 (3D3) and CR9505 (an antibody based on 131-2G, i.e. comprising the heavy and light chain variable region of 131-2G, as disclosed in WO 2009/055711) were used as reference.

Vero cells (ATCC, cat no: CCL-81; Manassas) were employed for host cell infection. Vero cells were grown in DMEM (HyClone, cat no: SH 30285.01) with 10% fetal bovine serum (FBS) (HyClone, cat no: SH30070.03), supplemented with 1% L-Glutamine (HyClone, cat no: SH30034.01) and 1% Penicillin-Streptomycin solution (HyClone, cat no: SV30010). The Vero cells were maintained in a 37° C. incubator with 5% C02 and passaged twice per week.

On day 1 of the experiment, Vero cells were cultured in 24-well cell culture plates. The cells were plated at a density (approximately 9x 10 ⁴ cells per well) which allows formation of a cell monolayers (>80% confluence) by day 2. On day 2, each antibody was serially diluted in plain Eagle's minimal essential medium (EMEM, ATCC, cat no: 30-2003) that contained 10% baby rabbit complement (AbD Serotec, cat no. C12CAX). The final antibody concentrations tested were: 10 μg/mL, 1.3 μg/mL, 156 ng/mL, 19.5 ng/mL, 2.4 ng/mL, and 0.3 ng/mL (with the exception of CB010.7, which used antibody concentrations: 2.5 μg/mL, 312.5 ng/mL, 39.1 ng/mL, 4.9 ng/mL, 0.61 ng/mL, and 0.08 ng/niL). The virus was also diluted in plain EMEM to a concentration of 2000-3000 pfu/mL (100-150 pfu/50 μί) and 85 of the diluted RSV was added to 85 of each diluted antibody solution and mixed by pipetting. For the virus control sample, 85 μΐ, of the diluted virus was added to 85 μΐ _^ plain EMEM. The antibody-virus or virus control mixtures were incubated at 37° C. for 2 hours. Following incubation, the culture media was decanted from the 24-well cell culture plates containing the Vera host cells and 150 μΐ, of the pre-incubated virus- antibody or virus-control mixture were then transferred to each well. Each test and control sample was prepared in triplicate. The cells were then incubated at 37° C. for one hour with mixing every 15 min.

Following the incubation period, 1 mL of overlay medium was added to each well (overlay medium contained EMEM, 2% FBS, 1% L-glutamine, 0.75% methylcellulose). The 24-well cell culture plates were then incubated at 37° C (with 5% C0 ₂ ) for approximately 96-120 hours. Cell plates were fixed with 10%> formalin for 1 hour at room temperature, washed 10 times with ddH ₂ 0 and blocked with 5% non-fat dry milk (NFDM) in PBS at 37° C for one hour. Following incubation, the blocking solution was decanted and 200 μΐ, of HRP-conjugated mouse anti-RSV antibody (ab20686, Abeam, 1 :750 dilution in 1% NFDM) was added to each well. The plates were incubated at 37° C for 2 hours, and washed 10 times with ddH ₂ 0. Following washing, 200 μΐ, of TrueBlue® peroxidase substrate (KPL Cat. No. 50-78- 02) was added to each well. The plates were developed for 10 min at room temperature. The plates were washed twice with ddH ₂ 0 and dried on a paper towel and the number of blue plaques was counted.

The IC50 (effective dilution for 50% neutralization of plaque formation) was calculated using SPSS for Windows. The plaque reduction rate was calculated according to the following formula:

Plaque Reduction Rate (percentile) = 1- [(average plaque number in each antibody dilution)/(average plaque number in virus control wells)]* 100 .

Table 13 lists the IC50 for a panel of antibodies for RSV strains A/A2 (ATCC Cat. No. VR-1540) and RSV B/18537 (ATCC Cat. no. VR-1580). Table 13. Neutralization assay results for the top RSV G protein-specific monoclonal antibodies

RSV A RSV B

Strain A/A2 B/18537

Assay Neutralization Neutralization

IC50 (ng/mL) IC50 (ng/mL)

CR9514 (3D3) 40.7 33.0

CB002.1 35.5 23.4

CB003.1 31.5 24.6

CB010.7 16.5 14.1

CB028.2 11.0 19.6

CB048.3 16.7 8.0

CB058.1 14.4 4.2

Table 13 shows that the IC50 (effective dilution for 50% neutralization of plaque formation) of the antibodies and antigen-binding fragments for RSV strain A/A2 (ATCC Cat. No. VR-1540) was below 40 ng/ml and/or the IC50 for RSV strains B/18537 (ATCC Cat. No. VR-1589) was below 30 ng/ml.

In addition, IC50 for antibodies CB003.1, CB010.7 and control antibodies CR9505 (131-2G) and CR9514 (3D3) for RSV strain A/Long (ATCC Cat.No. VR-26) were 16, 12, 18, and 17 ng/mL, respectively.

EXAMPLE 8

Construction of fully human immunoglobulin molecules (human monoclonal antibodies) including codon optimization and de-risking analysis

The heavy and light chain variable regions (VH and VL) for each antibody clone isolated in Example 5 above were examined for the presence of free cysteines and potential post-translational modification sites including glycosylation, deamidation and oxidation sites. To remove these sites, amino acid mutations consisting of structurally conservative and/or germline-based substitutions are used (Table 14). Non-conserved cysteines in the variable regions were mutated to serine. For glycosylation sites, several mutations can be used, including replacement of asparagine for the conservative glutamine or germline mutations. Modifications to the deamidation sites include replacement of aspartic acid for asparagine and serine or alanine for glycine. Sites of potential oxidation are not modified. The nucleotide and amino acid sequences obtained from each VH and VL of the antibody clones were then codon-optimized for expression in human cells at GeneArt/Invitrogen. The variable regions of these functional variants were subsequently cloned directly by restriction digest for expression in the IgG expression vectors pCP9-kappa (See SEQ ID: 127) and pCP9-gamma (See SEQ ID: 128). BamHI, Xhol and/or Srfl were used to clone the variable heavy chains and Notl and Ascl were used to clone the variable light chains. Nucleotide sequences for all constructs were verified according to standard techniques known to the skilled artisan.

Table 14. De-risking of RSV G protein specific monoclonal antibodies

IgG identification Variable Chain Mutation Reason

CB002.1 Heavy C102S C107S Free cysteine

CB003.1 Light N30D Deamidation

CB010.7 NA NA NA

CB028.2 Heavy C105S C110S C112S Free cysteine

C117S

CB048.3 Light N92D Glycosylation

CB058.1 Heavy C104S C109S Free cysteine

EXAMPLE 9

Peptide binding studies by ELISA and Octet

Detailed epitope mapping was performed for the RSV G protein specific mAbs identified such as CB010.7 and CB030.1. Peptides were synthesized by Fmoc chemistry and purified by reversed phase high-performance liquid chromatography (HPLC). For the peptide-peptide interaction studies, some peptides were N-terminally biotinylated via an aminohexanoic acid (Ahx) spacer. The peptides were analyzed for identity by electrospray mass spectrometry. Samples were analyzed by ultra- performance liquid chromatography (UPLC, Alliance, Waters, Milford, MA, USA) with a CI 8 reversed phase column and were detected with a photodiode array detector and a mass sensitive detector. A gradient at 25%/min for 25-100%) acetonitrile (ACN) with solvent A (H20 + 0.05% trifluoroacetic acid [TFA]) and solvent B (ACN + 0.05% TFA) was used. All reagents were at least HPLC grade.

The mAbs were tested for binding to biotinylated peptides that contain the central conserved region of RSV-G type A and B (Table 15). Avidin-coated 96-well microtiter plates were washed and incubated with 100 biotinylated peptide (2.37 xlO ^"7 M) in ELISA buffer (PBS + 1% FBS + 0.05% Tween20) for 1 hr at RT. Next, after washing, 180 μί of blocking buffer (PBS + 10% FBS) per well was transferred to the wells and incubated 1 hr at RT. Subsequently, plates were washed and incubated with anti-human-HRP (Jackson ImmunoResearch), for 1 hr at RT.

Following washing, 100 uL of o-Phenylenediamine horseradish peroxidase substrate (Thermo Scientific) was added to each well. The reaction was stopped after 10 min with 100 μΐ, 1 M H2S04. Absorption was read at 490 nm.

Table 15. RSV-G peptides used for antibody binding studies

Type A central region

biotin-

Sym-1705 I ₄₅ KQRQNKPPNKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGKKTTTKP TKK ₂₀₁

(SEQ ID NO: 129)

biotin-n ₅ KQRQNKPPNKPNNDFHFEVFNFVPCS ICSNNPTCWAICKR ₁₈₄ (SEQ ID NO:

Sym-1706

^J 125)

Type B central region

biotin-

Sym-1788 ₅ KPRPKSPPKKPKDDYHFEVFNFVPCSICGNNQLCKSICKTIPSNKPKKKPTIKP TNK2oi

(SEQ ID NO: 130)

biotin- _{1 5} KPRPKSPPKKPKDDYHFEVFNFVPCS ICGNNQLCKS ICKT ₁₈₄ (SEQ ID NO: Sym-1789

131)

Note: underlined residues correspond to unglycosylated central conserved domain All mAbs described above bind to the RSV Ga and Gb protein (Example 6) and to the central region type A and type B peptides (data not shown). Titration of the antibodies CB003.1 and CB010.7 showed that these mAbs have IC50s of -20 ng/mL for all four peptides (Figure 3). Binding of the mAbs to the RSV G peptides was also determined using Streptavidin sensor tips on the Octet Red384 (ForteBio). Again, the mAbs showed cross-reactivity to both type A and type B peptides (Table 16).

CB003.1 showed the highest response to both type A and type B peptides. CB010.7 showed slightly higher binding to type B, compared to type A peptides.

Table 16. Binding of RSV G specific mAbs to RSV-G peptides (Octet) [RU]

Peptide CB010.7 CB003.1

Sym-1705 1.25 3.48

Sym-1706 1.74 3.36

Sym-1788 1.94 3.28

Sym-1789 2.96 3.20

RU: responsive units EXAMPLE 10

Mapping of minimal epitopes (PepScan)

In order to map the minimal epitope recognized by the mAbs, the reactivity was tested for peptides of multiple length (5, 8, 10, 14, 18, 25, or 32-mer) corresponding to the central region of SV-G type A and B (residues 145-201) using PepScan analysis. The binding of antibodies to peptides was assessed in a PepScan- based ELISA. Each mAb was titrated to ensure that optimal binding was achieved and that nonspecific binding was avoided. Each of the credit-card-format polypropylene plates contained covalently linked peptides that were incubated overnight at 4°C with mAb, between 1 and 10 ng/mL in PBS containing 5% horse serum (v/v), 5% OVA (w/v), and 1% (v/v) Tween 80, or in an alternative blocking buffer of PBS containing 4% horse serum (v/v), and 1% (v/v) Tween 80. After washing, the plates were incubated with a HRP-linked rabbit anti-mAb (DakoCytomation) for 1 hour at 25°C. After further washing, peroxidase activity was assessed using ABTS substrate and color development quantified using a charge-coupled device camera and an image- processing system.

The analysis shows the minimal peptide that binds the antibody corresponding to the energetic core of the epitope and the peptide with the highest binding that contains extra adjacent residues that also contribute to binding and contains the complete epitope. The reactivity of the antibodies to the peptides is summarized in Table 17 (residues depicted as caps). While all antibodies bind the central conserved domain, the critical residues for their binding are different. For two antibodies (CB003.1 and CB010.7) the minimal epitope is limited to the N-terminal CCD region (similar to 3D3, disclosed in WO2009/055711).

EXAMPLE 11

Full substitution analysis (PepScan)

In order to identify the side chains critical for binding and to study the broadness of recognition for the known RSV strains, dedicated sets of peptides were synthesized. A full substitution analysis with a dedicated peptide array of 280 single substitution variant peptides for each position of the sequence FHFEVFNFVPCSIC (SEQ ID NO: 132) recognized by antibodies CB003.1 and CB010.7 was performed and revealed the residues important for binding to these antibodies (Figure 5). The epitope of these antibodies is comparable to the 3D3 epitope but recognized in a completely different manner. This is reflected by the substitution analysis which shows that the epitope of our antibodies have completely different essential residues compared to 3D3. Therefore, the recognition and mode of binding is very different. As shown in Example 7, the antibodies of the present invention have a higher neutralizing capacity than 3D3.

3D3: i5 ₉ FHFEVFNFVPCSICi ₇₂

CBO 10.7 : ₁₅₉ FHFEV FN FVPCSIC ₁₇₂

CB003.1 : ₁₅₉ FHFEVFNFVPCSIC ₁₇ 2

The conserved residues important for binding are also summarized in Table 17 (critical residues depicted in bold).

EXAMPLE 12

Alanine scanning (PepScan)

A set of peptides were tested in which each position was substituted by an Alanine residue (Figure 6). The side chains critical for binding antibodies are summarized in Table 17 (indicated in bold black).

EXAMPLE 13

Binding to natural variant peptides (PepScan)

Next, the antibodies were tested against the panel of 31 peptides that encompass the full diversity of the RSV-G central domain as it occurred in GenBank on January 1, 2012. As shown in Figure 7, almost all naturally occurring variant peptides of type A and B are recognized. CB003.1 shows lower binding to type A than to type B peptides. CBO 10.7 binds both type A and type B peptides equally well. The antibodies are critical to mutations at position 180 in the type A variant peptides. Mutation of Serl70Cys was not critical for CB010.7. Ilel 7 IThr mutation was critical for CB003.1 binding, and Glnl75Arg mutation was critical for CB003.1. The double mutation Ilel81Phe; Ilel84Ala was also critical for CB003.1. Naturally occurring variants critical for binding the four antibodies are summarized in Table 17 (indicated by underline). Table 17. Epitope mapping of RSV G protein specific monoclonal antibodies (PepScan) mAb Type Critical residues in central conserved domain Epitope

RSV-A ₁₅₈ DFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGKi92

CB048.3 RSV-A -FHFEVFNFVP

RSV-B -YHFEVFNFVP-s i-g-nqlc -ic-t- CB058.1 RSV-A -- HFEVFNFVP

RSV-B --HFEVFNFVPcsicgnnqlck-ic- ip

Legend: CAPS = minimal epitope (shortest reactive peptide), ITALIC CAPS = additional residues that contribute to binding, ^^^^^^^^ = critical residues identified using full substitution analysis, bold black = (additional) critical residues identified using alanine scanning, unde r l i ne = (additional) critical residues identified using available central region variant peptides.

EXAMPLE 14

Prophylactic efficacy of anti-G mAbs

To determine whether the anti-G mAbs show in vivo prophylactic efficacy, mAbs CB0003.1 and CB010.7 were tested in the RSV-A/Long cotton rat model. At 24 hr before challenge, male cotton rats, inbred, seronegative for paramyxoviruses, 6- 8 weeks old, weight range day-1 60-80g, were injected intramuscularly with 5 mg/kg of CB003.1, CB010.7, Synagis ^® , or vehicle (n=5 per group) in the upper hind leg (M. quadriceps). At day 0 the cotton rats were challenged with 10 ^5'4 pfu RSV-A/Long by intranasal instillation with 100 (50 each nostril). After 96 hr animals were sacrificed to collect lungs and nasal turbinates: the lingual lobe for isolation of total RNA for total viral RNA load determination by qPCR, the remaining lung and the nasal turbinates for infectious viral load determination by pfu test. Blood samples were collected at day 0 before challenge (24 hr after mAb administration) and at study termination (96 hr after challenge) to confirm adequate dosing. The G mAbs reduced lung and nasal turbinate infectious virus titers and lung RNA virus load compared to vehicle (Figure 8). Lung infectious virus titers (logio PFU/g) were reduced by 2.456 and 1.559 logio by antibodies CB003.1 and CB010.7, respectively, while prophylactic treatment with CR9 14 (3D3) only resulted in a 0.801 logio decrease.

EXAMPLE 15

Therapeutic efficacy of anti-G mAbs

To determine whether the anti-G mAbs show in vivo therapeutic efficacy, mAbs CB003.1 and CB010.7 were tested in the RSV-A/Long cotton rat model. At day 0, male cotton rats, inbred, seronegative for paramyxoviruses, 6-8 weeks old, weight range day-1 60-8 Og, were challenged with 10 ^6'1 pfu RSV-A/Long by intranasal instillation with 100 each nostril). After day 1 post challenge 50 mg/kg CB003.1 , CB010.7, Synagis ^® (n= 14 per group) or vehicle (n=23 per group) were administered by intra-cardic injection. At day 4, 5 animals per group, randomly picked, were sacrificed to collect lungs and nasal turbinates: the lingual lobe for isolation of total RNA for total viral RNA load determination by qPCR, the remaining lung and the nasal turbinates for infectious viral load determination by pfu test. At day 6, all remaining animals (n=9 or 18 per group) were sacrificed to collect lung for pulmonary histopathology. Blood samples were collected at day 2 post challenge (24 hr after mAb administration), and at study termination (day 4 or day 6 after challenge) to confirm adequate dosing. The G mAbs reduced lung and nasal turbinate infectious virus titers, but not lung RNA virus load, compared to vehicle (Figure 9). Lung infectious virus titers (logio PFU/g) were reduced by 2.348 and 1.736 logio by antibodies CB003.1 and CB010.7, respectively, while therapeutic treatment with CR9514 (3D3) only resulted in a 1.369 logio decrease. Moreover, the new G mAbs reduced histopathology scores for peri-bronchiolitis, peri-vasculitis, interstitial pneumonitis and alveolitis (Figure 10), while CR9514 (3D3) only reduced interstitial pneumonitis. SEQUENCES

>CB058.1 VH - SEQ ID NO: 37

EVQLVESGGGLVQPGGSLRLSCVASGFTFSSYAMSWVRQAPGKGLEWVSAIRGSVDNTYY ADSVKGRFT ISRDNSKNTLYLQMNSLRVEDTAVYYCAKDPALYCSGETCFSDLTDWGQGTLVTVSS

>CB058.1 VK-SEQID NO: 38

DIQMTQSPSSLSASVGDRVTITCRASQGINNYLAWFQQKPGKAPKSLIYAASTLPSGVPS RFSGSGSGTDF TLTISSLQPEDSATYFCQHYIRYPHTFGQGTKLEIK

>CB048.3VH-SEQID NO:39

QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHGMHWVRQAPGKGLEWVAVISYDGNKKYY ADSVKGR FTVSRDNSKNTLSLQMDSLRAEDTAIYYCAKTTFYFDDSNYYEYLDYWGQGTLVTVSS >CB048.3 VK-SEQID NO: 40

DIQLTQSPSFLSASVGDRVTITCRASQGIRSYLAWYQQKPGKAPKLLIYAASTLQSGVPS RFSGGGSGTEFT LTI SS LQPE DSATYYCQQLNTS P PYTFGQGTKLE I K

>CB010.7 VH - SEQ ID NO: 41

QVQLVESGGGVVQPGRSLRLSCAASGFTFNTHGMHWVRQAPGKGLEWVAVMSYDGTK KYHADSVKG RFTISRDNSKNTLYLQMNSLRVEDTAIYYCAKVGELRSFDWLLADGTAYYYYGMDVWGQG TTVTVSS

>CB010.7 VK-SEQID NO: 42

DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWFQQKPGQPPRLLINWASTR EFGVPDRFSG SGSGTDFTLTISSLQAEDVAIYYCHQYYSIPLTFGGGTKVEIK

>CB003.1VH-SEQID NO:43

QVQLVQSGPELRKPGASVTVSCKASGYTFTTYYIHWVRQAPGGGLDWMGMINTGSGVTSY AQKFQGR VAMTRDTSTSTVFMELSSLRFEDTALYYCARMYSGSWYPFDYWGQGALVTVSS

>CB003.1 VK-SEQID NO: 44

EIVLTQSPGTLSLSPGERATLSCRASQNINGNYLAWYQQKPGLAPRLLIYEASSRATGIP DRFSGSGSGTDF TLTISSLEPEDFGVYYCQQYGTSPFFTFGPGTKVDIK >CB028.2VH-SEQIDNO:45

QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYGITWVRQAPGQGLEWMGWISGDSDNTNY AQNLQG RVTLTTDISTRTAYMELRSLKPDDTAMYYCARALAKWYCSSSSCFCGGGSCYSDYWGQGT LVTVSS

>CB028.2 VK-SEQ ID NO: 46

DIQMTQSPSSLSASVGDRVTITCRASQGMSNYLNWYQQKPGKAPELLIYAASTLQSG VPSRFSGSGSGTD FTLTINSLQPEDFATYFCQQSFSTPLTFGGGTKVEIK

> CB002.1 VH - SEQ ID NO: 47

QVQLQESGPRLVKPSETLSLTCTVSGGSTSSYFWNWIRQPPGKGLEWIGYIYGSGSADYN PSLKSRVTISID TSKTQFSLKLTSVTAADTAVYYCARSGFCTNDACYRRGSWFDPWGQGTLVTVSS

> CB002.1 VK - SEQ ID NO: 48

DIQMTQSPSSLSASVGDRVTITCRASQSIDNYLNWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDF TLTVSSLHPED FATYYCQQSYSTLTWTFGQGTKVE I K SEP ID: 127 (pCP9-kappa sequence)

TACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCAT GAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTC CGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGATCGGGAGAT CTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCAT AGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGT GCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGC ATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCTAGGTGGTC AATATTGGCCATTAGCCATATTATTCATTGGTTATATAGCATAAATCAATA TTGGCTATTGGCCATTGCATACGTTGTATCCATATCATAATATGTACATTT ATATTGGCTCATGTCCAACATTACCGCCATGTTGACATTGATTATTGACTA GTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGG AGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCC AACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAAC GCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAA CTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTA TTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATG ACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCT ATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCG GTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAG TTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTC CGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATA TAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCA CGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGG CCGGGAACGGTGCATTGGAAGCTTGGTACCGAGCTCGGATCCTTAATTAA CTCGAGGCCCGAGCCCGGGCGAGCCCAGACACTGGACGCTGAACCTCGCG GACAGTTAAGAACCCAGGGGCCTCTGCGCCCTGGGCCCAGCTCTGTCCCA CACCGCGGTCACATGGCACCACCTCTCTTGCAGCCTCCACCAAGGGCCCA TCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTC GTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCC TACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCA GCAGCTTGGGCACCCAGACCTAC ATCTGC AACGTGAATC ACAAGCCCAGC AACACCAAGGTGGACAAGAGAGTTGGTGAGAGGCCAGCACAGGGAGGGA GGGTGTCTGCTGGAAGCCAGGCTCAGCGCTCCTGCCTGGACGCATCCCGG CTATGCAGTCCCAGTCCAGGGCAGCAAGGCAGGCCCCGTCTGCCTCTTCA CCCGGAGGCCTCTGCCCGCCCCACTCATGCTCAGGGAGAGGGTCTTCTGG CTTTTTCCCCAGGCTCTGGGCAGGCACGGGCTAGGTGCCCCTAACCCAGG CCCTGCACACAAAGGGGCAGGTGCTGGGCTCAGACCTGCCAAGAGCCATA TCCGGGAGGACCCTGCCCCTGACCTAAGCCCACCCCAAAGGCCAAACTCT CCACTCCCTCAGCTCGGACACCTTCTCTCCTCCCAGATTCCAGTAACTCCC AATCTTCTCTCTGCAGAGCCCAAATCTTGTGACAAAACTCACACATGCCCA CCGTGCCCAGGTAAGCCAGCCCAGGCCTCGCCCTCCAGCTCAAGGCGGGA CAGGTGCCCTAGAGTAGCCTGCATCCAGGGACAGGCCCCAGCCGGGTGCT GACACGTCCACCTCCATCTCTTCCTCAGCACCTGAACTCCTGGGGGGACCG TCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGA CAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGT CCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCA AGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAA GCCAAAGGTGGGACCCGTGGGGTGCGAGGGCCACATGGACAGAGGCCGG CTCGGCCCACCCTCTGCCCTGAGAGTGACCGCTGTACCAACCTCTGTCCCT ACAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGA GGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCT ATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAA CAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCT CTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTC TTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAA GAGCCTCTCCCTGTCTCCGGGTAAATGAGCTAGCGAATTCACCGGTACCA AGCTTAAGTTTAAACCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAG CCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCC ACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTG AGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGG GGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCT ATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCA CGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCA GCGTGACCGCTACACTTGCCAGCGCCTAGCGCCCGCTCCTTTCGCTTTCTT CCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCG GGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAA AAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGA CGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTT GTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTT ATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTA ACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTG TGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATC TCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGC AGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCC CCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCC GCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTC TGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAG GCTTTTGCAAAAAGCTCCCGGGAGCTTGGATATCCATTTTCGGATCTGATC AAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCAC GCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGC ACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGC AGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATG AACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTT CCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCT GCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCC TGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGC TTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAG CGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGA CGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGG CGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGC TTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGT GGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCG TGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCT TTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCT TGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGGTGCTACGAGATTTCGA TTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGA CGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGC CCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAG CATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGG TTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCT AGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGT TATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAA AGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTC ACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGAATTGCATGAAGA ATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCTAGGTGGTCAATATTG GCCATTAGCCATATTATTCATTGGTTATATAGCATAAATCAATATTGGCTA TTGGCCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGG CTCATGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTA ATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCG CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACC CCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATA GGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCA CTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGT CAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTAT GGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCA TGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGAC TCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTT TGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCC ATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCA GAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGT TTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGA ACGGTGCATTGGAAGCTTGGTACCGGTGAATTCGGCGCGCCAGATCTGCG GCCGCTAGGAAGAAACTCAAAACATCAAGATTTTAAATACGCTTCTTGGT CTCCTTGCTATAATTATCTGGGATAAGCATGCTGTTTTCTGTCTGTCCCTAA CATGCCCTGTGATTATCCGCAAACAACACACCCAAGGGCAGAACTTTGTT ACTTAAACACCATCCTGTTTGCTTCTTTCCTCAGGAACTGTGGCTGCACCA TCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCC TCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAG TGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCAC AGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACG CTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCA CCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAG TGTTAGTTAACGGATCGATCCGAGCTCGGTACCAAGCTTAAGTTTAAACC GCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCC CCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTT CCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTA TTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGA CAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGG AAAGAACCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGT TTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCG GTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACG GTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAA GGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTT CCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTC AGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCT GGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATAC CTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGC TGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTG CACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGT CTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCAC TGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCT TGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATC TGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTG ATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCA GCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTT CTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTG GTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAA TGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGT TACC AATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGT TCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAG GGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTC ACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGC GCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTT GCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTT GTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCT TCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATG TTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGT AAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCT CTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCA ACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCC GGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGC TCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCG CTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCA GCATCTTTTACTTTC ACC AGCGTTTCTGGGTGAGCAAAAAC AGGAAGGCA AAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTC A

SEP ID: 128 (pCP9-lambda sequence)

TACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCAT GAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTC CGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGATCGGGAGAT CTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCAT AGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGT GCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGC ATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCTAGGTGGTC AATATTGGCCATTAGCCATATTATTCATTGGTTATATAGCATAAATCAATA TTGGCTATTGGCCATTGCATACGTTGTATCCATATCATAATATGTACATTT ATATTGGCTCATGTCCAACATTACCGCCATGTTGACATTGATTATTGACTA GTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGG AGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCC AACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAAC GCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAA CTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTA TTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATG ACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCT ATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCG GTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAG TTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTC CGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATA TAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCA CGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGG CCGGGAACGGTGCATTGGAAGCTTGGTACCGAGCTCGGATCCTTAATTAA CTCGAGGCCCGAGCCCGGGCGAGCCCAGACACTGGACGCTGAACCTCGCG GACAGTTAAGAACCCAGGGGCCTCTGCGCCCTGGGCCCAGCTCTGTCCCA CACCGCGGTCACATGGCACCACCTCTCTTGCAGCCTCCACCAAGGGCCCA TCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTC GTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCC TACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCA GCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGC AACACCAAGGTGGACAAGAGAGTTGGTGAGAGGCCAGCACAGGGAGGGA GGGTGTCTGCTGGAAGCCAGGCTCAGCGCTCCTGCCTGGACGCATCCCGG CTATGCAGTCCCAGTCCAGGGCAGCAAGGCAGGCCCCGTCTGCCTCTTCA CCCGGAGGCCTCTGCCCGCCCCACTCATGCTCAGGGAGAGGGTCTTCTGG CTTTTTCCCCAGGCTCTGGGCAGGCACGGGCTAGGTGCCCCTAACCCAGG CCCTGCACACAAAGGGGCAGGTGCTGGGCTCAGACCTGCCAAGAGCCATA TCCGGGAGGACCCTGCCCCTGACCTAAGCCCACCCCAAAGGCCAAACTCT CCACTCCCTCAGCTCGGACACCTTCTCTCCTCCCAGATTCCAGTAACTCCC AATCTTCTCTCTGCAGAGCCCAAATCTTGTGACAAAACTCACACATGCCCA CCGTGCCCAGGTAAGCCAGCCCAGGCCTCGCCCTCCAGCTCAAGGCGGGA CAGGTGCCCTAGAGTAGCCTGCATCCAGGGACAGGCCCCAGCCGGGTGCT GACACGTCCACCTCCATCTCTTCCTCAGCACCTGAACTCCTGGGGGGACCG TCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGA CAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGT CCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCA AGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAA GCCAAAGGTGGGACCCGTGGGGTGCGAGGGCCACATGGACAGAGGCCGG CTCGGCCCACCCTCTGCCCTGAGAGTGACCGCTGTACCAACCTCTGTCCCT ACAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGA GGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCT ATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAA CAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCT CTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTC TTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAA GAGCCTCTCCCTGTCTCCGGGTAAATGAGCTAGCGAATTCACCGGTACCA AGCTTAAGTTTAAACCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAG CCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCC ACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTG AGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGG GGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCT ATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCA CGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCA GCGTGACCGCTACACTTGCCAGCGCCTAGCGCCCGCTCCTTTCGCTTTCTT CCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCG GGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAA AAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGA CGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTT GTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTT ATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTA ACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTG TGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATC TCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGC AGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCC CCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCC GCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTC TGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAG GCTTTTGCAAAAAGCTCCCGGGAGCTTGGATATCCATTTTCGGATCTGATC AAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCAC GCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGC ACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGC AGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATG AACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTT CCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCT GCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCC TGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGC TTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAG CGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGA CGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGG CGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGC TTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGT GGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCG TGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCT TTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCT TGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGGTGCTACGAGATTTCGA TTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGA CGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGC CCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAG CATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGG TTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCT AGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGT TATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAA AGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTC ACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGAATTGCATGAAGA ATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCTAGGTGGTCAATATTG GCCATTAGCCATATTATTCATTGGTTATATAGCATAAATCAATATTGGCTA TTGGCCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGG CTCATGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTA ATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCG CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACC CCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATA GGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCA CTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGT CAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTAT GGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCA TGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGAC TCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTT TGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCC ATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCA GAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGT TTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGA ACGGTGCATTGGAAGCTTGGTACCGGTGAATTCGGCGCGCCAGATCTGCG GCCGCTAGGAAGAAACTCAAAACATCAAGATTTTAAATACGCTTCTTGGT CTCCTTGCTATAATTATCTGGGATAAGCATGCTGTTTTCTGTCTGTCCCTAA CATGCCCTGTGATTATCCGCAAACAACACACCCAAGGGCAGAACTTTGTT ACTTAAACACCATCCTGTTTGCTTCTTTCCTCAGGTCAGCCCAAGGCTGCC CCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAG GCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGT GGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACC ACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGA GCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTC ACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTT CATAGAGTTAACGGATCGATCCGAGCTCGGTACCAAGCTTAAGTTTAAAC CGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGC CCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTT TCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCT ATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAG ACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCG GAAAGAACCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGG TTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCG GTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACG GTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAA GGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTT CCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTC AGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCT GGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATAC CTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGC TGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTG CACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGT CTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCAC TGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCT TGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATC TGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTG ATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCA GCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTT CTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTG GTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAA TGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGT TACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGT TCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAG GGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTC ACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGC GCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTT GCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTT GTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCT TCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATG TTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGT AAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCT CTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCA ACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCC GGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGC TCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCG CTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCA GCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCA AAATGCCGC AAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTC A