HK1146073B - Rsv-specific binding molecules and means for producing them - Google Patents

Description

RSV-specific binding molecules and means for producing them

The present invention relates to the fields of biology and medicine.

Respiratory Syncytial Virus (RSV) is a common cold virus and belongs to the family paramyxoviridae. RSV is virulent, readily transmitted, and the most common cause of lower respiratory tract disease in children under the age of 2. Up to 98% of daycare children will be infected during one RSV season. Hospitalization is required for 0.5% to 3.2% of children infected with RSV. In the united states, there are approximately 90,000 hospitalizations and 4500 deaths per year. The major risk factors for hospitalization due to RSV are preterm birth, chronic lung disease, congenital heart disease, immune insufficiency and other healthy children under 6 weeks of age. There is no effective treatment for RSV-positive bronchiolitis, but sufficient nutritional and oxygen-therapy forms of supportive care are available. Antiviral treatments such as ribavirin are ineffective in RSV infection. A monoclonal antibody palivizumab (also known as Synagis) is registered for the prevention of RSV infection. Palivizumab is a monoclonal antibody to a genetically engineered (humanized) RSV fusion protein. However, palivizumab is not always effective. Thus, there is a need in the art for additional antibodies and treatments to combat RSV.

It is an object of the present invention to provide means and methods for combating and/or preventing RSV-related diseases. It is another object of the present invention to provide alternative and/or improved antibodies to RSV or functional equivalents thereof and to provide stable cells capable of producing antibodies to RSV or functional equivalents thereof.

The present invention provides antibodies and functional equivalents thereof capable of specifically binding RSV. Such antibodies and/or functional equivalents, also referred to herein as "anti-RSV antibodies" or "RSV-specific antibodies", are capable of specifically binding at least one component of RSV, e.g., an epitope of an RSV protein. The term "specific binding" does not include non-specific adhesion. The anti-RSV antibodies and functional equivalents of the invention are particularly suitable for combating and/or at least partially preventing RSV infection and/or adverse effects of RSV infection. A particularly preferred anti-RSV antibody of the invention is the antibody designated "D25" which has a heavy chain region and a light chain region as shown in FIGS. 11A-D. The CDR sequences of D25 that specifically gave rise to the antigen binding properties of D25 are shown in FIG. 11D. Antibody D25 appears to have superior performance compared to the registered anti-RSV antibody palivizumab (fig. 8). For example, D25 has an IC50 value of about 0.4-1.5ng/ml in an in vitro neutralization experiment of HEp-2 cell infected RSV, while palivizumab has an IC50 value of about 453 ng/ml.

Functional equivalents of antibodies are defined herein as functional portions, derivatives or analogs of antibodies.

A functional portion of an antibody is defined as a portion that has at least one of the same properties (of the same species, but not necessarily in the same amount) as the antibody. The functional moiety is capable of binding to the same antigen as the antibody, but not necessarily to the same extent. The functional portion of the antibody preferably comprises a single domain antibody, a single chain variable fragment (scFv), a Fab fragment or a F (ab')₂And (3) fragment.

A functional derivative of an antibody is defined as an antibody that is engineered to have at least one property, preferably an antigen binding property, of substantially the same species, but not necessarily the same amount, of the resulting compound. Derivatives are provided in a number of ways, for example by conservative amino acid substitutions, in which an amino acid residue is substituted by another residue of substantially similar nature (size, hydrophobicity, etc.), such that overall function is less likely to be severely affected.

Analogous compounds to those of skill in the art that are capable of producing antibodies. This is done by screening peptide libraries or phage display libraries. Such analogs have at least one property that is substantially the same as the antibody in species and not necessarily in amount.

As is well known to those skilled in the art, an antibody heavy chain is the larger of the two chain types that make up an immunoglobulin molecule. The heavy chain includes a constant region and a variable region, wherein the variable region is involved in antigen binding. The light chain of an antibody is the smaller of the two chain types that make up an immunoglobulin molecule. Light chains include constant and variable regions. The variable regions are involved in antigen binding along with the heavy chain variable regions.

Complementarity Determining Regions (CDRs) are hypervariable regions in the heavy chain variable region and the light chain variable region. The CDRs of the heavy chain and the linked light chain of the antibody together form the antigen binding site.

Since the present invention recognizes that the CDR sequences shown in fig. 11 provide the desired RSV-binding properties, the skilled artisan is able to generate variants comprising at least one altered CDR sequence. For example, conservative amino acid substitutions are employed. Conservative amino acid substitutions include the substitution of one amino acid with another amino acid of substantially similar properties (size, hydrophobicity, etc.) such that overall function is less likely to be severely affected.

It is also possible to alter at least one of the CDR sequences depicted in fig. 11 in order to generate a variant antibody or functional equivalent thereof having an altered at least one property compared to D25. Preferably, an antibody or functional equivalent is provided comprising a CDR sequence which is at least 70% identical to the CDR sequence as depicted in fig. 11, in order to at least partially maintain, or even improve, the advantageous binding properties of D25. The CDR sequences depicted in fig. 11 are preferably altered such that the resulting antibody or functional equivalent comprises at least one improved property, such as improved binding affinity, selectivity and/or stability, compared to D25. Accordingly, variant antibodies comprising an amino acid sequence at least 70% identical to the CDR sequence depicted in fig. 11 or functional equivalents thereof are within the scope of the present invention. Various methods of altering amino acid sequences are available in the art. For example, heavy or light chain sequences having the desired CDR sequences are synthesized. Preferably, the nucleic acid sequences encoding the CDRs are mutated, e.g., using random or site-directed mutagenesis.

Accordingly, the present invention provides in a first aspect an isolated, synthetic or recombinant antibody or functional equivalent thereof capable of specifically binding respiratory syncytial virus, which comprises:

-a heavy chain CDR1 sequence comprising a sequence at least 70% identical to the sequence NYIIN, and/or

-a heavy chain CDR2 sequence comprising a sequence at least 75% identical to sequence GIIPVLGTVHYAPKFQG, and/or

-a heavy chain CDR3 sequence comprising a sequence at least 70% identical to sequence ETALVVSTTYLPHYFDN, and/or

-a light chain CDR1 sequence comprising a sequence at least 85% identical to sequence QASQDIVNYLN, and/or

-a light chain CDR2 sequence comprising a sequence at least 70% identical to the sequence VASNLET.

Preferably, the antibody further comprises a light chain CDR3 sequence comprising a sequence at least 70% identical to the sequence QQYDNLP.

Preferably, an antibody or functional equivalent of the invention comprises a CDR sequence which is at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% identical to at least one CDR sequence as depicted in fig. 11D. Most preferably, an antibody or functional equivalent of the invention comprises a CDR sequence which is at least 95% identical to at least one of the CDR sequences depicted in fig. 11D. The above-described particularly preferred antibody D25 comprises a CDR sequence consisting of the CDR sequence shown in FIG. 11D. Accordingly, a particularly preferred embodiment of the invention provides an isolated, synthetic or recombinant antibody or functional equivalent thereof capable of specifically binding respiratory syncytial virus, which comprises:

-a heavy chain CDR1 sequence comprising the sequence NYIIN, and/or

-a heavy chain CDR2 sequence comprising the sequence GIIPVLGTVHYAPKFQG, and/or

-a heavy chain CDR3 sequence comprising the sequence ETALVVSTTYLPHYFDN, and/or

-a light chain CDR1 sequence comprising the sequence QASQDIVNYLN, and/or

-a light chain CDR2 sequence comprising the sequence VASNLET.

Preferably, the antibody further comprises a light chain CDR3 sequence comprising the sequence QQYDNLP.

In one embodiment, an antibody or functional equivalent is provided comprising three heavy chain CDR sequences and three light chain CDR sequences as depicted in fig. 11D, or sequences at least 70%, preferably at least 80%, more preferably at least 85% identical thereto. Thus, there is further provided an isolated, synthetic or recombinant antibody or functional equivalent thereof comprising a heavy chain CDR1 sequence comprising a sequence at least 70% identical to the sequence NYIIN, a heavy chain CDR2 sequence comprising a sequence at least 70% identical to the sequence GIIPVLGTVHYAPKFQG, a heavy chain CDR3 sequence comprising a sequence at least 70% identical to the sequence ETALVVSTTYLPHYFDN, a light chain CDR1 sequence comprising a sequence at least 70% identical to the sequence QASQDIVNYLN, a light chain CDR2 sequence comprising a sequence at least 70% identical to the sequence VASNLET and a light chain CDR3 sequence comprising a sequence at least 70% identical to the sequence QQYDNLP. Preferably, the antibody or functional equivalent comprises CDR sequences at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% identical to the heavy chain CDR sequences and the light chain CDR sequences depicted in fig. 11D. Antibodies or functional equivalents comprising the above-described heavy chain CDR1, CDR2, and CDR3 sequences and the above-described light chain CDR1, CDR2, and CDR3 sequences are also provided.

Also provided is an antibody or functional equivalent thereof comprising a heavy chain variable region amino acid sequence at least 70% identical to the heavy chain sequence set forth in figure 11. Such heavy chain sequences provide the desired RSV-binding properties, as demonstrated by antibody D25. Accordingly, there is also provided an antibody or functional equivalent thereof, the heavy chain sequence of which comprises a sequence at least 70% identical to sequence QVQLVQSGAEVKKPGSSVMVSCQASGGPLRNYIINWLRQAPGQGPEWMGGIIPVLGTVHYAPKFQGRVTITADESTDTAYIHLISLRSEDTAMYYCATETALVVSTTYLPHYFDNWGQGTLVTVSS. Moreover, a light chain variable region amino acid sequence at least 70% identical to the light chain sequence set forth in fig. 11 provides the desired RSV binding properties, as demonstrated by antibody D25. Accordingly, there is also provided an antibody or functional equivalent thereof having a light chain sequence at least 70% identical to sequence DIQMTQSPSSLSAAVGDRVTITCQASQDIVNYLNWYQQKPGKAPKLLIYVASNLETGVPSRFSGSGSGTDFSLTISSLQPEDVATYYCQQYDNLPLTFGGGTKVEIKRTV. The antibody or functional portion of the invention preferably comprises a heavy chain variable region sequence and/or a light chain variable region sequence which is at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% identical to the heavy chain sequence and/or the light chain sequence as depicted in figure 11. The higher the homology, the closer the antibody or functional portion is to antibody D25. Preferably, the antibody or functional part of the invention comprises a heavy chain like the heavy and light chain of D25 as well as a light chain. Accordingly, there is also provided an antibody or functional part comprising a heavy chain sequence and a light chain sequence which are at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% identical to the heavy chain sequence and the light chain sequence as depicted in figure 11.

One embodiment provides an antibody or functional equivalent thereof comprising a heavy chain sequence consisting of the heavy chain sequence shown in figure 11 and a light chain sequence consisting of the light chain sequence shown in figure 11. Alternatively, as is well known to those skilled in the art, it is possible to produce heavy or light chain sequences that are shortened, but maintain the binding properties of interest. Preferably, such shortened heavy or light chains are produced having a shorter constant region than the original heavy or light chain. Preferably, the variable region is kept constant. For example, Fab fragments or F (ab')₂And (3) fragment. Thus, also provided is a kit comprising at least the sequence shown in FIG. 11Antibody functional equivalents of functional moieties. The functional portion is at least 20 amino acids in length and comprises a sequence that is at least 70% identical to a heavy chain CDR1 sequence shown in fig. 11D, and/or a sequence that is at least 75% identical to a heavy chain CDR2 sequence shown in fig. 11D, and/or a sequence that is at least 70% identical to a heavy chain CDR3 sequence shown in fig. 11D, and/or a sequence that is at least 85% identical to a light chain CDR1 sequence shown in fig. 11D, and/or a sequence that is at least 70% identical to a light chain CDR2 sequence shown in fig. 11D. Preferably, the functional portion further comprises a sequence that is at least 70% identical to the light chain CDR3 sequence shown in fig. 11D.

Another particularly preferred anti-RSV antibody of the invention is the antibody designated "AM 14" which has a heavy chain region and a light chain region as shown in figure 14A. The CDR sequences of AM14 that specifically produced the antigen binding properties of AM14 are also seen in fig. 14A.

Since the present invention recognizes that the CDR sequences shown in fig. 14A provide the desired RSV-binding properties, the skilled artisan is able to generate variants comprising at least one altered CDR sequence. For example, conservative amino acid substitutions are employed. Conservative amino acid substitutions include the substitution of one amino acid with another amino acid of substantially similar properties (size, hydrophobicity, etc.) such that overall function is less likely to be severely affected.

It is also possible to alter at least one of the CDR sequences depicted in fig. 14A in order to generate a variant antibody or functional equivalent thereof having an altered at least one property compared to AM 14. Preferably, an antibody or functional equivalent is provided comprising a CDR sequence which is at least 70% identical to the CDR sequence as depicted in fig. 14A, in order to at least partially retain, or even improve, the advantageous binding properties of AM 14. Preferably, the CDR sequences depicted in fig. 14A are altered such that the resulting antibody or functional equivalent comprises at least one improved property, such as improved binding affinity, selectivity and/or stability, compared to AM 14. Accordingly, variant antibodies comprising amino acid sequences at least 70% identical to the CDR sequences depicted in fig. 14A or functional equivalents thereof are within the scope of the present invention. Various methods of altering amino acid sequences are available in the art. For example, heavy or light chain sequences having the desired CDR sequences are synthesized. Preferably, the nucleic acid sequences encoding the CDRs are mutated, e.g., using random or site-directed mutagenesis.

Accordingly, the present invention provides in one aspect an isolated, synthetic or recombinant antibody or functional part, derivative and/or analogue thereof capable of specifically binding respiratory syncytial virus, which comprises:

-a heavy chain CDR1 sequence comprising a sequence at least 70% identical to the sequence GFSFSHYA, and/or

-a heavy chain CDR2 sequence comprising a sequence at least 70% identical to the sequence ISYDGENT, and/or

-a heavy chain CDR3 sequence comprising a sequence at least 70% identical to the sequence ardridvddyyyygmdv, and/or

A light chain CDR1 sequence comprising a sequence at least 70% identical to sequence QDIKKY, and/or

-a light chain CDR2 sequence comprising a sequence at least 70% identical to the sequence DAS, and/or

A light chain CDR3 sequence comprising a sequence at least 70% identical to the sequence QQYDNLPPLT.

Preferably, an antibody or functional equivalent of the invention comprises a CDR sequence which is at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% identical to at least one CDR sequence as depicted in fig. 14A. Most preferably, an antibody or functional equivalent of the invention comprises a CDR sequence which is at least 95% identical to at least one of the CDR sequences depicted in fig. 14A. The above-described particularly preferred antibody AM14 comprises a CDR sequence consisting of the CDR sequence shown in fig. 14A. Accordingly, a particularly preferred embodiment of the invention provides an isolated, synthetic or recombinant antibody or functional equivalent thereof capable of specifically binding respiratory syncytial virus, which comprises:

-a heavy chain CDR1 sequence comprising the sequence GFSFSHYA, and/or

-a heavy chain CDR2 sequence comprising the sequence ISYDGENT, and/or

-a heavy chain CDR3 sequence comprising the sequence ardridvddyyyygmdv, and/or

A light chain CDR1 sequence comprising the sequence QDIKKY, and/or

-a light chain CDR2 sequence comprising the sequence DAS, and/or

A light chain CDR3 sequence comprising the sequence QQYDNLPPLT.

In one embodiment, an antibody or functional equivalent is provided comprising three heavy chain CDR sequences and three light chain CDR sequences as depicted in fig. 14A, or sequences at least 70% identical thereto. Thus, there is further provided an isolated, synthetic or recombinant antibody or functional equivalent thereof comprising a heavy chain CDR1 sequence comprising a sequence at least 70% identical to the sequence GFSFSHYA, a heavy chain CDR2 sequence comprising a sequence at least 70% identical to the sequence ISYDGENT, a heavy chain CDR3 sequence comprising a sequence at least 70% identical to the sequence ardriddyyyyygmdv, a light chain CDR1 sequence comprising a sequence at least 70% identical to the sequence QDIKKY, a light chain CDR2 sequence comprising a sequence at least 70% identical to the sequence DAS and a light chain CDR3 sequence comprising a sequence at least 70% identical to the sequence qydqnlpplt. Preferably, the antibody or functional equivalent comprises CDR sequences at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% identical to the heavy chain CDR sequences and the light chain CDR sequences depicted in fig. 14A. Also provided are antibodies or functional equivalents comprising the heavy chain CDR1, CDR2, and CDR3 sequences of fig. 14A described above and the light chain CDR1, CDR2, and CDR3 sequences of fig. 14A described above.

Also provided is an antibody or functional equivalent thereof comprising a heavy chain amino acid sequence at least 70% identical to the heavy chain sequence set forth in figure 14A. Such heavy chain sequences provide the desired RSV-binding properties, as demonstrated by antibody AM 14. Accordingly, there is also provided an antibody or functional equivalent thereof, the heavy chain sequence of which comprises a sequence at least 70% identical to sequence EVQLVESGGGVVQPGRSLRLSCAASGFSFSHYAMHWVRQAPGKGLEWVAVISYDGENTYYADSVKGRFSISRDNSKNTVSLQMNSLRPEDTALYYCARDRIVDDYYYYGMDVWGQGATVTVSS. Moreover, a light chain amino acid sequence at least 70% identical to the light chain sequence set forth in fig. 14A provides the desired RSV binding characteristics, as evidenced by antibody AM 14. Accordingly, there is also provided an antibody or functional equivalent thereof having a light chain sequence at least 70% identical to sequence DIQMTQSPSSLSASVGDRVTITCQASQDIKKYLNWYHQKPGKVPELLMHDASNLETGVPSRFSGRGSGTDFTLTISSLQPEDIGTYYCQQYDNLPPLTFGGGTKVEIKRTV. Preferably, the antibody or functional portion of the invention comprises a heavy chain variable region sequence and/or a light chain variable region sequence which is at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% identical to the heavy chain sequence and/or the light chain sequence as depicted in figure 14A. The higher the homology, the closer the antibody or functional portion is to antibody AM 14. Preferably, the antibody or functional part of the invention comprises a heavy chain and a light chain similar to the heavy and light chains of AM 14. Accordingly, there is also provided an antibody or functional part comprising a heavy chain sequence and a light chain sequence which are at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% identical to the heavy chain sequence and the light chain sequence as set out in figure 14A.

One embodiment provides an antibody or functional equivalent thereof comprising a heavy chain sequence consisting of the heavy chain sequence shown in figure 14A and a light chain sequence consisting of the light chain sequence shown in figure 14A. Alternatively, as is well known to those skilled in the art, it is possible to produce heavy or light chain sequences that are shortened, but maintain the binding properties of interest. Preferably, such shortened heavy or light chains are produced having a shorter constant region than the original heavy or light chain. Preferably, the variable region is kept constant. For example, Fab fragments or F (ab')₂And (3) fragment. Accordingly, functional equivalents of antibodies comprising at least a functional portion of the sequence shown in figure 14A are also provided. The functional portion is at least 20 amino acids in length and comprises a sequence at least 70% identical to at least one of the CDR sequences depicted in fig. 14A.

Another particularly preferred anti-RSV antibody of the invention is the antibody designated "AM 16" which has a heavy chain region and a light chain region as shown in figure 14B. The CDR sequences of AM16 that specifically produced the antigen binding properties of AM16 are also seen in fig. 14B.

Since the present invention recognizes that the CDR sequences shown in fig. 14B provide the desired RSV-binding properties, the skilled artisan is able to generate variants comprising at least one altered CDR sequence. For example, conservative amino acid substitutions are employed. Conservative amino acid substitutions include the substitution of one amino acid with another amino acid of substantially similar properties (size, hydrophobicity, etc.) such that overall function is less likely to be severely affected.

It is also possible to alter at least one of the CDR sequences depicted in fig. 14B in order to generate a variant antibody or functional equivalent thereof having an altered at least one property compared to AM 16. Preferably, an antibody or functional equivalent is provided comprising a CDR sequence which is at least 70% identical to the CDR sequence as depicted in fig. 14B, in order to at least partially retain, or even improve, the advantageous binding properties of AM 16. Preferably, the CDR sequences depicted in fig. 14B are altered such that the resulting antibody or functional equivalent comprises at least one improved property, such as improved binding affinity, selectivity and/or stability, compared to AM 16. Accordingly, variant antibodies or functional equivalents thereof comprising an amino acid sequence at least 70% identical to the CDR sequences depicted in fig. 14B are within the scope of the present invention. Various methods of altering amino acid sequences are available in the art. For example, heavy or light chain sequences having the desired CDR sequences are synthesized. Preferably, the nucleic acid sequences encoding the CDRs are mutated, e.g., using random or site-directed mutagenesis.

Accordingly, the present invention provides in one aspect an isolated, synthetic or recombinant antibody or functional part, derivative and/or analogue thereof capable of specifically binding respiratory syncytial virus, which comprises:

-a heavy chain CDR1 sequence comprising a sequence at least 70% identical to the sequence GFTFSSYN, and/or

-a heavy chain CDR2 sequence comprising a sequence at least 70% identical to the sequence isagssy, and/or

-a heavy chain CDR3 sequence comprising a sequence at least 70% identical to the sequence aredyygpgnyyspnwfdp, and/or

-a light chain CDR1 sequence comprising a sequence at least 70% identical to the sequence SSNIGAGYD, and/or

A light chain CDR2 sequence comprising a sequence at least 70% identical to the sequence GNT, and/or

A light chain CDR3 sequence comprising a sequence at least 70% identical to the sequence HSYDRSLSG.

Preferably, an antibody or functional equivalent of the invention comprises a CDR sequence which is at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% identical to at least one CDR sequence as depicted in fig. 14B. Most preferably, an antibody or functional equivalent of the invention comprises a CDR sequence which is at least 95% identical to at least one of the CDR sequences depicted in fig. 14B. The above-described particularly preferred antibody AM16 comprises a CDR sequence consisting of the CDR sequence shown in fig. 14B. Accordingly, a particularly preferred embodiment of the invention provides an isolated, synthetic or recombinant antibody or functional equivalent thereof capable of specifically binding respiratory syncytial virus, which comprises:

-a heavy chain CDR1 sequence comprising the sequence GFTFSSYN, and/or

-a heavy chain CDR2 sequence comprising the sequence ISAGSSYI, and/or

-a heavy chain CDR3 sequence comprising the sequence aredyygpgnyyspnwfdp, and/or

-a light chain CDR1 sequence comprising the sequence SSNIGAGYD, and/or

A light chain CDR2 sequence comprising the sequence GNT, and/or

-a light chain CDR3 sequence comprising the sequence HSYDRSLSG.

In one embodiment, an antibody or functional equivalent is provided comprising three heavy chain CDR sequences and three light chain CDR sequences as depicted in fig. 14B, or sequences at least 70% identical thereto. Thus, there is further provided an isolated, synthetic or recombinant antibody or functional equivalent thereof comprising a heavy chain CDR1 sequence comprising a sequence at least 70% identical to the sequence GFTFSSYN, a heavy chain CDR2 sequence comprising a sequence at least 70% identical to the sequence ISAGSSYI, a heavy chain CDR3 sequence comprising a sequence at least 70% identical to the sequence aredyygpgnyyspnwfdp, a light chain CDR1 sequence comprising a sequence at least 70% identical to the sequence SSNIGAGYD, a light chain CDR2 sequence comprising a sequence at least 70% identical to the sequence GNT and a light chain CDR3 sequence comprising a sequence at least 70% identical to the sequence HSYDRSLSG. Preferably, the antibody or functional equivalent comprises a CDR sequence which is at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% identical to the above-described heavy chain CDR sequence and the above-described light chain CDR sequence as depicted in fig. 14B. Also provided are antibodies or functional equivalents comprising the heavy chain CDR1, CDR2, and CDR3 sequences of fig. 14B described above and the light chain CDR1, CDR2, and CDR3 sequences of fig. 14B described above.

Also provided is an antibody or functional equivalent thereof comprising a heavy chain amino acid sequence at least 70% identical to the heavy chain sequence set forth in figure 14B. Such heavy chain sequences provide the desired RSV-binding properties, as demonstrated by antibody AM 16. Accordingly, there is also provided an antibody or functional equivalent thereof, the heavy chain sequence of which comprises a sequence at least 70% identical to sequence EVQLVETGGGLAQPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSHISAGSSYIYYSDSVKGRFTVSRDNVRNSVYLQMNSLRAADTAVYYCAREDYGPGNYYSPNWFDPWGQGTLVTVSS. Moreover, a light chain amino acid sequence at least 70% identical to the light chain sequence set forth in fig. 14B provides the desired RSV binding characteristics, as evidenced by antibody AM 16. Accordingly, there is also provided an antibody or functional equivalent thereof having a light chain sequence at least 70% identical to sequence QSVVTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNTNRPSGVSDRFSGSKSGTSASLAITGLQAEDEADYYCHSYDRSLSGSVFGGGTKLTV. Preferably, the antibody or functional portion of the invention comprises a heavy chain variable region sequence and/or a light chain variable region sequence which is at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% identical to the heavy chain sequence and/or the light chain sequence as depicted in figure 14B. The higher the homology, the closer the antibody or functional portion is to antibody AM 16. Preferably, the antibody or functional part of the invention comprises a heavy chain and a light chain similar to the heavy and light chains of AM 16. Accordingly, there is also provided an antibody or functional part comprising a heavy chain sequence and a light chain sequence which are at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% identical to the heavy chain sequence and the light chain sequence as set out in figure 14B.

One embodiment provides an antibody or functional equivalent thereof comprising a heavy chain sequence consisting of the heavy chain sequence shown in figure 14B and a light chain sequence consisting of the light chain sequence shown in figure 14B. Alternatively, as is well known to those skilled in the art, it is possible to produce heavy or light chain sequences that are shortened, but maintain the binding properties of interest. Preferably, such shortened heavy or light chains are produced having a shorter constant region than the original heavy or light chain. Preferably, the variable region is kept constant. For example, Fab fragments or F (ab')₂And (3) fragment. Accordingly, functional equivalents of antibodies comprising at least a functional portion of the sequences shown in figure 14B are also provided. The functional portion is at least 20 amino acids in length and comprises a sequence at least 70% identical to at least one of the CDR sequences set forth in fig. 14B.

Another particularly preferred anti-RSV antibody of the invention is the antibody designated "AM 23" which has a heavy chain region and a light chain region as shown in figure 14C. The CDR sequences of AM23 that specifically produced the antigen binding properties of AM23 are also seen in fig. 14C.

Since the present invention recognizes that the CDR sequences shown in fig. 14C provide the desired RSV-binding properties, the skilled artisan is able to generate variants comprising at least one altered CDR sequence. For example, conservative amino acid substitutions are employed. Conservative amino acid substitutions include the substitution of one amino acid with another amino acid of substantially similar properties (size, hydrophobicity, etc.) such that overall function is less likely to be severely affected.

It is also possible to alter at least one of the CDR sequences depicted in fig. 14C in order to generate a variant antibody or functional equivalent thereof having an altered at least one property compared to AM 23. Preferably, an antibody or functional equivalent is provided comprising a CDR sequence which is at least 70% identical to the CDR sequence as depicted in fig. 14C, in order to at least partially retain, or even improve, the advantageous binding properties of AM 23. Preferably, the CDR sequences depicted in fig. 14C are altered such that the resulting antibody or functional equivalent comprises at least one improved property, such as improved binding affinity, selectivity and/or stability, compared to AM 23. Accordingly, variant antibodies comprising amino acid sequences at least 70% identical to the CDR sequences depicted in fig. 14C or functional equivalents thereof are within the scope of the present invention. Various methods of altering amino acid sequences are available in the art. For example, heavy or light chain sequences having the desired CDR sequences are synthesized. Preferably, the nucleic acid sequences encoding the CDRs are mutated, e.g., using random or site-directed mutagenesis.

Accordingly, the present invention provides in one aspect an isolated, synthetic or recombinant antibody or functional part, derivative and/or analogue thereof capable of specifically binding respiratory syncytial virus, which comprises:

-a heavy chain CDR1 sequence comprising a sequence at least 70% identical to the sequence GFNFHNYG, and/or

-a heavy chain CDR2 sequence comprising a sequence at least 70% identical to the sequence VWYDGSKK, and/or

-a heavy chain CDR3 sequence comprising a sequence at least 70% identical to sequence VRDKVGPTPYFDS, and/or

-a light chain CDR1 sequence comprising a sequence at least 70% identical to the sequence NIGSET, and/or

-a light chain CDR2 sequence comprising a sequence at least 70% identical to sequence DDD, and/or

A light chain CDR3 sequence comprising a sequence at least 70% identical to sequence QVWDRSNYHQV.

Preferably, an antibody or functional equivalent of the invention comprises a CDR sequence which is at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% identical to at least one CDR sequence as depicted in fig. 14C. Most preferably, an antibody or functional equivalent of the invention comprises a CDR sequence which is at least 95% identical to at least one of the CDR sequences depicted in fig. 14C. The above-described particularly preferred antibody AM23 comprises a CDR sequence consisting of the CDR sequence shown in fig. 14C. Accordingly, a particularly preferred embodiment of the invention provides an isolated, synthetic or recombinant antibody or functional equivalent thereof capable of specifically binding respiratory syncytial virus, which comprises:

-a heavy chain CDR1 sequence comprising the sequence GFNFHNYG, and/or

-a heavy chain CDR2 sequence comprising the sequence VWYDGSKK, and/or

-a heavy chain CDR3 sequence comprising the sequence VRDKVGPTPYFDS, and/or

-a light chain CDR1 sequence comprising the sequence NIGSET, and/or

-a light chain CDR2 sequence comprising the sequence DDD, and/or

A light chain CDR3 sequence comprising the sequence QVWDRSNYHQV.

In one embodiment, an antibody or functional equivalent is provided comprising three heavy chain CDR sequences and three light chain CDR sequences as depicted in fig. 14C, or sequences at least 70% identical thereto. Thus, there is further provided an isolated, synthetic or recombinant antibody or functional equivalent thereof comprising a heavy chain CDR1 sequence comprising a sequence at least 70% identical to the sequence GFNFHNYG, a heavy chain CDR2 sequence comprising a sequence at least 70% identical to the sequence VWYDGSKK, a heavy chain CDR3 sequence comprising a sequence at least 70% identical to the sequence VRDKVGPTPYFDS, a light chain CDR1 sequence comprising a sequence at least 70% identical to the sequence NIGSET, a light chain CDR2 sequence comprising a sequence at least 70% identical to the sequence DDD and a light chain CDR3 sequence comprising a sequence at least 70% identical to the sequence QVWDRSNYHQV. Preferably, the antibody or functional equivalent comprises a CDR sequence which is at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% identical to the above-described heavy chain CDR sequence and the above-described light chain CDR sequence as depicted in fig. 14C. Also provided are antibodies or functional equivalents comprising the heavy chain CDR1, CDR2, and CDR3 sequences of fig. 14C above and the light chain CDR1, CDR2, and CDR3 sequences of fig. 14C above.

Also provided is an antibody or functional equivalent thereof comprising a heavy chain amino acid sequence at least 70% identical to the heavy chain sequence set forth in figure 14C. Such heavy chain sequences provide the desired RSV-binding properties, as demonstrated by antibody AM 23. Accordingly, there is also provided an antibody or functional equivalent thereof, the heavy chain sequence of which comprises a sequence at least 70% identical to sequence EVQLVESGGNVVKPGTSLRLSCAATGFNFHNYGMNWVRQAPGKGLEWVAVVWYDGSKKYYADSVTGRFAISRDNSKNTLYLQMNSLRVEDTAVYYCVRDKVGPTPYFDSWGQGTLVTVSS. Moreover, a light chain amino acid sequence at least 70% identical to the light chain sequence set forth in fig. 14C provides the desired RSV binding characteristics, as evidenced by antibody AM 23. Accordingly, there is also provided an antibody or functional equivalent thereof having a light chain sequence at least 70% identical to sequence SYVLTQPPSVSLAPGGTAAITCGRNNIGSETVHWYQQKPGQAPVLVVYDDDDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDRSNYHQVFGGGTKLTV. Preferably, the antibody or functional portion of the invention comprises a heavy chain variable region sequence and/or a light chain variable region sequence which is at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% identical to the heavy chain sequence and/or the light chain sequence as depicted in figure 14C. The higher the homology, the closer the antibody or functional portion is to antibody AM 23. Preferably, the antibody or functional part of the invention comprises a heavy chain and a light chain similar to the heavy and light chains of AM 23. Accordingly, there is also provided an antibody or functional part comprising a heavy chain sequence and a light chain sequence which are at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% identical to the heavy chain sequence and the light chain sequence as set out in figure 14C.

One embodiment provides an antibody or functional equivalent thereof comprising a heavy chain sequence consisting of the heavy chain sequence shown in figure 14C and a light chain sequence consisting of the light chain sequence shown in figure 14C. Alternatively, as is well known to those skilled in the art, it is possible to produce heavy or light chain sequences that are shortened, but maintain the binding properties of interest. Preferably, such shortened heavy or light chains are produced having a shorter constant region than the original heavy or light chain. Preferably, the variable region is kept constant. For example, Fab fragments or F (ab')₂And (3) fragment. Therefore, tooFunctional equivalents of antibodies comprising at least a functional portion of the sequence shown in figure 14C are provided. The functional portion is at least 20 amino acids in length and comprises a sequence at least 70% identical to at least one CDR sequence as set forth in fig. 14C.

The present invention provides RSV-specific antibodies or functional equivalents thereof having improved properties compared to prior art antibodies. The present inventors succeeded in producing RSV-specific antibodies with low IC50 values. Such antibodies have a particularly high or particularly strong affinity for RSV and are therefore particularly suitable for combating and/or at least partially preventing RSV infection and/or adverse effects of RSV infection. One embodiment provides antibodies having an IC50 value of less than 10ng/ml in an in vitro neutralization assay of HEp-2 cell infected RSV, as well as functional equivalents of such antibodies. The IC50 value of the antibody or functional equivalent is preferably less than 5ng/ml, more preferably less than 2 ng/ml. In the in vitro neutralization experiments described in the examples, the IC50 value of antibody D25 is preferably about 0.5-1.5ng/ml (see FIG. 8).

The antibody of the invention is preferably a human antibody. The use of human antibodies in the treatment of humans reduces the chance of side effects due to the immune response of human individuals to non-human sequences. In another preferred embodiment, the antibody or functional part, derivative or analogue of the invention is a chimeric antibody. In this way, sequences of interest, e.g., binding sites of interest, may be included within an antibody or functional equivalent of the invention.

The invention also provides an isolated, synthetic or recombinant nucleic acid sequence encoding an antibody or functional equivalent of the invention or a functional part, derivative or analogue thereof. For example, such nucleic acids are isolated from B cells capable of producing the antibodies of the invention, as described in more detail below. Preferred embodiments provide a nucleic acid sequence comprising a sequence at least 70% homologous to a functional portion of the nucleic acid sequence depicted in figure 11, figure 12, figure 14A, figure 14B and/or figure 14B. The nucleic acid sequence preferably comprises a sequence which is at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% homologous to a functional part of the nucleic acid sequence as depicted in figure 11, figure 12, figure 14A, figure 14B and/or figure 14B. The functional moiety is at least 30 nucleotides, preferably at least 50 nucleotides, more preferably at least 75 nucleotides in length. Preferably, the functional portion encodes at least one nucleic acid sequence as shown in figure 11D, figure 12, figure 14A, figure 14B and/or figure 14B. The sequence is preferably a CDR sequence.

The antibodies or functional equivalents of the invention are particularly suitable for use as medicaments or prophylactic agents. Also provided herein are antibodies of the invention, or functional portions, derivatives or analogs thereof, for use as a medicament and/or prophylactic. In a particularly preferred embodiment, the antibody comprises antibody D25, AM14, AM16 and/or AM23, or a functional part, derivative or analogue thereof. The pharmaceutical or prophylactic agent is preferably used to combat or at least partially prevent RSV infection, or to combat or at least partially prevent the adverse effects of RSV infection. Thus, there is also provided the use of an antibody, functional part, derivative or analogue of the invention for the manufacture of a medicament and/or prophylactic agent for the at least partial treatment and/or prevention of an RSV-associated disease, as well as a method for the at least partial treatment and/or prevention of an RSV-associated disease, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or functional equivalent of the invention. The antibody preferably comprises antibody D25, AM14, AM16 and/or AM23, or a functional part, derivative or analogue thereof.

For protection against RSV, the antibody or functional equivalent of the invention is preferably administered to an individual prior to the occurrence of RSV infection. Alternatively, an antibody or functional equivalent of the invention is administered when an individual has been infected with RSV. The antibody or functional equivalent is preferably administered to individuals at increased risk of RSV-related diseases, such as preterm infants, individuals with chronic lung disease, congenital heart disease and/or immune insufficiency, and children under 6 weeks of age. The risk of RSV-related disease in the elderly is also increased. The antibodies or functional equivalents of the invention are preferably administered orally or by one or more injections. The dosage range of the antibodies and/or functional equivalents of the invention for the therapeutic applications described herein is designed according to clinical dose escalation studies in clinical trials requiring rigorous protocols. Typical doses are from 0.1 to 10mg/kg body weight. In therapeutic applications, the antibodies or functional equivalents of the invention are generally used in combination with pharmaceutically acceptable carriers, adjuvants, diluents and/or excipients. Examples of suitable carriers include, for example, Keyhole Limpet Hemocyanin (KLH), serum albumin (e.g., BSA or RSA), and ovalbumin. Those skilled in the art are aware of many suitable oil-based and water-based adjuvants. In one embodiment, the excipient comprises Specol. In another embodiment, the suitable carrier comprises a solution, such as saline.

In another embodiment, a nucleic acid encoding an antibody or functional portion of the invention is used. After administration of such nucleic acids, the antibodies or functional equivalents are produced by the cellular machinery of the host. The antibodies or functional equivalents produced are capable of preventing and/or combating RSV infection and/or adverse effects of RSV infection. Accordingly, also provided herein are nucleic acid sequences, functional moieties, derivatives and/or analogues of the invention for use as a medicament and/or prophylactic agent. The nucleic acids are preferably used against RSV. Accordingly, there is also provided the use of a nucleic acid sequence, functional part, derivative and/or analogue of the invention in the manufacture of a medicament and/or prophylactic agent for the at least partial treatment and/or prevention of an RSV-associated disease.

At least a functional part of a nucleic acid of the invention refers to a part of said nucleic acid, having a length of at least 30 base pairs, preferably at least 50 base pairs, more preferably at least 100 base pairs, which comprises at least one expression feature (of the same kind, but not necessarily the same amount) identical to a nucleic acid of the invention. The functional portion encodes at least an amino acid sequence comprising a sequence at least 70% identical to a CDR sequence as depicted in fig. 11D, fig. 14A, fig. 14B and/or fig. 14C.

The invention also provides a cell producing an isolated antibody, which is capable of producing an antibody, functional portion, derivative or analogue of the invention. Possible (but non-limiting) ways to obtain such antibody-producing cells are described in detail in the examples. The present inventors have developed and used novel methods to improve the stability of cells producing RSV-specific antibodies. RSV-specific antibody producing cells produced using this method are stable for at least 6 months. Accordingly, the present invention also provides an RSV-specific antibody producing cell of the invention which is stable for at least 9 weeks, preferably at least 3 months, more preferably at least 6 months.

The present inventors have recognized that stability of an RSV-specific antibody-producing cell can be achieved by affecting the amount of BCL6 and/or Blimp-1 expression product in the antibody-producing cell. The amount of BCL6 and/or Blimp-1 expression product is affected directly or indirectly. Preferably, the amount of both BCL6 and Blimp-1 expression products in the antibody-producing cell is modulated, as both expression products are involved in the stability of the antibody-producing cell. Stability of an antibody-producing cell is defined as the ability of the antibody-producing cell to remain at a certain developmental stage (preferably after the cell has entered that stage). The different developmental stages of the cells comprise at least one different characteristic of said cells. For example, it is known that memory B cells differentiate into antibody-secreting plasma cells after stimulation through a stage called plasmablasts by some researchers. Memory B cells, plasmablasts, and plasma cells are different stages of development of B cells in which B cells have different characteristics. Memory B cells have low proliferation and low antibody secretion properties. Plasmablasts have higher proliferation levels and higher antibody secretion levels than memory B cells, while plasma cells secrete high antibody levels but are unable to proliferate. It is possible to modulate the replicative life span of antibody-producing cells using the methods described by the present inventors. Herein, the replicative life span of an antibody-producing cell is defined as the time during which the B cell and its progeny are able to replicate and maintain their ability to produce and/or develop into an antibody-producing cell. It is preferred to extend the replicative life span of antibody-producing cells, meaning that the antibody-producing cells will not undergo terminal differentiation or will undergo terminal differentiation only after a longer period of time than currently used antibody-producing cells of the same species and continue to proliferate in vitro. According to the present invention, it is possible to modulate the amount of BCL6 and/or Blimp-1 expression product in an antibody-producing cell such that the antibody-producing cell enters and/or remains in a predetermined developmental state in which the cell continues to proliferate. Thus, using the methods of the invention it is possible to extend the replicative life span of antibody producing cells, as it is possible to maintain B cells at a certain developmental stage where replication occurs. See PCT/NL2006/000625 filed by the present applicant. The present invention provides means and methods for generating stable RSV-specific antibody producing cells.

An antibody-producing cell is defined as a cell capable of producing and/or secreting an antibody or functional equivalent thereof, and/or a cell capable of developing into a cell capable of producing and/or secreting an antibody or functional equivalent thereof. Herein, an RSV-specific antibody producing cell is defined as a cell capable of producing and/or secreting an antibody or a functional equivalent thereof capable of specifically binding to RSV and/or an RSV component, such as an epitope of the RSV F (fusion) protein, the RSV G (attachment) protein or the RSV SH (small hydrophobic) protein. Preferably, the RSV-specific antibody producing cells comprise B cells and/or B cell-derived plasma cells. B cells are referred to herein as antibody-producing cells, even when the B cells are in a stage where antibody production is low or no antibody production at all, such as activated or unactivated naive B cells or memory B cells, as such cells are capable of developing into antibody-producing cells, such as plasmablasts and/or plasma cells.

The RSV-specific antibody producing cell of the invention preferably comprises a mammalian cell. Non-limiting examples include antibody producing cells derived from human individuals, rodents, rabbits, camels, pigs, cows, goats, horses, apes, gorillas. The antibody-producing cells preferably comprise human cells, murine cells, rabbit cells and/or camel cells.

BCL6 encodes a transcriptional repressor required for the development and maturation of normal B and T cells and for the formation of germinal centers. (Ye, 1997). BCL6 was highly expressed in germinal center B cells, and hardly expressed in plasma cells. BCL6 inhibits the differentiation of activated B cells into plasma cells. Transcription repressor B lymphocyte-induced mature protein-1 (Blimp-1) is a factor essential for B cell development into plasma cells. A human variant of Blimp-1 is designated Prdm 1. As used herein, any reference to Blimp-1 includes Prdm 1. Blimp-1 drives plasma cell differentiation. BCL6 and Blimp-1 mutually repressed expression; thus in natural conditions, one of them is forced to a certain differentiation state when it reaches a higher expression level than the other. In humans, differentiation into plasma cells from activated naive B cells or memory B cells involves down-regulation of BCL6 and up-regulation of Blimp-1. At germinal center, the cell BCL6 expression level is high, and the Blimp-1 expression level is low. In resting memory cells, BCL6 and Blimp-1 were expressed at a lower level on average. The signal triggering differentiation causes Blimp-1 to be upregulated, and this Blimp-1 antagonizes the expression of BCL 6. Both BCL6 and Blimp-1 were expressed in a short period of time, called plasmablasts. With increasing levels of Blimp-1, BCL6 expression disappeared, resulting in the formation of plasma cells.

In one embodiment of the invention, an RSV-specific antibody-producing cell is provided in which BCL6 and Blimp-1 are co-expressed (meaning that both BCL6 and Blimp-1 are expressed in said antibody-producing cell for at least 1 day, preferably at least 1 week, more preferably at least 6 weeks, and most preferably at least 3 months). The RSV-specific antibody producing cell is capable of proliferating when provided with an appropriate signal. It has been found that co-expression of BCL6 and Blimp-1 results in antibody-producing cells that are capable of proliferation and antibody production. BCL6 and Blimp-1 are preferably co-expressed in B cells, preferably human B cells. Co-expression of BCL6 and Blimp-1 in B cells resulted in the B cells being stable in the plasmablast stage. Plasmablasts, like plasma cells, are capable of secreting antibodies. However, plasmablasts are still able to proliferate, while plasma cells have lost their proliferative capacity. Therefore, plasma cells are not suitable for culturing antibody-producing cell lines.

One preferred embodiment provides an RSV-specific antibody producing cell comprising an exogenous nucleic acid sequence encoding BCL6 or a functional part, derivative and/or analogue thereof. An exogenous nucleic acid is defined herein as a nucleic acid sequence that does not naturally belong to the genome of a cell. Using such exogenous nucleic acid molecules, it is possible to modulate BCL6 concentrations in antibody-producing cells without relying on endogenous BCL6 expression. Thus, even if endogenous BCL6 is expressed at a low level or is not expressed (e.g., by Blimp-1), the exogenous nucleic acid sequence encoding BCL6, or functional portions, derivatives, and/or analogs thereof, is still capable of producing BCL6 at a concentration sufficient to affect the stability of the antibody-producing cell. The nucleic acid sequence encoding BCL6 or a functional part, derivative and/or analogue thereof is preferably constitutively active, such that BCL6 expression is maintained even when endogenous BCL6 expression of said cell is inhibited by an endogenous repressor, such as Blimp-1. Most preferably, the expression of said nucleic acid sequence encoding BCL6 or a functional part, derivative and/or analogue thereof is regulated by an exogenous inducer of a repressor, such that the extent of BCL6 expression can be arbitrarily regulated.

Preferably, the RSV-specific antibody producing cells of the invention comprise an exogenous nucleic acid sequence encoding Bcl-xL or a functional portion, derivative and/or analog thereof, as described in detail below. If Bcl-xL or a functional part, derivative and/or analogue thereof is present, it is possible to culture plasmablasts under conditions of low cell density. Preferably, the expression of said nucleic acid sequence encoding Bcl-xL or a functional part, derivative and/or analogue thereof is regulated by an exogenous inducer of the repressor, such that the extent of Bcl-xL expression can be arbitrarily regulated. Accordingly, a preferred embodiment provides an RSV-specific antibody producing cell comprising:

-an exogenous nucleic acid sequence encoding BCL6 or a functional part, derivative and/or analogue thereof, and/or

-an exogenous nucleic acid sequence encoding Bcl-xL or a functional part, derivative and/or analogue thereof.

The RSV-specific antibody-producing cell preferably comprises both an exogenous nucleic acid sequence encoding BCL6 or functional portions, derivatives, and/or analogs thereof and an exogenous nucleic acid sequence encoding BCL-xL or functional portions, derivatives, and/or analogs thereof. Preferably, the expression of said nucleic acid sequence encoding BCL6, Bcl-xL or a functional part, derivative and/or analogue of BCL6 or Bcl-xL is regulated by an activator and/or repressor induced by an exogenous compound. For example, inducible promoter systems, such as the Tet-on or Tet-off systems, are used.

The stable RSV-specific antibody producing cells of the present invention are preferably produced by co-expressing BCL6 and Blimp-1 in RSV-specific antibody producing cells. The RSV-specific antibody-producing cell is preferably obtained from an individual who has been exposed to RSV. Methods of isolating antibody-producing cells are known in the art. For example, an RSV-derived compound labeled with a label or tag is incubated with a sample of an individual exposed to RSV, the sample comprising antibody producing cells. RSV-specific antibody producing cells recognizing the labeled RSV-derived compound are isolated while unbound cells are washed away. Subsequently, the resulting RSV-specific antibody-producing cells were stabilized by co-expression of BCL6 and Blimp-1.

One embodiment involves first stabilizing all antibody producing cells from an RSV contact donor and then isolating cells that recognize the labeled RSV-derived compound. In another embodiment, the antibody-producing cells have a (fluorescent) label downstream of their B-cell receptor (BCR, membrane-expressed form of the antibody) that signals when the antibody-producing cells bind unlabeled/unlabeled antigen via the BCR. Antibody-producing cells with this marker inverted were selected and subsequently stabilized by co-expression of BCL6 and Blimp-1. In another embodiment, where no antigen-derived compounds are available, but where experiments are available to screen for unique antibodies, all/bulk antibody producing cells are stabilized by co-expressing BCL6 and Blimp-1, optionally also expressing Bcl-XL. According to this embodiment, the cells are cultured at low density in the presence of L cells, preferably 10 to 100 cells per well in a 96-well plate (small batch culture, MBC). The culture supernatant can be used directly in screening assays, e.g., ELISA, Western blot or functional assays such as ELISPOT, neutralization assays or cell migration assays.

In one embodiment, MBC is selected and the cultures are subjected to limiting dilution, preferably after 2-3 weeks, in order to obtain a monoclonal cell line of antibody-producing cells of interest, and the supernatants of these cultures are screened again with the preferred assay.

As is well known to those skilled in the art, many alternatives are available in the art. The above embodiments are not limitative.

Accordingly, there is also provided a method of producing an antibody producing cell which is stable for at least three months and is capable of producing an RSV-specific antibody or functional equivalent thereof, the method comprising:

-increasing the level of Blimp-1 expression in a cell capable of producing an RSV-specific antibody or functional equivalent thereof; and

-increasing and/or maintaining BCL6 expression levels in said cell.

With the method of the invention it is possible to convert RSV-specific memory B cells into plasmablast-like cells and to stabilize said cells, and thus not to differentiate rapidly into plasma cells. This is in contrast to the natural development of plasma cells, where expression of Blimp-1 in memory B cells results in rapid development into plasma cells, thereby inhibiting BCL6 expression, and the resulting plasma cells therefore hardly express BCL 6. Thus, one embodiment of the invention includes co-expression of BCL6 and Blimp-1 in RSV-specific B cells, resulting in cells capable of proliferation and antibody production. Preferably, the level of BCL6 expression in the RSV-specific B cells is adjusted to or maintained at the same level as or higher than that of plasmablasts. In this manner, stable cultures of RSV-specific B cells are produced that retain the ability to produce RSV-specific antibodies. These RSV-specific B cells co-expressing BCL6 and Blimp-1 are further stabilized, preferably by the addition of an anti-apoptotic gene, Bcl-xL. By introducing Bcl-xL it is now possible to culture plasmablasts under low cell density conditions. Accordingly, the present invention also provides a method of culturing plasmablasts under conditions of low cell density, the method comprising producing RSV-specific antibody-producing cells having levels of BCL6, Blimp-1, and BCL-xL expression using any of the methods described herein.

The level of BCL6 expression product (preferably BCL6 protein) in RSV-specific antibody producing cells is regulated in various ways.

In one embodiment, the antibody-producing cell is provided with a compound capable of directly or indirectly affecting expression of BCL 6. Preferably, antibody producing cells are provided with compounds capable of enhancing expression of BCL6 to counteract down-regulation of BCL6 during Blimp-1 expression. Such compounds preferably comprise signal transducer and activator of transcription 5(STAT5) or a functional part, derivative and/or analogue thereof, and/or nucleic acid sequences encoding the same. STAT5 is a signaling protein capable of enhancing BCL6 expression. STAT5 has two known forms, STAT5a and STAT5b, which are encoded by two different tandem genes. Administration and/or activation of STAT5 resulted in increased levels of BCL 6. Thus, the upregulation of BCL6 expression by STAT5 or a functional portion, derivative and/or analog thereof at least partially compensates for the downregulation of BCL6 by Blimp-1. Thus, STAT5 or a functional part, derivative and/or analogue thereof is capable of directly affecting BCL6 expression. BCL6 expression may also be indirectly affected. This may be achieved, for example, by modulating the amount of compound capable of activating STAT5 directly or indirectly and/or modulating STAT5 expression. Thus, in one embodiment, the expression and/or activity of endogenous and/or exogenous STAT5 is increased. For example, BCL6 expression may be indirectly enhanced by culturing antibody-producing cells in the presence of Interleukin (IL)2 and/or IL4 capable of activating STAT 5.

In one embodiment, the RSV-specific antibody producing cell is provided with a nucleic acid sequence encoding STAT5 or a functional part, derivative and/or analogue thereof, wherein said nucleic acid sequence is constitutively active, meaning that STAT5 is expressed persistently independent of the presence of a (endogenous) regulator. In the case of low or no expression of endogenous STAT5, preferably application of an exogenous constitutively active nucleic acid sequence encoding STAT5 or a functional part, derivative and/or analogue thereof results in a concentration of STAT5 or a functional part, derivative and/or analogue thereof sufficient to increase BCL6 expression. Most preferably, the RSV-specific antibody producing cell is provided with a nucleic acid sequence encoding a compound, preferably a fusion protein, comprising STAT5 or a functional part, derivative and/or analogue thereof, the activity of which is regulated by an exogenous inducer of the repressor and thus the degree of activation of BCL6 expression is optionally regulated. Another Tet-on system for induction of BCL-6 is provided, in which tetracycline and/or tetracycline derivatives are added to induce the activity of transactivators which induce transcription of the BCL6 gene, followed by BCL protein synthesis. In a preferred embodiment, the antibody-producing cell is provided with a nucleic acid sequence encoding the fusion protein ER-STAT5 of the Estrogen Receptor (ER) and STAT 5. Such a fusion protein is inactive because it forms a complex with a heat shock protein in the cytoplasm. STAT5 could not reach the nucleus in this way and could not increase BCL6 expression. Following administration of the exogenous inducer 4 hydroxy-tamoxifen (4HT), the fusion protein ER-STAT5 dissociates from heat shock proteins, thus STAT5 is able to enter the nucleus and activate BCL6 expression.

Additionally or alternatively, the antibody-producing cells are cultured in the presence of a compound capable of directly or indirectly enhancing expression of BCL6, thereby increasing expression of BCL6 in RSV-specific antibody-producing cells.

Accordingly, one embodiment provides a method of producing an RSV-specific antibody producing cell, the method comprising:

-providing an RSV-specific antibody producing cell with a compound capable of directly or indirectly enhancing BCL6 expression; and/or

-culturing an RSV-specific antibody producing cell in the presence of a compound capable of directly or indirectly enhancing BCL6 expression.

Said compound capable of directly or indirectly enhancing BCL6 expression preferably comprises STAT5 or a functional part, derivative and/or analogue thereof. Accordingly, the present invention provides a method comprising providing STAT5 or a functional part, derivative and/or analogue thereof, or a nucleic acid sequence encoding STAT5 or a functional part, derivative and/or analogue thereof, to said RSV-specific antibody producing cell. In one embodiment, the antibody-producing cell is cultured after introducing into the cell a nucleic acid sequence encoding STAT5 or a functional portion, derivative and/or analog thereof. For example, the nucleic acid sequence is introduced into the cell by transfection and/or virus-mediated gene transfer. Many alternative methods of introducing nucleic acid sequences into cells are available in the art and need not be further explained here.

It is possible to increase the expression of endogenous BCL6 using compounds capable of directly or indirectly enhancing BCL6 expression. However, in a preferred embodiment, the antibody-producing cell is provided with a nucleic acid sequence encoding BCL6 or a functional part, derivative and/or analogue thereof. As previously mentioned, exogenous nucleic acids encoding BCL6 are preferred because this enables the intracellular BCL6 concentration to be regulated without relying on endogenous BCL6 expression. Thus, even if endogenous BCL6 is expressed at a low level or is not expressed (e.g., by Blimp-1), the exogenous nucleic acid sequence encoding BCL6, or functional portions, derivatives, and/or analogs thereof, is still capable of producing BCL6 at a concentration sufficient to affect the stability of the antibody-producing cell. Accordingly, the present invention also provides a method comprising providing to an RSV-specific antibody producing cell a nucleic acid sequence encoding BCL6 or a functional part, derivative and/or analogue thereof. Preferably, the antibody-producing cell is provided with a constitutively active nucleic acid sequence encoding BCL6 or a functional part, derivative and/or analogue thereof, whereby BCL6 expression is maintained when endogenous BCL6 expression of said cell is inhibited by an endogenous repressor, such as Blimp-1. Most preferably, the expression of said nucleic acid sequence encoding BCL6 or a functional part, derivative and/or analogue thereof is regulated by an exogenous inducer of the repressor, thereby optionally regulating the degree of expression of BCL 6. For example, an inducible promoter system such as the Tet-on or Tet-off system is used, as is well known.

In another preferred embodiment, the present invention provides a method wherein BCL6 levels are indirectly modulated by providing an RSV-specific antibody producing cell with a nucleic acid sequence encoding E47 or a functional part, derivative and/or analogue thereof. The transcription factor encoded by E47 belongs to the helix-loop-helix protein family, i.e. the E-protein family. There are four E-proteins, E12, E47, E2-2 and HEB, involved in lymphocyte development. E12 and E47 are encoded by one gene, E2A, but the splicing is different. The E-protein can be inhibited by the E protein inhibitors Id2 and Id3, as well as ABF-1 (Mathas s., 2006). The E protein is described as a tumor suppressor, whose overexpression induces apoptosis. One of the specific targets of E47 is the Socs1 and Socs3 genes. These Socs genes are called negative regulators of STAT5b and are thus indirect regulators of BCL 6. In other words, expression of E47 in B cells enhanced Blimp-1 expression, which resulted in differentiation of B cells towards an antibody-producing phenotype (plasma cells).

The expression level of Blimp-1 in RSV-specific antibody producing cells is also regulated in various ways. In one embodiment, an RSV-specific antibody producing cell is provided with a compound capable of directly or indirectly affecting Blimp-1 expression. Additionally or alternatively, the antibody-producing cell is cultured in the presence of a compound capable of directly or indirectly affecting expression of Blimp-1. Accordingly, the invention also provides a method comprising providing to an RSV-specific antibody producing cell a compound capable of directly or indirectly affecting Blimp-1 expression. The invention also provides a method comprising culturing the antibody-producing cell in the presence of a compound capable of directly or indirectly affecting Blimp-1 expression. Preferably, compounds capable of enhancing expression of Blimp-1 are used to counteract the down-regulation of Blimp-1 during expression of BCL 6. Most preferably, the compound comprises IL-21.

In a preferred embodiment, the compound capable of directly or indirectly affecting Blimp-1 expression comprises a signal transducer and activator of transcription 3(STAT3) protein or a functional part, derivative and/or analogue thereof, and/or a nucleic acid sequence encoding the same. STAT3 is a signaling protein involved in B cell development and differentiation. STAT3 was able to upregulate Blimp-1 expression. Thus, the present invention also provides a method wherein said compound capable of directly or indirectly affecting Blimp-1 expression comprises STAT3 or a functional part, derivative and/or analogue thereof, or a nucleic acid sequence encoding STAT3 or a functional part, derivative and/or analogue thereof. Most preferably, the expression of said nucleic acid sequence encoding STAT3 or a functional part, derivative and/or analogue thereof is regulated by an exogenous inducer of the repressor, thereby optionally regulating the extent of expression of STAT 3. For example, inducible promoter systems, such as the Tet-on or Tet-off systems, are used. In one embodiment, a fusion product comprising STAT3, a derivative or analog, and ER is introduced into the cell so as to modulate STAT3 expression by hydroxytamoxifen.

Since STAT3 is capable of affecting Blimp-1 expression, it is also possible to indirectly modulate Blimp-1 expression by administering compounds capable of directly or indirectly modulating the activity and/or expression of STAT 3. In one embodiment, antibody producing cells are provided with a compound that enhances STAT3 activity so as to indirectly increase the expression of Blimp-1. Accordingly, the present invention also provides a method wherein a compound capable of directly or indirectly enhancing STAT3 activity is provided to an antibody-producing cell.

Thus, in one embodiment, antibody producing cells are provided with a compound capable of directly or indirectly activating STAT3 so as to increase Blimp-1 expression.

STAT3 is activated in various ways. Preferably, STAT3 is activated by providing a cytokine to the antibody-producing cell. Cytokines that are naturally involved in B cell differentiation are very effective in modulating STAT proteins. Very potent activators of STAT3 are IL-21 and IL-6, and IL-2, IL-7, IL-10, IL-15, and IL-27 are also known to activate STAT 3. Furthermore, Toll-like receptors (TLRs) involved in innate immunity are also capable of activating STAT 3. Accordingly, one embodiment of the present invention provides a method wherein said compound capable of directly or indirectly affecting the expression of Blimp-1 comprises IL-21, IL-2, IL-6, IL-7, IL-10, IL-15 and/or IL-27. IL-21 is most preferably used, since IL-21 is particularly suitable for influencing the stability of antibody-producing cells. IL-21 was able to up-regulate Blimp-1 expression even when BCL6 was directed against Blimp-1 expression.

Additionally or alternatively, a mutant janus kinase (JAK) was used to activate STAT 3. Under natural conditions, JAKs themselves, activated by at least one cytokine, are able to phosphorylate STAT 3. A mutated janus kinase capable of activating STAT3 independent of the presence of cytokines is particularly suitable for use in the methods of the invention.

As previously mentioned, a compound capable of enhancing Blimp-1 expression in one embodiment includes a nucleic acid sequence encoding STAT3 or a functional portion, derivative, and/or analog thereof. The presence of an exogenous nucleic acid sequence encoding STAT3 or a functional part, derivative and/or analogue thereof allows the persistence of STAT3 or a functional part, derivative and/or analogue thereof even when the expression of endogenous STAT3 is very low or not expressed.

It is also possible to decrease expression and/or activity of STAT5, up-regulating Blimp-1. If the level and/or activity of STAT5 is reduced, activation of BCL6 expression is also reduced, which results in a reduction in the level of BCL6 expression products. Since BCL6 and Blimp-1 were expressed against each other, a decrease in the amount of BCL6 expression product resulted in an increase in the amount of Blimp-1 expression product. Thus, compounds that down-regulate STAT5 activity are capable of indirectly up-regulating Blimp-1. For example, such compounds include cytokine signaling inhibitory protein (SOCS). Thus, in one embodiment, the level of Blimp-1 expression product in an RSV-specific antibody producing cell is upregulated by providing the cell with, and/or activating, a SOCS protein in the cell.

In a preferred embodiment, STAT5 expression and/or activity is decreased when a nucleic acid sequence encoding E47 or a functional part, derivative and/or analogue thereof is provided to an RSV-specific antibody producing cell. Expression of E47 in B cells expressing STAT5B at high levels interferes with differentiation and proliferation, i.e., blockade of STAT5 by E47 and SOCS results in decreased levels of BCL6, followed by increased levels of Blimp-1. Upregulation of the level of Blimp-1 results in a decrease in proliferation levels and in differentiation of the cells involved into antibody producing cells. In other words, expression of E47 in B cells enhances Blimp-1 expression, which results in differentiation of B cells towards an antibody-producing phenotype (plasma cells).

At least the functional portion of STAT5 protein, STAT3 protein, Bcl-xL and/or Bcl6 refers to a proteinaceous molecule that has the same ability to affect the stability of an antibody-producing cell-the same kind, not necessarily the same amount-as STAT5 protein, STAT3 protein, Bcl-xL and/or Bcl 6. For example, a functional portion of the STAT5 protein or STAT3 protein contains no or few amino acids involved in the ability. Derivatives of STAT5 protein, STAT3 protein, Bcl-xL, and/or Bcl6 are defined as proteins that have been altered in essentially the same kind of ability to affect the stability of antibody-producing cells, but not necessarily in the same amount. Derivatives are provided in a number of ways, for example by conservative amino acid substitutions, where one amino acid is substituted by another amino acid of substantially similar nature (size, hydrophobicity, etc.), such that the overall function is less likely to be severely affected. For example, derivatives include fusion proteins such as STAT5-ER or STAT3-ER fusion proteins, the activity of which is dependent on the presence of 4 hydroxy-tamoxifen (4 HT). Analogs of STAT5 protein, STAT3 protein, Bcl-xL, and/or Bcl6 are defined as molecules that are of the same kind and not necessarily the same amount in their ability to affect the stability of an antibody-producing cell. The analogs need not be derived from the STAT5 protein, STAT3 protein, Bcl-xL, and/or Bcl 6.

In a preferred embodiment, the RSV-specific antibody producing cell is cultured in the presence of IL-21 prior to providing the nucleic acid sequence encoding BCL6 or a functional portion, derivative and/or analog thereof to the antibody producing cell. Preferably, the RSV-specific antibody producing cell, preferably a B cell, is cultured in the presence of IL-21 prior to providing said cell with a nucleic acid sequence encoding BCL6 or a functional part, derivative and/or analogue thereof, since in these embodiments stability, proliferation and/or antibody production is particularly improved.

In a preferred embodiment, the present invention provides a method of affecting the stability of an RSV-specific antibody producing cell described herein, further comprising directly or indirectly increasing the level of a Bcl-xL expression product in said antibody producing cell. This may be accomplished, for example, by providing the antibody-producing cell with a nucleic acid sequence encoding Bcl-xL or functional portions, derivatives and/or analogs thereof, or a nucleic acid sequence encoding other anti-apoptotic genes, including but not limited to Bcl-2. In another embodiment, this is achieved by providing said antibody producing cells with a compound capable of directly or indirectly increasing expression of Bcl-xL, said compound comprising APRIL, BAFF, CD40, BCR stimulation, cytokines, growth factors or downstream effector-like JNK and akt (pkb).

Bcl-xL is an anti-apoptotic Bcl-2 family member, and Bcl2 protein interacts or opposes so-called family members containing only Bcl-2 homeodomain 3(BH3), such as Bax, Bak, Bim, and Bad, and induces cytochrome c release upon receiving intrinsic death stimuli (Boise, l.h., 1993). Therefore, protection of mitochondrial membrane integrity by proteins such as Bcl-xL is critical for cell survival.

STAT5 activation has been shown to protect cells from cell death. STAT5 was able to modulate Bcl-xL expression, supporting the anti-apoptotic effect of STAT 5. STAT5 upregulates Bcl-xL expression via STAT binding elements within the Bcl-xL promoter. In vivo, there was no Bcl-xL expression in the bone marrow of STAT 5A/B-double deficient mice. Moreover, STAT 5-mediated erythroblast survival is dependent on the upregulation of Bcl-xL. In recent years, transgenic overexpression of Bcl-xL in mouse B cells has been demonstrated to promote B cell survival and non-malignant plasma cell foci.

The methods of the invention are particularly suited for producing cell cultures comprising RSV-specific antibody-producing cells capable of proliferating and secreting antibody. In one embodiment, RSV-specific memory B cells are used to generate ex vivo B cell cultures. The memory B cells are preferably human cells, such that human antibodies are produced. Preferably, the B cells are derived from an individual who has previously been exposed to respiratory syncytial virus. In one embodiment, RSV-specific B cells are isolated from peripheral blood samples and/or tonsil samples using methods known in the art. For example, memory B cells are isolated by selecting (magnetic bead sorting) the B cell markers CD19 and/or CD22 and (subsequently) cell surface IgG and/or CD27 and/or negative selection IgM, IgD and/or IgA. In germinal center B cells, BCL6 expression was high, while Blimp-1 expression was low. The natural development of antibody-secreting cells involves upregulation of Blimp-1 expression. Since Blimp-1 inhibits BCL6 expression, Blimp-1 up-regulation naturally leads to BCL6 down-regulation. In a preferred embodiment of the invention, Blimp-1 expression is upregulated, while BCL6 expression is at least partially maintained. This resulted in the generation of RSV-specific antibody producing cells co-expressing BCL6 and Blimp-1. The RSV-specific antibody-producing cells are capable of proliferating and secreting anti-RSV antibodies and are therefore suitable for ex vivo B cell culture. In another preferred embodiment, the antibody-producing cells are protected from apoptosis by Bcl-xL. The RSV-specific antibody-producing cells of the present invention offer the advantage of being stable for a long period of time and not undergoing terminal differentiation. The antibody-producing cells of the present invention are stable for at least one week, preferably at least one month, more preferably at least three months, and most preferably at least six months. It is preferred to culture the B cells of the invention in the presence of CD40L, since CD40L favors the proliferation of the majority of B cells. In one embodiment, BCL6 expression is maintained at substantially the same or higher level compared to germinal center B cells, as significant BCL6 expression, as well as Blimp-1 expression, results in antibody producing cells with preferred proliferation and antibody production characteristics and/or stability. In preferred embodiments, said expression of BCL6 and/or expression of Blimp-1 is concomitant with expression of Bcl-xL, resulting in more preferred proliferation and antibody production characteristics and/or stability.

Accordingly, one embodiment provides a method of producing an RSV-specific antibody producing cell which is stable for at least one week, preferably at least one month, more preferably at least three months, more preferably at least six months, comprising:

-providing RSV-specific memory B cells;

-increasing the level of expression of Blimp-1 in said cell; and

-increasing and/or maintaining BCL6 expression levels in said cell.

Also provided is an ex vivo method of producing an RSV-specific antibody producing cell, comprising increasing the expression level of Blimp-1 in an RSV-specific memory B cell, and increasing and/or maintaining the expression level of BCL6 in said cell. Preferably, the levels of BCL6 and Blimp-1 expression are regulated to and/or maintained at substantially the same or higher levels as compared to plasmablasts. In a preferred embodiment, the B cells are transduced with BCL6 and Bcl-xL. Accordingly, there is also provided a method of producing an RSV-specific antibody producing cell that is stable for at least three months, the method comprising:

-providing BCL6 or a functional part, derivative and/or analogue thereof to a B-cell capable of producing an RSV-specific antibody.

-providing Bcl-xL or a functional part, derivative and/or analogue thereof to said B-cell; and

-culturing said B-cells.

Preferably, the B-cells are provided with a nucleic acid sequence encoding BCL6 or a functional part, derivative and/or analogue thereof, and a nucleic acid sequence encoding BCL-xL or a functional part, derivative and/or analogue thereof.

Preferably, said B cells are cultured in the presence of a compound capable of increasing expression of Blimp-1, such as IL-21, IL-2, IL-6, Il-7, IL-10, IL-15, IL-27 or a mutated Janus kinase. IL-21 is preferred because this cytokine is particularly suitable for enhancing Blimp-1 expression and stabilizing antibody producing cells using the methods of the invention. Furthermore, in order to increase the transduction efficiency, it is preferred to culture the B-cells in the presence of IL-21 prior to transducing the B-cells with a nucleic acid sequence encoding BCL6 and/or BCL-xL, or a functional part, derivative and/or analogue thereof.

In one embodiment, the B cell is provided with a SOCS protein or a functional part, derivative and/or analogue thereof, or a nucleic acid encoding same, as the SOCS protein or a functional part, derivative and/or analogue thereof is capable of indirectly enhancing Blimp-1 expression. In another alternative or additional embodiment, the B cell is provided with E47 or a functional part, derivative and/or analogue thereof, or a nucleic acid encoding same. As previously described, the Socs protein function is enhanced and Blimp-1 expression is indirectly increased as a result of the increased levels of E47 or a functional portion, derivative and/or analog thereof.

Particularly preferred embodiments are described in the examples. According to a particularly preferred embodiment, RSV-specific B-cells are first cultured in the presence of IL-21. Next, the B cells are transduced with a nucleic acid encoding BCL6 and a nucleic acid encoding Bcl-xL. Preferably, centrifugation is used for transduction (spin transduction). Most preferably, the B cells comprising at least one nucleic acid of interest and the virus are mixed and the mixture is then centrifuged to achieve high transduction efficiency. Following transduction, B cells were cultured for 3-5 days in the absence of IL-21 and in the presence of IL-4 and L-cells to express BCL 6. Next, according to this preferred embodiment, the B cells are again transduced with a nucleic acid encoding BCL6 and a nucleic acid encoding Bcl-xL. Then, B cells were again cultured for 3-5 days in the absence of IL-21 and in the presence of IL-4 and L-cells to express BCL 6. Next, cells expressing BCL6 and Bcl-xL were isolated and IL-21 was again administered to the culture to enhance proliferation and antibody production. Preferably, culture supernatants are screened for neutralizing capacity/activity/reactivity to RSV in vitro of antibodies secreted by Bcl-6, Blimp1, and Bcl-XL expressing cells. Antibody-producing cells producing these antibodies are preferably further selected by, for example, limiting dilution culture. Thus, stable RSV-specific B cells expressing both BCL6 and Blimp-1 were obtained. Under in vitro culture conditions, the B cells are capable of proliferating and producing antibodies for a period of at least six months.

One embodiment provides a method of the invention further comprising selecting and/or isolating an RSV-specific antibody or functional equivalent thereof. In one embodiment, IgM producing cells and IgG producing cells are selected and isolated. Preferably, IgG-producing cells are selected and/or isolated.

The RSV-specific antibody-producing cells produced by the method of the present invention are suitable for producing antibodies against RSV. However, in a preferred embodiment, the genes encoding the Ig heavy and/or light chains are isolated from the cell and expressed in a second cell, such as a Chinese Hamster Ovary (CHO) cell line or 293(T) cell. The second cell, also referred to herein as a producer cell, is preferably suitable for commercial antibody production. Propagation of the producer cells results in a producer cell line capable of producing RSV-specific antibodies. Preferably, the producer cell line is suitable for producing compounds for use in humans. Thus, the producer cell line is preferably pathogen free, e.g. pathogenic microorganisms.

The method of the present invention is preferably used to produce antibody producing cells that are stable for at least one week, preferably at least one month, more preferably at least three months, more preferably at least six months, for commercial antibody production. Most preferably, a stable cell line capable of producing monoclonal antibodies is produced. Such manipulation is preferably performed by selecting CD19 and/or CD22(B cell markers) and cell surface IgG and/or CD27 (to label memory cells) and/or by negative selection for IgM, IgD and/or IgA, using memory B cells isolated from, for example, a sample. Furthermore, RSV-specific antibody producing cells are selected in a binding assay using RSV or components of RSV from source, such as the RSV F protein, G protein and/or SH protein. Subsequently, according to this preferred embodiment, Blimp-1 and BCL6 are co-expressed in said RSV-specific antibody producing cells, resulting in a culture of cells capable of specifically binding (a component of) RSV. In another preferred embodiment, the B-cell is also provided with Bcl-xL or a functional part, derivative and/or analogue thereof.

If only one memory B cell is used, a cell line of the invention producing monoclonal antibodies is obtained. It is also possible to obtain a cell line capable of producing monoclonal antibodies from B cells capable of producing anti-RSV antibodies. Following production of a stable B cell culture by the method of the invention, B cells capable of producing antibodies against RSV-specific antigens are isolated, preferably by expressing at least a functional part of the genes encoding the Ig heavy and/or light chains from said B cells in a second cell line. Preferably, at least a functional part of the Ig heavy chain encoding gene and at least a functional part of the Ig light chain encoding gene from said B cell are expressed in a second cell line.

In one embodiment, antibody-producing cells, preferably but not necessarily memory B cells, obtained from an individual who has been exposed to RSV are used in the methods of the invention. In this way, it is possible to produce human antibodies of interest ex vivo.

Accordingly, there is also provided a method of producing an antibody capable of specifically binding to and/or neutralizing respiratory syncytial virus, the method comprising:

-producing antibody producing cells capable of producing RSV-specific antibodies using the method of the invention; and

-obtaining the antibody produced by the antibody producing cell.

Also provided are isolated or recombinant antibodies obtainable by the methods of the invention, as well as isolated or recombinant antibody producing cells, or functional equivalents thereof. The antibody preferably comprises antibody D25, AM14, AM16 and/or AM23, or a functional part, derivative or analogue thereof.

Once the RSV-specific antibody producing cell of the invention has been obtained, it is preferred that at least a functional part of the genes encoding the Ig light and/or heavy chains of said cell is isolated and/or produced artificially. In one embodiment, a nucleic acid sequence is provided comprising at least a functional portion of the nucleic acid sequence set forth in figure 11, figure 12, figure 14A, figure 14B, and/or figure 14C. Preferably, the functional portion comprises at least one nucleic acid sequence as depicted in figure 11D, figure 12, figure 14A, figure 14B and/or figure 14C. The functional portion preferably encodes at least one CDR as shown in fig. 11D, fig. 12, fig. 14A, fig. 14B and/or fig. 14C.

Also provided is an isolated, synthetic or recombinant nucleic acid sequence comprising a heavy chain sequence at least 70%, preferably at least 80%, more preferably at least 90% homologous to at least a portion of sequence CAGGTGCAGCTGGTACAGTCTGGGGCTGAAGTGAAGAAGCCTGGGTCCTCGGTGATGGTCTCCTGCCAGGCCTCTGGAGGCCCCCTCAGAA, ACTATATTATCAAC, TGGCTACGACAGGCCCCTGGACAAGGCCCTGAGTGGATGGGA, GGGATCATTCCTGTCTTGGGTACAGTACACTACGCACCGAAGTTCCAGGGC, AGAGTCACGATTACCGCGGACGAATCCACAGACACAGCCTACATCCATCTGATCAGCCTGAGATCTGAGGACACGGCCATGTATTACTGTGCGACG, GAAACAGCTCTGGTTGTATCTACTACCTACCTACCACACTACTTTGACAAC, TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG and/or CAGGTGCAGCTGGTACAGTCTGGGGCTGAAGTGAAGAAGCCTGGGTCCTCGGTGATGGTCTCCTGCCAGGCCTCTGGAGGCCCCCTCAGAAACTATATTATCAACTGGCTACGACAGGCCCCTGGACAAGGCCCTGAGTGGATGGGAGGGATCATTCCTGTCTTGGGTACAGTACACTACGCACCGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACAGACACAGCCTACATCCATCTGATCAGCCTGAGATCTGAGGACACGGCCATGTATTACTGTGCGACGGAAACAGCTCTGGTTGTATCTACTACCTACCTACCACACTACTTTGACAACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG, said portion having at least 15 nucleotides. The heavy chain sequence is preferably derived from antibody D25. Preferably, the heavy chain sequence comprises a sequence which is at least 70%, preferably at least 80%, more preferably at least 90% homologous to the sequence shown in figure 11D. Also provided herein are isolated, synthetic or recombinant nucleic acid sequences comprising a heavy chain sequence consisting of any of the above heavy chain sequences.

Also provided is an isolated, synthetic or recombinant nucleic acid sequence comprising a light chain sequence at least 70%, preferably at least 80%, more preferably at least 90% homologous to at least a portion of sequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCAGCTGTAGGAGACAGAGTCACCATCACTTGC, CAGGCGAGTCAGGACATTGTCAACTATTTAAAT, TGGTATCAACAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTAC, GTTGCATCCAATTTGGAGACA, GGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTAGTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACATATTATTGT, CAACAATATGATAATCTCCCA, CTCACATTCGGCGGAGGGACCAAGGTTGAGATCAAAAGA and/or GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCAGCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTGTCAACTATTTAAATTGGTATCAACAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGTTGCATCCAATTTGGAGACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTAGTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACATATTATTGTCAACAATATGATAATCTCCCA CTCACATTCGGCGGAGGGACCAAGGTTGAGATCAAAAGA, said portion having at least 15 nucleotides. The light chain sequence is preferably derived from antibody D25.

The light chain sequence preferably comprises a sequence that is at least 70%, preferably at least 80%, more preferably at least 90% homologous to the sequence set forth in fig. 11D. Also provided herein are isolated, synthetic or recombinant nucleic acid sequences comprising a heavy chain sequence consisting of any of the above light chain sequences.

Also provided is an isolated, synthetic or recombinant nucleic acid sequence comprising a heavy chain sequence at least 70%, preferably at least 80%, more preferably at least 90% homologous to at least a portion of sequence GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCGGCCTCT, GGATTCAGCTTCAGTCACTATGCC, ATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCAGTT, ATATCTTATGATGGAGAAAATACA, TATTACGCAGACTCCGTGAAGGGCCGATTCTCCATCTCCAGAGACAATTCCAAGAACACAGTGTCTCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTCTATATTACTGT, GCGAGAGACCGCATAGTGGACGACTACTACTACTACGGTATGGACGTC, TGGGGCCAAGGGGCCACGGTCACCGTCTCCTCAG and/or GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCGGCCTCTGGATTCAGCTTCAGTCACTATGCCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCAGTTATATCTTATGATGGAGAAAATACATATTACGCAGACTCCGTGAAGGGCCGATTCTCCATCTCCAGAGACAATTCCAAGAACACAGTGTCTCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTCTATATTACTGTGCGAGAGACCGCATAGTGGACGACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGGCCACGGTCACCGTCTCCTCA, said portion having at least 15 nucleotides. The heavy chain sequence is preferably derived from antibody AM 14. Also provided herein are isolated, synthetic or recombinant nucleic acid sequences comprising a heavy chain sequence consisting of any of the above heavy chain sequences.

Also provided is an isolated, synthetic or recombinant nucleic acid sequence comprising a light chain sequence at least 70%, preferably at least 80%, more preferably at least 90% homologous to at least a portion of sequence GACATCCAGATGACCCAGTCTCCATCTTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGT, CAGGACATTAAGAAGTAT, TTAAATTGGTATCATCAGAAACCAGGGAAAGTCCCTGAGCTCCTGATGCAC, GATGCATCC, AATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGCAGGGGATCTGGGACAGATTTTACTCTCACCATTAGCAGCCTGCAGCCTGAAGATATTGGAACATATTACTGT, CAACAGTATGATAATCTGCCTCCGCTCACT, TTCGGCGGAGGGACCAAGGTGGAGATCAAAC and/or GACATCCAGATGACCCAGTCTCCATCTTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTAAGAAGTATTTAAATTGGTATCATCAGAAACCAGGGAAAGTCCCTGAGCTCCTGATGCACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGCAGGGGATCTGGGACAGATTTTACTCTCACCATTAGCAGCCTGCAGCCTGAAGATATTGGAACATATTACTGTCAACAGTATGATAATCTGCCTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGAACTGTG, said portion having at least 15 nucleotides. The light chain sequence is preferably derived from antibody AM 14. Also provided herein are isolated, synthetic or recombinant nucleic acid sequences comprising a heavy chain sequence consisting of any of the above light chain sequences.

Also provided is an isolated, synthetic or recombinant nucleic acid sequence comprising a heavy chain sequence at least 70%, preferably at least 80%, more preferably at least 90% homologous to at least a portion of sequence GAGGTGCAGCTGGTGGAGACCGGGGGAGGCCTGGCCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCT, GGATTCACATTCAGTAGTTATAAC, ATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCACAC, ATTAGTGCGGGTAGTAGTTACATA, TACTACTCAGACTCAGTGAAGGGCCGATTCACCGTCTCCAGAGACAACGTCAGGAACTCAGTATATCTGCAAATGAACAGCCTGAGAGCCGCTGACACGGCTGTGTATTACTGT, GCGAGAGAGGATTATGGTCCGGGAAATTATTATAGTCCTAACTGGTTCGACCCC, TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG and/or GAGGTGCAGCTGGTGGAGACCGGGGGAGGCCTGGCCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACATTCAGTAGTTATAACATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCACACATTAGTGCGGGTAGTAGTTACATATACTACTCAGACTCAGTGAAGGGCCGATTCACCGTCTCCAGAGACAACGTCAGGAACTCAGTATATCTGCAAATGAACAGCCTGAGAGCCGCTGACACGGCTGTGTATTACTGTGCGAGAGAGGATTATGGTCCGGGAAATTATTATAGTCCTAACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA, said portion having at least 15 nucleotides. The heavy chain sequence is preferably derived from antibody AM 16. Also provided herein are isolated, synthetic or recombinant nucleic acid sequences comprising a heavy chain sequence consisting of any of the above heavy chain sequences.

Also provided is an isolated, synthetic or recombinant nucleic acid sequence comprising a light chain sequence at least 70%, preferably at least 80%, more preferably at least 90% homologous to at least a portion of sequence CAGTCTGTCGTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGAGTCACCATCTCCTGCACTGGGAGC, AGCTCCAACATCGGGGCAGGTTATGAT, GTACACTGGTACCAGCAGCTTCCAGGAACAGCCCCCAAACTCCTCATCTAT, GGCAACACT, AATCGGCCCTCAGGGGTCTCCGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGACTCCAGGCTGAGGATGAGGCTGATTATTACTGC, CACTCCTATGACAGAAGCCTGAGTGGT, TCAGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAG and/or CAGTCTGTCGTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGAGTCACCATCTCCTGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGATGTACACTGGTACCAGCAGCTTCCAGGAACAGCCCCCAAACTCCTCATCTATGGCAACACTAATCGGCCCTCAGGGGTCTCCGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGA CTCCAGGCTGAGGATGAGGCTGATTATTACTGCCACTCCTATGACAGAAGCCTGAGTGGTTCAGTATTCGGCGGAGGGACCAAGCTGACCGTC, said portion having at least 15 nucleotides. The light chain sequence is preferably derived from antibody AM 16. Also provided herein are isolated, synthetic or recombinant nucleic acid sequences comprising a heavy chain sequence consisting of any of the above light chain sequences.

Also provided is an isolated, synthetic or recombinant nucleic acid sequence comprising a heavy chain sequence at least 70%, preferably at least 80%, more preferably at least 90% homologous to at least a portion of sequence CAGGTGCAACTGGTGGAGTCTGGGGGAAATGTGGTCAAGCCTGGGACGTCCCTGAGACTGTCCTGTGCAGCGACT, GGATTCAACTTCCATAACTACGGC, ATGAACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCGGTT, GTTTGGTATGATGGAAGTAAGAAA, TACTATGCAGACTCCGTGACGGGCCGATTCGCCATCTCCAGAGACAATTCCAAGAACACTCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCTGTTTATTATTGT, GTGAGAGATAAAGTGGGACCGACTCCCTACTTTGACTCC, TGGGGCCAGGGAACCCTGGTCACCGTATCCTCAG and/or GAGGTGCAGCTGGTGGAGTCTGGGGGAAATGTGGTCAAGCCTGGGACGTCCCTGAGACTGTCCTGTGCAGCGACTGGATTCAACTTCCATAACTACGGCATGAACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCGGTTGTTTGGTATGATGGAAGTAAGAAATACTATGCAGACTCCGTGACGGGCCGATTCGCCATCTCCAGAGACAATTCCAAGAACACTCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCTGTTTATTATTGTGTGAGAGATAAAGTGGGACCGACTCCCTACTTTGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT, said portion having at least 15 nucleotides. The heavy chain sequence is preferably derived from antibody AM 23. Also provided herein are isolated, synthetic or recombinant nucleic acid sequences comprising a heavy chain sequence consisting of any of the above heavy chain sequences.

Also provided is an isolated, synthetic or recombinant nucleic acid sequence comprising a light chain sequence at least 70%, preferably at least 80%, more preferably at least 90% homologous to at least a portion of sequence TCCTATGTGCTGACTCAGCCACCCTCGGTGTCACTGGCCCCAGGAGGGACGGCCGCGATCACCTGTGGAAGAAAC, AACATTGGAAGTGAAACT, GTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCGTCTAT, GATGATGAC, GACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAGGCCGGGGATGAGGCCGACTATTACTGT, CAGGTGTGGGATAGGAGTAATTATCATCAGGTA, TTCGGCGGAGGGACCAAGTTGACCGTCCTAG and/or TCCTATGTGCTGACTCAGCCCCCCTCGGTGTCACTGGCCCCAGGAGGGACGGCCGCGATCACCTGTGGAAGAAACAACATTGGAAGTGAAACTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCGTCTATGATGATGACGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAGGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGATAGGAGTAATTATCATCAGGTATTCGGCGGAGGGACCAAGCTGACCGTC, said portion having at least 15 nucleotides. The light chain sequence is preferably derived from antibody AM 23. Also provided herein are isolated, synthetic or recombinant nucleic acid sequences comprising a heavy chain sequence consisting of any of the above heavy chain sequences.

Also provided are nucleic acid sequences encoding amino acid sequences at least 70%, preferably at least 80%, more preferably at least 90% identical to a functional part of at least the amino acid sequences shown in figure 11, figure 14A, figure 14B and/or figure 14C, said part having at least 5 amino acid residues. The nucleic acid sequence preferably encodes an amino acid sequence which is at least 80% identical to the heavy chain CDR sequences 1, 2 and/or 3 and/or the light chain CDR sequences 1 or 2 as set out in fig. 11D. In another preferred embodiment, the nucleic acid sequence encodes an amino acid sequence that is at least 80% identical to at least one of the CDR sequences depicted in fig. 14A, fig. 14B and/or fig. 14C. In a preferred embodiment, the nucleic acid sequence encodes an amino acid sequence that is at least 70% identical to the heavy chain sequence shown in FIG. 11A, to the heavy chain sequence shown in FIG. 14A, to the heavy chain sequence shown in FIG. 11B, to the heavy chain sequence shown in FIG. 14C, to the light chain sequence shown in FIG. 11A, to the light chain sequence shown in FIG. 14B, and/or to the light chain sequence shown in FIG. 14C.

Accordingly, there is also provided an isolated, synthetic or recombinant nucleic acid sequence comprising a sequence encoding an amino acid sequence which is at least 70%, preferably at least 80%, more preferably at least 85% identical to the amino acid sequence shown in figures 11A-D. The nucleic acid sequence preferably encodes an amino acid sequence which is at least 80% identical to the heavy chain CDR sequences 1, 2 and/or 3 and/or the light chain CDR sequences 1 or 2 as set out in fig. 11A-D. One embodiment provides an isolated, synthetic or recombinant nucleic acid sequence comprising a sequence encoding an amino acid sequence at least 70% identical to the amino acid sequence NYIIN, and/or at least 75% identical to sequence GIIPVLGTVHYAPKFQG, and/or at least 70% identical to sequence ETALVVSTTYLPHYFDN, and/or at least 85% identical to sequence QASQDIVNYLN, and/or at least 70% identical to sequence VASNLET, and/or at least 70% identical to sequence QVQLVQSGAEVKKPGSSVMVSCQASGGPLRNYIINWLRQAPGQGPEWMGGIIPVLGTVHYAPKFQGRVTITADESTDTAYIHLISLRSEDTAMYYCATETALVVSTTYLPHYFDN WGQGTLVTVSS, and/or at least 70% identical to sequence DIQMTQSPSSLSAAVGDRVTITCQASQDIVNYLNWYQQKPGKAPKLLIYVASNLETGVPSRFSGSGSGTDFSLTISSLQPEDVATYYCQQYDNLPLTFGGGTKVEIKRTV.

The nucleic acid sequence of the invention is preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% homologous to any of the above sequences.

Also provided is an isolated, synthetic or recombinant nucleic acid sequence comprising a sequence encoding an amino acid sequence at least 70%, preferably at least 80%, more preferably at least 85% identical to the amino acid sequence set forth in figures 14A-C. The nucleic acid sequence preferably encodes an amino acid sequence which is at least 70% identical to a CDR sequence as depicted in fig. 14A, 14B and/or 14C. One embodiment provides an isolated, synthetic or recombinant nucleic acid sequence comprising a sequence encoding an amino acid sequence at least 70% identical to an amino acid sequence selected from the group consisting of seq id no: GFSFSHYA, ISYDGENT, ARDRIVDDYYGMDV, QDIKKY, DAS, QYDNLPPLT, EVQLVESGGGVQPGRSLRLSCASFSHYAMHWVRQVRQKGLEWVIVYDGEDYSYDSVKVKSRDKNTVKSQMLNSQRLDNDLPHTHRADLCATALDETHRADDTARGDAVDTVDDDVTVSS, DIQQQSYSPSSDLTSLVTSLVTSLVTSLVTSVQKPGKWATVSQKQKQKQKQKQKQVPELLMHDRALVESGVGGVGGVGTGTGTGTGTFTFTSTGTSTGTSTGTSVGTVGTYTGVQLVTSVQVYGVGGVSVQVQVYGVQVYGVQVYGVGVGGVGGVGVGVGVGVGVGVGVQVQVGVGVGVGVGVGVGVGVGVGVGVGVGVGVVGVVGVGVGVGVGVVVVVVGVGVGVVVVVDD, ISVGVGVGVGVGVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVGTVVGVVGVVGVVGVVGVVGVGTVVGVVGVVGVVGVGTVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVGTVVGVVGVVGVVGVVGVVGVGTVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVGTVVGVVGVGTVVGVVGVVGVVGVGTVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVGVVG.

The nucleic acid sequence of the invention is preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% homologous to any of the above sequences.

As mentioned above, the nucleic acid sequences of the invention are particularly suitable for expressing the antibodies of the invention or functional parts, derivatives or analogues thereof, preferably D25, AM14, AM16, AM23 or functional parts, derivatives and/or analogues thereof in a nucleic acid expression system. The nucleic acid sequences of the invention are preferably expressed in cells, more preferably in producer cells suitable for antibody production.

The invention is further explained in the following examples. These examples do not limit the scope of the invention, but are merely illustrative thereof.

Examples

Materials and methods

Maintenance and isolation of human B cells

CD19 positive human B cells were isolated from buffy coats derived from blood banks using standard methods (other sources could be fresh blood containing anticoagulant factors, or lymphoid organs such as tonsils or spleens). Briefly, total Peripheral Blood Mononuclear Cells (PBMCs) were isolated using ficoll density fractionation (Amersham, Buckinghamshire, UK). B cells were positively selected using CD 22-labeled beads by the manufacturer's described MACS cell sorting technique (Miltenyi, urtrecht, Netherlands, miky-specific, udlerley, Netherlands). Cells were then stained with the appropriate combination of monoclonal antibodies (mabs) to CD19, CD27, IgD, IgM, and IgA (Becton Dickinson, BD, Franklin Lakes, NJ, USA). Memory B cells positive for CD19 and CD27 and negative for IgM, IgA, and IgD were then sorted with facsaria (bd) (fig. 1). In addition to memory B cells, other B cell subsets can be isolated using suitable markers, such as blasts, follicular cells, memory cells, antibody producing cells, central blasts, central cells, germinal central cells, plasmablasts, plasma cells, marginal zone cells, sinusoid pericytes or transitional B cells (many of these subsets have been tested only in mice).

Cell culture

Sorted cells were washed and plated with 80-Gray irradiated L cells expressing CD40L (5X 10)⁴Individual cells/ml; culture (1.5-2X 10) in complete Medium (Iscove's Modified D minimum Essential Medium containing 8% Fetal Calf Serum (FCS) and penicillin/streptomycin) on 24-well plates from Schering Plough France (Dr. J.Banchereau), Inc.)⁵Individual cells/ml). Unless otherwise stated, these CD40L expressing L cells were always cultured in medium containing 8% FCS. To prepare B cells for retroviral transduction, IL-21(50ng/ml, R of Minneapolis, Midland, Minn., USA) was used in mice&Company D (R)&D, Minneapolis, MN, USA)) for 36 hours. After transduction, the cells are preferably cultured in the presence of IL-21, however the cells respond to IL-4, IL-15 and IL-10 (other cytokines are not excluded). For example, IL-4-induced B cell expansion is lower than IL-21, and lower levels of cell division may be desirable in certain experiments.

Retroviral constructs and production of recombinant retroviruses

Constitutively active mutants of STAT5a and b have been previously described. DNA encoding these mutants and wild-type STAT5b was obtained from t. In a screen for senescent rescue of murine fibroblasts, Bcl-6 was identified as an inhibitor of antiproliferative p19ARF-p53 signaling. Bcl-XL was identified as an anti-apoptotic factor, which was kindly provided by Korsmeyer (Boston, HH Medical Institute of Boston, USA). These DNAs were ligated into the previously described LZRS-linker-IRES-GFP (or IRES-YFP or IRES-NGFR) vectors (Heemskerk et al, 1997; Heemskerk et al, 1999). IRES-YFP (yellow fluorescent protein) or IRES-NGFR (nerve growth factor receptor) may also be used in place of the IRES-GFP (green fluorescent protein) marker. NGFR is a signal transduction disabling mutant of NGFR, kindly provided by doctor c. Cells expressing NGFR were observed using monoclonal antibodies to NGFR (Chromaprobe, Mountain View, CA, US) or Mitsubishi (Miltenyi).

To generate recombinant retroviruses, retroviral plasmids were transfected into the helper virus-free amphotropic producer cell line Phoenix-A, a derivative of the human embryonic kidney cell line 293 (Kinsella and Nolan, 1996) (friend gifted by Dr. G. Nolan, Stanford University, Palo Alto, CA) using Fugene-6 (Roche Diagnostics Netherlands, Allerger, Netherlands) following the manufacturer's protocol. Two days later, selection of transfected cells was initiated by the addition of 2 μ g/ml puromycin (Becton Dickinson Clontech Laboratories, Pa., Calif.). 10-14 days after transfection, 6X10⁶Cells were seeded into a 10cm petri dish (Becton Dickinson Discovery laboratory facility, Bedford, Mass.) containing 10ml of complete medium without puromycin. The next day, fresh medium was changed, and on the third day, retroviral supernatants were harvested, centrifuged and frozen at-70 ℃ in cell-free aliquots. This method provides a reproducible, rapid, large-scale and high titer retroviral production process with yields exceeding 3x10⁶Individual infectious viral particles per ml.

Retroviral transduction

The recombinant human fibronectin fragment CH-296 transduction method (RetroNectin) was performed as previously described (Heemskerk et al, 1997; Heemskerk et al, 1999)^TM(ii) a Takara, Otsu, Japan). Non-tissue culture treated 24-well plates (Costar, baddoevedorp, Netherlands) were coated with 0.3ml of 30 μ g/ml recombinant human fibronectin fragment CH-296 for 2 hours at room temperature or overnight at 4 ℃. Using different sizesThe lung tissue culture plate of (1) is prepared by using the reagents in proportion. The CH-296 solution was removed and then incubated with 2% Human Serum Albumin (HSA) in Phosphate Buffered Saline (PBS) for 30 minutes at room temperature, followed by one wash with PBS. 5x10 prepared for retroviral transduction⁵Individual B cells were seeded in 0.25ml RPMI without FCS and L-cells and mixed with 0.25ml of thawed retroviral supernatant. For Bcl-6Bcl-XL double transduction, 125. mu.l of Bcl-6-IRES-NGFR (or IRES-YFP) (Shvarts A. et al, Genes Dev., 2002) and 125. mu.l of Bcl-XL-IRES-GFP (supplied by S.Korsmeyer of the HH Medical Institute of Boston Children's Howarding Medical Institute, Childrens Hospital, Boston, USA) were mixed and added to the cells. Then, the culture was centrifuged at 1800rpm at 25 ℃ for 60 minutes and incubated at 37 ℃ for 6 hours. Next, 0.25ml of the supernatant was removed and 0.25ml of fresh retroviral supernatant was added. The culture was again centrifuged at 1800rpm at 25 ℃ for 60 minutes and incubated overnight at 37 ℃. The following morning, cells were transferred to 24-well tissue culture plates (Costar) in the presence of human IL-4(50ng/ml) or mouse IL-21(50ng/ml, R of minneapolis, minnesota, usa)&Company D (R)&D, Minneapolis, MN, USA)) for 3-5 days under normal conditions. Transduction efficiency was determined by antibody staining of signal transduction disabling mutants of truncated nerve growth factor receptors (Δ NGFR, provided by c.bonini of st. raphael Hospital, Milan, Italy) or (co-) expression of GFP and/or YFP. Cells containing the transgene of interest were selected for further experiments.

Flow cytometry

Antibodies to human molecules IgD, IgG, CD3, CD19, CD20, CD27, CD38, CD40, CD45, CD56, CD70, CD80, CD86, HLA-dr (bd), directly labeled with FITC, PE, PERCP, PE-Cy5, APC or APC-Cy7, and IgM, kappa light chain, lambda light chain, CD138 directly labeled with PE (dako), were used for flow cytometry analysis. Stained cells were analyzed by lsrii (bd) and FACS data was processed using FlowJo computer software (TS corporation (Tree Star, Inc.).

Proliferation assay

Primary and memory B cells were isolated from fresh PBMCs on FACSAria:

primary B cells: CD19-Pe-Cy7pos, CD27-APC neg, IgD-PE pos

Memory B-cells: CD19-Pe-Cy7pos, CD27-APC pos, IgD-PE neg, IgA-FITCNeg

Cells were washed with PBS and resuspended in 0.5ml RPMI without FCS (37 ℃). An equal amount of IMDM containing 2. mu.M carboxyfluorescein succinimidyl ester (CFSE) was added to the cell mixture and incubated at 37 ℃ for 7 minutes. Cells were washed with ice-cold FCS to stop labeling of cells. Cells were resuspended in 500. mu.l IMDM-8% FCS and cultured with L cells in the presence or absence of IL-21. Unlabeled cells were used as controls.

After 36 hours (just before transduction), a portion of the cells were analyzed for CFSE content. The remaining cells were transduced by centrifugation with Bcl-6-IRES-NGFR, cultured for 3 days, and analyzed for CFSE content using LSRII. The data were analyzed with FlowJo software (TS).

Isolation of antigen-specific human B cells by high-speed single-cell sorting

In addition to the memory B cell isolation method described above starting with MBC (i.e., 100 cells/culture well), human memory B cells can also be incubated with fluorescently labeled antigen and sorted for antigen recognition. An example is the isolation of Phycoerythrin (PE) -labeled tetanus toxoid-binding B cells (supplied by a. radbruch, Berlin, Germany) (fig. 4). Cells were cultured at 1 cell/well and examined for TT binding. However, any other labeled antigen may be used.

Determination of altered B Cell Receptor (BCR) expression resulting from Long-term culture of Bcl-6 and Bcl-XL transduced cells

It is known that membrane expression of BCR disappears in B cells differentiated during in vitro culture, and this phenomenon is also observed in EBV-transformed B cells. Thus, B cells transduced with Bcl-6 and Bcl-XL and cultured in the presence of IL-21 were stained with GFP, NGFR, CD19, kappa and/or lambda or IgG, or with labeled tetanus toxoid. To demonstrate the usefulness of BCR expression, we sorted TT-pe (radbruch) bound cells at 1 cell/well (96 well plate) using facsaria (bd) and then seeded in media containing L cells and IL-21. After three weeks, the colonies were tested for tetanus toxoid binding using a FACS Canto (BD). To this end, cells were harvested and stained with GFP, NGFR, CD19 and TT-PE in 96-well plates.

Development of antibody secreting Bcl-6 and Bcl-XL double positive B cell lines

B cell lines were established which produced monoclonal antibodies and were 100% Bcl-6 and Bcl-XL double positive. First, IL-21 is used to induce proliferation and differentiation to achieve this goal. At the same time, these cells were transduced with Bcl-6-IRES-NGFR and Bcl-XL-IRES-GFP retroviruses. These cells were maintained on IL-4 for 3-4 days. Subsequently, cells transduced with one or both of the retroviruses express the transgene and, thus, the NGFR or GFP proteins. Expression of NGFR and/or GFP can be observed using lsrii (bd). If necessary, the cells can be transduced again to obtain a greater number of cells expressing both transgenes. Whether or not the second transduction was performed, cells expressing both transgenes were sorted using FACS Aria (BD) and cultured in 96-well plates at a cell density ranging from 10-500 cells/well in the presence of IL-21 and 2500-. By day 5, these small batch cultures (MBCs) secreted considerable antibodies in the culture supernatant, which could then be used for screening purposes. The screening can be based on available techniques for the antigen of interest, such as ELISA/EIA/RIA, Western blotting or direct functional assays, such as neutralizing cytokine blocking assays. After screening and selection of MBCs that recognize the antigen of interest (TT and RSV in our experiment), cells were subcloned in 96-well plates in the presence of IL-21 at a density of 0.5-1 cells/well. Subcloning is usually performed for 2-3 weeks, either by Limiting Dilution (LD) culture or flow cytometry (FACSAria) single cell sorting.

RSV A-2 Virus stocks and HEp2 cell line

RSV a-2 virus (kindly provided by g.vanblek of udlerian branch WKZ (WKZ, urtecht)) and HEp2 cell line (clinical laboratory of Amsterdam AMC) were cultured in large quantities and frozen in liquid nitrogen.

HEp2 adherent cell line was cultured in normal medium in a T175 Falcon flask, and aliquots were frozen.

To obtain higher titers of RSV stock, HEp2 cells were inoculated and cultured to reach 50-60% confluence. The original RSV stock (1/20 diluted in a total volume of 5ml) was added to HEp2 cells at room temperature using 45'. 15ml of fresh medium was added, and the cells were placed at 37 ℃ in 5% CO with the cap opened₂O/n. The following morning, the culture supernatant was carefully removed and 15ml of medium containing 1% FCS was added. The bottle cap was tightened and the cells were placed at 37 ℃ in 5% CO₂And (4) incubating for 24-36 hours. While RSV-induced syncytia were clearly visible, most of the syncytia remained complete, the medium was collected, filtered (0.22 μm), centrifuged at 1450rpm at room temperature, and the samples were snap frozen and stored in liquid nitrogen. A second harvest can be obtained by adding fresh medium containing 1% FCS immediately and freezing after 4-6 hours.

RSV lysates for ELISA

Virus stocks obtained by infection of HEp2 cells with RSV A-2 can be used to isolate RSV proteins. The first well was carefully washed with PBS and digested with trypsin. Trypsin (Gibbo) was washed off and the cell pellet was lysed with 1% octyl glucoside (cell pellet from one T175 flask was treated with 2 ml octyl glucoside). The suspension was homogenized with a syringe and needle (10 times up and down), incubated on ice for 1 hour, and then dialyzed at 4 ℃ against 2L TBS buffer pH 7.4, o/n. After centrifugation to remove cell debris, a supernatant was obtained. The protein content was determined to be 3.6mg/ml and used in ELISA at a concentration of 20. mu.g/ml (50. mu.l).

Determination of TCID of RSV stock₅₀And PFU

To determine TCID50, 10⁴HEp2 were seeded in 96-well plates and serially diluted in 2 or 10 stepsThe RSV of (2) infected HEp2 with 4-plo. After 2-3 days, the culture supernatant was removed and the cells were fixed with 80% acetone at room temperature 10'. After removal of acetone, the fixed cell layer was dried and either maintained at 4 ℃ or frozen at-20 ℃. To stain RSV HEp2 cells, plates were first blocked with 5% milk powder in PBS 0.1% tween 20. The plates were then washed 3 times, followed by incubation with polyclonal goat anti-RSV-HRP (1: 500, Biodesign, Saco, ME, US) at 37 ℃ for 3-5 hours, and washed thoroughly. Next, the wells were incubated with AEC substrate 30' at room temperature. Infected foci stained red, and were visually observed and counted by an optical microscope. Determination of TCID Using Standard Excel software₅₀。

To determine the Plaque Forming Unit (PFU) value of the virus, 1 × 10 was plated in 24-well plates⁵Perlhep 2 cells were serially diluted 10-fold with 1% FCS (10%^-3-10^-7) The RSV virus stock (E) was incubated at 37 ℃ for 45' (200. mu.l) together, and then the cells and virus were overlaid with 0.5ml of 0.25% sea-spot (seaplaque) agar (Biozyme) by Bayer). The agarose layer prevents the spread of the virus through the medium onto uninfected cells. Thus, the virus can only infect neighboring cells, which are eventually killed by the virus, forming plaques in the HEp2 cell monolayer. Fixed cells (96% ethanol-100% acetic acid-10% formaldehyde 6: 2: 1) were stained with 1% crystal violet solution to visualize these plaques. Plaques were counted (from at least two different individuals) and PFU values were determined.

Selection of Respiratory Syncytial Virus (RSV) neutralizing antibodies

To obtain anti-Respiratory Syncytial Virus (RSV) B cell clones, two donor peripheral blood cells (PBMCs) (donor B62 and B63) were isolated from buffy coats derived from blood banks. Sorting CDs 19 with FACSAria (BD)^posIgM^negIgD^negIgA^negCD27^posPrior to the cells (FIG. 1), the CD22+ cells were isolated using MACS beads and columns (Mich-Tech Co.). Cells were cultured with L cells unless otherwise indicated. Cells were cultured in the presence of IL-21 for 36 hours and then used aloneBcl-6-IRES-NGFR transduction. After 12 hours the cells were harvested, cultured for 3 days in the presence of IL-4, and then NGFR-expressing cells were sorted using MACS beads (MIANTIARY Co.) and immediately transduced with Bcl-XL-IRES-GFP. B cells that did not bind MACS beads were washed and transduced simultaneously with Bcl-6 and Bcl-XL. Cells were harvested after 12 hours, pooled and cultured in the presence of IL-4 for 3 days, and then sorted on FACSAria for GFP and NGFR expression. Cells were washed and cultured in 96-well plates (Costar) at a density of 100 cells/well in the presence of IL-21.

RSV binding was screened for Bcl-6 and Bcl-XL dual transduced B cell cultures using an ELISA of HEp2 cell lysates infected with RSV, and tested in parallel using RSV microneutralization experiments. Briefly, 10 will be described⁴Individual HEp2 cells were seeded in flat bottom 96-well plates (firm costa) containing complete medium. The following day, the medium was replaced with a 30 minute pre-incubation mixture of RSV virus and cell culture supernatant at 37 ℃ for 1 hour at room temperature. The total volume was 25. mu.l, and the final RSV concentration was 0.1 MOI. After 1 hour, the virus supernatant mixture was diluted 9-fold with PBS and replaced with 100 μ Ι imdm/5% FCS. After 2 days, the cells were fixed with 80% acetone and stained with polyclonal anti-RSV-HRP (Biodesign). Using H₂O₂And AEC, cells infected with RSV produce a red color. Infected cells were observed using an optical microscope and counted if necessary. Goat polyclonal anti-RSV (Abcam corporation, Cambridge, MA) was used as a control for RSV neutralization.

RT-PCR and cloning of VH and VL regions

By usingMinikits (Qiagen, Venlo, the Netherlands) from about 5X10⁵Total RNA was isolated from individual B cells. 250ng total RNA was reverse transcribed in 20. mu.l of a system containing 1 Xfirst strand buffer, 500. mu.M dNTPs, 250ng random hexamers, 5mM DTT, 40U RNase (Promega) and 200U SuperScript III RT (Invitrogen). C is mixed with ultrapure waterDNA was diluted 10X and contained 20mM Tris-HCl, 50mM KCL, 2.5mM MgCl in 50. mu.l₂PCR was performed on 2.5. mu.l of cDNA in a solution of 250. mu.M dNTP, 1U AmpliTaq gold-labeled DNA polymerase (Applied Biosystems Inc.) and 25pmol of each primer. The PCR conditions were as follows: the denaturation step was carried out at 96 ℃ for 8 minutes, followed by 35 cycles of: 30 seconds at 96 ℃, 30 seconds at 60 ℃ and 1 minute at 72 ℃; finally, extension was carried out at 72 ℃ for 10 minutes.

The PCR product was electrophoresed on an agarose gel, purified and cloned into pCR2.1TA cloning vector according to the manufacturer's recommended protocol. Sequence analysis was performed using BigDye terminator chemistry (applied biosystems) and Vector-NTI software (Invitrogen).

To exclude reverse transcriptase and/or DNA polymerase induced mutations, several independent cDNA transformation and PCR reactions were performed, and individual cloning and sequence analysis were performed. The consensus sequence was determined using Vector-NTI Contig Express (Vector-NTI Contig Express) software.

To express recombinant protein antibodies in 293T cells, full-length heavy and light chain constructs were produced using pcdna3.1(+) Zeo (invitrogen). The heavy chain leader sequence and VH region of clone D25 were amplified by PCR, introducing a 5 '-NheI site and a 3' -XhoI site, to construct a heavy chain expression vector. The IgG1 constant region (CH 1-hinge-CH 2-CH3) was amplified from the same cDNA, with the introduction of 5 '-XhoI and 3' -NotI sites. The full-length heavy chain expression vector was obtained by three-point ligation into NheI/NotI digested pCDNA3.1(+) Zeo. The light chain leader sequence, VL region and light chain constant region were amplified by PCR with primers to introduce 5 '-NheI and 3' -NotI sites, thereby generating a full-length light chain expression construct. The latter product was cloned into NheI/NotI digested pCDNA3.1(+) Zeo to obtain a full-length light chain expression vector.

Sequence analysis was performed to confirm the correctness of the expression construct.

293T cells were subjected to transient double transfection (Fugene-6, Roche, Germany) or the lipofectamine LTX (Lipofectamine LTX), Invitrogen) with heavy and light chain expression vectors to obtain recombinant monoclonal antibodies. FACS staining (48 hours) was performed with culture supernatants of Hep2 cells infected with RSV to show functional binding of the antibody to the RSV F-protein.

The oligonucleotides used for PCR amplification were:

VH region:

VH 1-Forward 5'-AAATCGATACCACCATGGACTGGACCTGGAGG-3'

VH 1B-Forward 5 '-AAATCGATACCACCATGGACTGGACCTGGAGM-3'

VH 2A-Forward 5 '-AAATCGATACCACCATGGACACACTTTGCTMCAC-3'

VH 2B-Forward 5'-AAATCGATACCACCATGGACATACTTTGTTCCAAC-3'

VH 3-Forward 5'-AAATCGATACCACCATGGAGTTTGGGCTGAGC-3'

VH 3B-Forward 5 '-AAATCGATTACCCATGCATGGARYTKKGRCTBHGC-3'

VH 4-Forward 5'-AAATCGATACCACCATGAAACACCTGTGGTTCTT-3'

VH 5-Forward 5'-AAATCGATACCACCATGGGGTCAACCGCCATC-3'

VH 6-Forward 5'-AAATCGATACCACCATGTCTGTCTCCTTCCTC-3'

Cy-reverse 5'-GGGTCTAGACAGGCAGCCCAGGGCCGCTGTGC-3'

V kappa region:

vk 1-Forward 5 '-AAATCGATACCACCATGGACATGAGGGTCCCY-3'

Vk 1B-Forward 5 '-AAATCGATACCACCATGGACATGAGRGTCCYY-3'

Vk 2-Forward 5'-AAATCGATACCACCATGAGGCTCCCTGCTCAG-3'

Vk 3-Forward 5 '-AAATCGATACCACCATGGAARCCCCAGCGCA-3'

Vk 4-Forward 5'-AAATCGATACCACCATGGTGTTGCAGACCCAG-3'

Ck-reverse 5'-GATCGCGGCCGCTTATCAACACTCTCCCCTGTTGAAGCTCTT-3'

V lambda region:

Vl1aecb 5’-AAATCGATACCACCATGGCCTGGTCCCCTCTCCTCC-3’

Vl1g 5’-AAATCGATACCACCATGGCCGGCTTCCCTCTCCTCC-3’

Vl2/10 5’-AAATCGATACCACCATGGCCTGGGCTCTGCTCCTCC-3’

Vl3jpah 5’-AAATCGATACCACCATGGCCTGGACCGCTCTCCTGC-3’

Vl5/7 5’-AAATCGATACCACCATGGCCTGGACTCCTCTCCTTC-3’

Vl6/9 5’-AAATCGATACCACCATGGCCTGGGCTCCTCTCCTTC-3’

Vl3rm 5’-AAATCGATACCACCATGGCCTGGATCCCTCTCCTCC-3’

Vl3l 5’-AAATCGATACCACCATGGCCTGGACCCCTCTCTGGC-3’

Vl3e 5’-AAATCGATACCACCATGGCCTGGGCCACACTCCTGC-3’

Vl4c 5’-AAATCGATACCACCATGGCCTGGGTCTCCTTCTACC-3’

Vl8a 5’-AAATCGATACCACCATGGCCTGGATGATGCTTCTCC-3’

Cl2/7 5’-GATCGCGGCCGCTTATCAWGARCATTCTGYAGGGGCCACTG-3’

the oligonucleotides used to construct the expression vectors were:

heavy chain expression vector:

VH1-L-NheI： 5’-GCGGCTAGCCACCATGGACTGGACCTGGAGG-3’

JH4/5-XhoI： 5’-GCGCTCGAGACGGTGACCAGGGTTCCCTG-3’

CHfw-XhoI： 5’-CGCGCTCGAGTGCCTCCACCAAGGGCCCATCGGTC-3’

CHrev-NotI：5’-GATCGCGGCCGCTTATCATTTACCCGGRGACAGGGAGAGGC-3’

light chain expression vector:

VK1-L-NheI： 5’-GCGGCTAGCCACCATGGACATGAGGGTCCCY-3’

CK-NotI：5’-GATCGCGGCCGCTTATCAACACTCTCCCCTGTTGAAGCTCTT-3’

EBV RT-PCR

to test whether the potent proliferation reaction was associated with the presence of EBV, EBV RT-PCR was performed. The RT procedure is as described above. The PCR conditions were as follows: a denaturation step at 94 ℃ for 7 minutes followed by 30 cycles of: 94 ℃ for 30 seconds, 62 ℃ (HPRT1), 52 ℃ (LMP-1) and 58 ℃ (EBNA1/2) for 30 seconds, 72 ℃ for 30 seconds; finally extension was carried out at 72 ℃ for 7 minutes. The oligonucleotides used for RT-PCR were as follows: HPRT1 Forward (5 '-TATGGACAGGACTGAACGTCTTGC-3') and HPRT1 reverse (5 '-GACACAAACATGATTCAAATCCCTGA-3'); LMP-1 forward direction: (5 '-GCGACTCTGCTGGAAATGAT-3') and LMP-1 reverse (5 '-GACATGGTAATGCCTAGAAG-3'); EBNA1/2 forward (5 '-AGCAAGAAGAGGAGGTGGTAAG-3') and EBNA1/2 reverse (5 '-GGCTCAAAGTGGTCTCTAATGC-3').

In addition to RT-PCR, we also performed PCR directly on cell pellet and supernatant DNA isolated with QIAmp isolation kit (Qiagen).

Example 1

Results

B cell phenotype

The use of human memory B cells as a platform for the isolation of therapeutic drugs relies on the ability to culture and test these cells over a considerable period of time. Human B cells can be cultured and maintained under laboratory conditions, but for a time insufficient to expand, select, and clone an individual B cell line for an antigen of interest. We developed immortalization techniques based on genetic modification of human B cells. We investigated downstream targets of STAT 5. One target is Bcl-6. Bcl-6 inhibits differentiation of B cells into proliferation-arrested plasma cells. The overexpression of Bcl-6 is in balance with BLIMP1, a transcription factor that potently enhances B cell expression (via the effect of STAT3) by stimulation of B cells with IL-21. Blip 1 is essential for the induction of Ig producing cell development (CD20-CD38+) and Bcl-6 prevents this process (cells maintain CD20 expression, the so-called germinal center phenotype).

To investigate the possible time lag (skewing) of certain cell populations within the B cell region, CFSE labeling before stimulation of fresh memory and naive human B cells revealed that all cells began to divide and all B cell populations were transduced equally (fig. 2). Memory B cells transduced with Bcl-6 and cultured in the presence of IL-21 and IL-4 are shown. At 36 hours, the transduction levels of naive B cells were lower and the division rate was slower, but after further 3 days of culture, identical to memory B cells (data not shown).

Next, we demonstrated that Bcl-6, as well as Bcl-XL (an anti-apoptotic downstream target of STAT5), CD40L signaling, and IL-21, maintain the CD20+ CD38dull phenotype of human IgG memory B cells for long periods (> 3 months) (FIG. 3).

Furthermore, the phenotype of Bcl-6Bcl-XL B cells corresponds to activated B cells (see Table 1, e.g., FACS staining of 3TT + B cell clones) because of the high expression of CD80, CD86, and HLA-DR in these cells.

Three different Bcl-6Bcl-XL B cells cultured with IL-21 and CD40L signaling

Determination of clones

Dyeing results

CD 2-negative, CD 69-negative

CD 5-negative CD 70-positive

CD 7-negative CD 71-positive

CD10 positive, CD73 negative

CD20 positive CD80 positive/high

CD21 positive CD86 positive

CD22 positive CD95 positive/high

CD23 negative/5% positive CD126 negative

CD24 negative, CD132 (common gamma) positive

CD25 positive, CD138 negative/2% positive

CD27 negative/Low CD154(CD40L) 8% positive

CD28 negative ICOSL positive

Positive for CD30 (56-74%) and negative for IgM

CD38 positive/moderate IgG positive

CD40 positive HLA-DR positive (high)

CD44 positive kappa positive/negative

CD45 positive lambda positive/negative

CD45RA positive/high IL21-R positive

Antibody membrane expression

Bcl-6Bcl-XL transduced EBV negative cells remained positive for BCR expression as determined by antigen binding or kappa and lambda staining (FIGS. 3 and 4). Such cells are therefore particularly suitable for isolating and/or screening for the desired specificity after long-term culture, for example using a labelled antigen, since such cells will bind the labelled antigen via their BCR. Validation was performed by single cell sorting of PE-labeled TT-binding Bcl-6 and Bcl-XL double-transduced B cells with FACSAria. Three weeks later, single cell sorted clones were stained with the appropriate markers and TT-PE in 96-well plates and binding was determined with facscan (bd) (fig. 4). In conclusion, in situations where the presence of a B cell receptor on a B cell is desired, such as in screening assays, it is preferred to transduce B cells with Bcl-6 and Bcl-XL, rather than with EBV infection.

Cell division and growth curves

Bcl-6Bcl-XL transduced B cells divide 0.6 times per day on average. The rate of division varies with donor and culture cell density (figure 5 a). The split rate of anti-RSV clone D25 was 0.47 times daily (fig. 5 b). Cells can be cultured in less than 1 cell/96 well plate for cloning purposes.

Antibody secretion from Bcl-6Bcl-XL B cells

Bcl-6Bcl-XL transduced B cells secreted on average 1. mu.g/ml antibody, which was sufficient to supply the requirements of preclinical studies (FIG. 6). Surprisingly, the D25 anti-RSV clone produced three times more antibody than the other cell lines tested.

Determination of the EBV content

EBV RT-PCR was performed on mRNA from a Bcl-6Bcl-XL cell line cultured with IL-21 and CD40L signaling. EBV gene transcripts were not detected in cell lines obtained with this immortalization technique (data not shown).

Selection step

Due to the stability of growth and BCR expression, these cells are particularly suitable for isolating antigen-specific B cells. It gives us the opportunity to use several different selection and cloning methods. One is to increase the probability of generating multiple antigen-specific B cell clones by obtaining antigen-specific cells immediately after introduction of Bcl-6 and Bcl-XL by FACS or magnetic bead sorting of the labeled antigen of interest. Another option is to culture purified Bcl-6 Bcl-XL-transduced memory (or any other) B cells in batches at low cell density (e.g., 100 cells/well). Supernatants from these 100 cell/well cultures can be collected and tested for specificity. The 100 cell/well cultures were found to recognize antigens and were then subcloned by limiting dilution of the cultures to obtain monoclonal cell lines. Using both methods, we could isolate over 40B cell clones recognizing Tetanus Toxoid (TT). Thus, these clones were selected with FACSAria based on TT binding to BCR, or selected by screening a series of cultures by ELISA until a single anti-TT monoclonal cell line was isolated (not shown).

Selection of RSV neutralizing antibodies

Cultures of 25 100 cells/well from donor B63 completely blocked RSV infection and replication. Neutralization of one of the 100 cell/well cultures, D10, produced potent anti-RSV antibodies, which we cloned by limiting dilution culture. One of the monoclonal antibodies, D25, was used for subsequent studies. Monoclonal antibody D25 (FIG. 7) with IgG1 heavy and kappa light chains was very effective in blocking RSV infection as determined by a commercially available ELISA (Amsterdam, Sanquin, not shown) with Amsterdam, the IC of which₅₀Values of 0.5-1.5ng/ml (+ -10 pM), and IC of clinically used standard anti-RSV antibodies (palivizumab developed by Midaminini Corp.)₅₀At 0.453. mu.g/ml (3.02nM) (H.Wu et al, 2005J.mol.biol., and A.Mejias et al, 2005 antimicrob.Agents Chemother.) (FIG. 8).

Antigen recognition

In addition to the neutralization experiments, binding of D25 to HEp2 cells infected with RSV was also determined. HEp2 cells were infected using conventional virus production protocols. HEp2 cells infected with RSV were trypsinized and incubated with 25-50. mu.l culture supernatant. Cells were washed and stained with mouse-anti-human IgG-PE (BD or Jackson) to determine binding of the D25 antibody to infected cells. The r-Biopharm ELISA control antibody was used as an internal standard. Figure 9a shows binding of D25 to intact HEp2 cells infected with RSV.

Since the RSV envelope (membrane) protein includes two proteins, i.e., the G and F proteins, D25 was tested for binding to cells infected with VSV virus (provided friendly to John K Rose) that did not have a pseudotype of RSV F or RSV G protein. As shown in FIG. 9b, D25 bound strongly to EL-4 cells infected with VSV-F protein. In an attempt to investigate the epitope recognized by D25 with palivizumab, EL-4 cells infected with VSV-F protein were incubated with increasing amounts of D25 or palivizumab. Cells were washed and stained with a mixture of 3 mouse-anti-RSV-F antibodies (Dako). In contrast to palivizumab, which competes for binding of the mouse-anti-RSV-F antibody to infected VSV-F cells, D25 binding was unaffected (data not shown).

Fig. 9c shows that palivizumab (Synagis) and D25 bound to infected HEp2 cells in a concentration-dependent manner. Since both antibodies bound their target proteins 1: 1, there was no difference in binding to infected HEp2 cells.

Frequency of RSV antigen binding and neutralizing clones

We calculated that the frequency of antigen-specific memory B cells that bound RSV was 17% and the frequency of antigen-specific cells that neutralized RSV was 6% for donor B63. The conformational epitope to which D25 binds is different from the epitope recognized by palivizumab. This can be seen in figure 10, where D25 does not bind the denatured linear epitope presented by lysates of lysed RSV infected cells coated on ELISA plates, whereas palivizumab binds to denatured (F) protein.

Isolation and purification of antibody fragments

We were able to obtain volumes up to 500ml from several B cell lines, including the high RSV neutralizing clone D25. These culture supernatants contain at least 2. mu.g/ml, therefore, we should be able to obtain enough purified antibody for preclinical (animal) studies. Purification was performed using a Montage (Montage) antigen purification kit (Millipore, Billerica, MA, USA) and a HiTrap protein a HP column (GE healthcare, Diegem, Belgium, USA).

In addition, 293T cells were transfected with heavy and light chains of D25 subcloned in the pcda3.1 protein expression vector by Lipofectamine LTX (invitrogen). The IgG content of the supernatant was about 22. mu.g/ml (total volume 50 ml). The cloned D25B cell line expressed the nucleotide sequence of the antibody and produced such an antibody that also recognized infected HEp2 cells (data not shown).

Antibody sequences

FIG. 11a shows the heavy and light chain nucleotide and amino acid sequences of clone B63D 10-D25. The heavy chain (Vh1-69) and light chain (VkIO8/O18) sequences were determined using standard RT-PCR and antibody specific primers. The entire antibody sequence was cloned using a TOPO vector and, after sequence control, subcloned into the pcdna3.1 mammalian protein expression vector (invitrogen). FIGS. 11B and 11c depict the VH and VL4 chains of this clone, with asterisks indicating mutations compared to the Vh1-69 germline sequences that must occur during affinity maturation and further B-cell selection.

In summary, we show the isolation, characterization and long-term culture of human memory B cells using transgenic Bcl-6 and Bcl-XL. They are tools necessary for our isolation of antibodies with unique properties, such as the anti-RSV monoclonal antibody B63D 10-B25. Since B cells are derived from human sources, they are readily developed into therapeutic drugs.

Example 2

The D25 heavy and light chains were cloned into standard expression vectors as described previously (p44 'antibody sequence'). To generate an expression construct that maximizes protein expression, the D25 heavy and light chain sequences were codon optimized by GENEART (Regensburg, Germany). In this approach, additional restriction sites are created to simplify future cloning steps, but most importantly, nucleotide codons that are translated into amino acid sequences are optimized for maximum translation into protein. Thus, the nucleotide sequence is optimized but the amino acid sequence remains unchanged. Example 4 shows the neutralizing capacity of D25, recombinant D25, and GENEART optimized D25 derived from purified B cell supernatant. They are both effective in neutralizing RSV.

The modifications made by GENEART compared to the original D25 sequence are shown in FIG. 12.

Example 3

After in vitro RSV neutralization experiments, we tested the D25 monoclonal antibody in an in vivo model. The models that have been described for in vivo anti-RSV testing are BALB/c mice and cotton rats (Sigmodon hispidus) (Mejias A et al, Antimicrobial Agents and chemotherapy 2004; p 1811, Johnson S et al, JID 1997; p 1215 and Wu H et al, JMB 2007: p 652). It is clear that the BALB/c mouse model is the weakest model, but since cotton rats are difficult to obtain and breed, we first set up the D25 test in BALB/c mice.

The scheme is as follows: RSV-specific antibodies in BALB/c, 5 days

Experiment design:

day-1. I.P. injection of 100. mu.l antibody

I.N infection on day 0 1x10⁷pfu RSV A2(50μl)

On days 1-5, mice were checked for overall health and weighed

Day 5, autopsy, collection of BAL, blood and lungs

Blood drawing by venipuncture

2.0ml BAL was collected by trachea

Collecting lung

TCID was started immediately on BAL material (1ml)₅₀。

1ml of BAL material (ELISA cytokine/RT-PCR) was frozen at-80C

TCID was performed on the prepared lung material (1ml)₅₀

1ml of lung material (ELISA cytokine/RT-PCR) was frozen at-80C

Blood was collected/centrifuged, and serum was subjected to hIgG ELISA and stored at-80C

The results are shown in FIG. 13:

(A) RSV stimulation by nasal spray (1X 10)⁷RSV-a2 pellets) animals were IP injected with varying amounts of Synagis (midgmuini), purified D25, or IgG1 control antibody (Eureka) (table 3). (FIG. 13B) the level of human IgG in mouse serum was measured from day 5 and the antibody serum level decreased over 5 days; table 4 shows a half-life summary. Figure 13D depicts viral titers in lung lavage (BAL) in treated and untreated animals on day 5, while figure 13E depicts T and B cell numbers in peripheral blood of treated and untreated mice. Fig. 13F shows a tissue section of lung and bronchial infiltrates (usually predominantly eosinophils) in untreated and treated animals.

Conclusion/results:

the estimated D25 half-life was 5-9 days according to (linear) calculations: on day 0, 60 and 30 μ g of antibody (2 and 1mg/kg, respectively) were injected, and on day 5, 33 or 16 μ g (total volume of mice 1,5) were detected. The Ig level dropped from 15. mu.g to 11. mu.g at day 5, beginning with 0,5mg/kg per animal on day 0, indicating a half-life of 9 days (Table 4).

TABLE 4

TCID₅₀The experimentally determined virus titer showed that 1x10 could be detected in control animals⁴PFU, whereas no virus could be detected in animals treated with Synagis (2mg/kg) or D25(2, 1 and 0,5 mg/kg).

Animals treated with Synagis or D25 maintained a higher percentage of peripheral blood CD4T cells and B220B cells. Synagis (2mg/kg) treated animals had less% CD4T cells than D25 treated animals. Although this may not be significant, it is noted that animals treated with low dose D25(1 and 0,5mg/kg) maintained high levels of B and T cells compared to control treated animals.

Although the histological data (fig. 13F) were not quantitative, it can be seen that Synagis and D25 reduced the influx of immune cells into the lung and around the bronchi compared to the control. When comparing D25 with Synagis, there was less cell infiltration into the lungs and around the bronchi in animals treated with D25.

To detect D25 in cotton rats, experiments were set up in NVI (holthoven, Netherlands) to compare animals pre-treated with Synagis and D25 prior to RSV-X virus stimulation.

Example 4

With the exception of B63-D10-D25, three new potent RSV neutralizing antibodies (AM14, AM16, and AM23) were isolated from the same donor (B63). Cultures of 100 cell/well batches of B cells initially selected for neutralization based on RSV and cryopreserved in liquid nitrogen were thawed and culture supernatants were tested for binding to HEp2 cells infected with RSV. We detected binding to infected Hep2 cells because it is a marker for antibodies that recognize natural oligomeric RSV membrane proteins, such as F and G proteins, and can be used as a good predictor of neutralization. When the binding is detected, the cells are cultured in single cells, and binding is screened to obtain clones. All three antibodies were cloned into GENEART vector originally constructed for D25. In addition, antibodies such as D25 recognize RSV-F protein (not shown). After cloning and expression in 293T cells, the recombinant protein was purified (nucleotide and amino acid sequences see fig. 14A, B and C). Neutralization of antibodies to several major RSV isolates was detected on Vero and HEp2 cells (fig. 15). All three antibodies were of the IgG1 isotype. AM14 has a kappa light chain, while AM16 and AM23 have a lambda light chain. All three antibodies, such as D25, contained somatic hypermutations in their antibody variable regions, suggesting that they have undergone affinity maturation during the germinal center reaction in vivo, a process that generates unique antibody sequences.

The results are shown in FIGS. 15-I and 15-II: RS virus neutralization experiments were performed with D25(sD25), recombinant purified D25(rD25), recombinant GENEART codon optimized D25(rD25GA), AM14, AM16, AM23 (all purified recombinant proteins), and Synagis derived from purified B cell line supernatant. The antibody neutralizing capacity of the virus was tested on two different cell lines (FIG. 15-I) Vero and (FIG. 15-II) Hep2 cells with different antibodies: a2(a), x (B), and 2006/1(C) are RSV subgroup a, while viruses z (d) and 2007-2(E) are subgroup B. Will 100TCID₅₀Each virus of (4) was added to an antibody solution serially diluted with DMEM/1% FCS, incubated at 37 ℃ for 1 hour, and then 100ul of Vero or HEp2 cells (1X 10)⁶In ml). The virus antibody mixture was not washed away. After three days, the supernatant was removed and the cells were fixed with 80% acetone 10' at room temperature. After removal of acetone, the fixed cell layer was dried and either maintained at 4 ℃ or frozen at-20 ℃. To stain HEp2 cells infected with RSV, the plates were first blocked with 5% milk powder in PBS 0.1% Tween 20, then washed 3 times, followed by incubation with polyclonal goat anti-RSV-HRP (1: 500, Soxhlet biosignature, Maine, USA) at 37 ℃ for 3-5 hours and washed thoroughly. Next, all wells were incubated with AEC substrate 30' at room temperature. Infected foci stained red, and were visually observed and counted by an optical microscope.

Results/conclusions

All antibodies were able to neutralize RSV a and B strains (table 5). In general, different D25 antibodies were effective in neutralizing RSV virus, but minor inter-experimental differences were observed. AM14 was as potent as D25, while AM16 was as potent as Synagis. However, AM23 was very effective at neutralizing the RSV a strain, but it was less effective at neutralizing the RSV B strain, but still comparable to Synagis.

TABLE 5IC50 values (ng/ml)

The IC50 value for RS virus subtype a for each antibody on Vero or HEp2 cells was calculated as the mean 50% neutralization value for the three virus strains (a2, X and 2006-1). IC50 values for RS virus subtype B for each antibody on Vero or HEp2 cells were calculated as mean 50% neutralization values for both strains (2007-2 and Z). Each neutralization experiment was performed in triplicate and repeated twice (also shown in fig. 15A and B).

sD25 ═ purification of culture supernatants produced by B cells

rD25 ═ purified recombinant D25

rD25GA 293T cell supernatant and GENEART codon optimized recombinant D25

Example 5

Synergistic and blocking effects of anti-RSV antibodies

To analyze whether the D25, Synagis, or new AM antibody panels mutually interfered with recognition of the RSV F protein, we preincubated RSV-infected HEp2 cells with increasing concentrations of unlabeled antibody until they reached the maximum binding plateau. We determined a plateau phase for each antibody, where there was no further increase in binding when the Ig amount increased. (not shown). After washing, the samples were incubated with a standard dose (3pmol) of PE-labeled D25 or APC-labeled Synagis. The dose also reached maximum binding.

Results

As shown in fig. 16, when HEp2 cells infected with RSV were preincubated with unlabeled Synagis or D25, the level of binding of labeled Synagis and D25 to these cells was reduced. Moreover, Synagis slightly reduced AM 16-induced binding. D25 binding was strongly blocked by AM23, but in contrast, pre-incubation with AM14 strongly enhanced D25 binding. This indicates that the epitope recognized by D25 is not normally fully exposed, but that AM14 enhances its exposure after binding to its native epitope. This demonstrates that these two antibodies can work together and enhance neutralization.

Brief description of the drawings

FIG. 1 shows a schematic view of a

Isolation of human IgG positive memory B cells. PBMC separated from buffy coat by Ficoll density separation (Amazonia) were incubated with anti-CD 22 magnetic beads and then separated by MACS column (Mitsuba). CD22 positive cells were then incubated with antibodies to human CD19, CD27, IgM, IgD and IgA (BD). Cells that were IgM, IgD, IgA negative and CD19, CD27 positive were sorted using high speed single cell sorting technology (FACSAria, BD).

FIG. 2

CFSE staining. Fresh human memory B cells were isolated, labeled with CSFE, stimulated with IL-21 for 36 hours, and then transduced with Bcl-6-IRES-NGFR. Cells were incubated with IL-21 for an additional 3 days and then assayed for CFSE content. CFSE dye was diluted at each cell division.

FIG. 3

Examples of human B cells transduced with Bcl-6 and Bcl-XL or only Bcl-XL. Cells were maintained on irradiated L cells expressing CD40L and the cytokine IL-21. The left panel shows BCR expression as determined by κ and λ staining (93% of κ λ positive cells are IgG isotypes, not shown). The X-axis of the right panel represents CD38 expression and the Y-axis represents CD20 expression. CD38^dullCD20⁺Staining indicates memory or germinal center B cells; CD38⁺CD20^-Staining indicated plasmablasts.

FIG. 4

Isolation of immortalized antigen-specific human B cells. Human memory B cells were isolated as shown in FIG. 1 and subsequently transduced with Bcl-6-IRES-NGFR and Bcl-XL-IRES-GFP. Cells expressing NGFR, GFP and capable of binding PE-labelled tetanus toxin were isolated using FACSAria. Cells were cultured as single cells in the presence of irradiated L cells and IL-21 in 96-well flat-bottom plates, and then selected for TT-PE binding using FACS Canto (BD).

FIG. 5

Cumulative cell growth and division rates of 6XL B cell clones. B cells from (A) two anti-TT clones and (B) one anti-RSV clone (B63D10-D25) were cultured in the presence of IL-21 and irradiated L cells.

FIG. 6

Fresh cultures were grown starting with 200,000 cells/24 well plate using 1,0ml of IMDM containing 8% FCS and cyan/streptomycin. The FCS used was either normal (Haimaichong (Hyclone)) or ultra-low bovine IgGFCS (Gibbo). After 3 days, the culture supernatant was replaced to adjust the cell number to 200,000 cells/ml. Mean IgG yields over 3 days measured at3 consecutive time points are shown with no significant difference (p value of 0.2).

FIG. 7

To determine the light chain phenotype of the D25 anti-RSV clones, the D25B cell line was stained with either kappa-phycoerythrin or lambda-phycoerythrin (BD) antibodies. Only the kappa-phycoerythrin antibody bound to this cell line, indicating that the antibody has a kappa light chain.

FIG. 8

100 cells/well cultures from donor B63 were cultured with Bcl-6Bcl-XL positive human memory B cells. One of the cultures D10 showed a strong neutralization. A monoclonal cell line derived from LD was prepared, and one D25 was effective in neutralizing RSV a-2 virus. Comparison of D25 with palivizumab (synagis) and polyclonal goat anti-RSV is shown here. Irrelevant culture supernatants from Bcl6Bcl-XL transduced cultured with IL-21 and CD40L signaling, which produce high levels of antibody but do not block RSV infection, are not shown. The D25 clone was used for further characterization.

FIG. 9

In fig. 9 a: HEp2 cells were seeded with IMDM/5% FCS at a density of 10-12e6 cells/T175 flasks (Nunc). The following day, the medium was replaced with 5ml of medium containing RSV virus (1.0MOI), incubated at room temperature for 45', and then 20ml of fresh medium was added to incubate the cells at 37 ℃ o/n. On the third day, the medium was replaced with IMDM/1% FCS and the lid was closed and incubated at 37 ℃ o/n. On day four, cells were washed with PBS and trypsinized. To stain infected cells, a preliminary incubation with culture supernatant was performed. A second incubation was performed with anti-human IgG-PE (BD). Cells were analyzed with LSRII (BD). As a positive control, a positive control of a commercially available ELISA kit (r-Biopharm) was used.

In fig. 9 b: EL-4 cells were infected with VSV virus with RSV F or G protein pseudotype (kindly supplied by John Rose) and incubated with D25 culture supernatant. Cells were washed and incubated with anti-human IgG-PE (Jackson) to determine the binding of D25 to infected cells. Only binding of D25 to VSV virus infected cells with the RSV F protein pseudotype was detected.

Fig. 9c shows that palivizumab (Synagis) and D25 bound to infected HEp2 cells in a concentration-dependent manner. Mean Fluorescence Intensity (MFI) is shown.

FIG. 10 shows a schematic view of a

Polyclonal goat anti-RSV (positive control), palivizumab (synagis) and D25 binding to coated HEp2 infected cell lysates.

FIG. 11

Sequence analysis of clone D25. 11a shows the nucleotide and predicted amino acid sequences of the heavy and light chain variable regions (SEQ ID NOS: 7-10). 11b/c show the D25 heavy and light chain sequences compared to the predicted germline. Asterisks indicate mutations that may occur during selection and in vivo affinity maturation of B cell clones (SEQ ID NOS: 7-8, 55 and 57).

FIG. 12

B cell lines transduced by BCL6BCL-xL clone and express recombinant human antibodies. This process has been described in relation to the D25 antibody (FIG. 11). The GENEART nucleotide modifications compared to the original D25 sequence are shown here, taking care that these mutations do not alter the amino acid composition of the D25 antibody (SEQ ID NO: 139-142).

FIG. 13

BALB/c mice were stimulated with D25 and Synagis derived from purified B-cell supernatants. (A) Animals were IP injected with different amounts of Synagis (midgmuini), purified D25 or IgG1 control antibody (yaleka) (Eureka)) the day prior to RSV stimulation by nasal spray (1x107RSV-a2 particles) (table 3). (B) Measuring human IgG levels in mouse serum from day 5, antibody serum levels decreasing in 5 days (C); table 4 shows a half-life summary. Figure 13D depicts viral titers in lung lavage (BAL) in treated and untreated animals on day 5, while figure 13E depicts T and B cell numbers in peripheral blood of treated and untreated mice. (F) Tissue sections showing lung and bronchial infiltrates (usually predominantly eosinophils) of untreated and treated animals.

FIG. 14

Three novel potent RSV neutralizing antibodies (a) AM14, (B) AM16, and (C) the nucleotide and amino acid sequence of AM 23.

FIG. 15 shows a schematic view of a

RS virus neutralization experiments were performed with D25(sD25), recombinant purified D25(rD25), recombinant GENEART codon optimized D25(rD25GA), AM14, AM16, AM23 (all purified recombinant proteins), and Synagis derived from purified B cell line supernatant. The antibody neutralizing capacity of the virus was tested on two different cell lines (FIG. 15-I) Vero and (FIG. 15-II) Hep2 cells with different antibodies: a2(a), x (B), and 2006/1(C) are RSV subgroup a, while viruses z (d) and 2007-2(E) are subgroup B. Will 100TCID₅₀Each virus of (4) was added to an antibody solution serially diluted with DMEM/1% FCS, incubated at 37 ℃ for 1 hour, and then 100ul of Vero or HEp2 cells (1X 10)⁶/ml)。

FIG. 16

Relative binding of fixed amounts (3pmol) of APC-labeled Synagis and PE-labeled rD25 to RSV-infected HEp2 cells, HEp2 cells preincubated with increasing concentrations of the indicated unlabeled antibody.

Reference to the literature

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Boise, l.h., m.gonzalez-Garcia, c.e.postema, l.ding, t.lindsten, l.a.turka, x.mao, g.nunez and c.b.thompson (1993). "Bcl-x, Bcl-2-related genes as a major regulator of apoptotic Cell death" (Bcl-x, a Bcl-2-related genes as a dominant regulator of apoptotic Cell death). Cell 74: 597.

dadgostar, h., Zarnegar, B., Hoffmann, a., Qin, x.f., Truong, u., Rao, g., Baltimore, d. and Cheng, g. (2002). "Cooperation of multiple signaling pathways in CD40 regulated gene expression in B lymphocytes" (collaboration of multiple signaling pathways in CD40-regulated gene expression in B lymphocytes) proc.

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Mathas S, Janz M, Hummel F, Hummel M, Wollet-Wulf B, Lusatis S, Anagnostopoulos I, Lietz A, Sigvardson M, Jundt F, Johns K, BommertK, Stein H, Dorken B (2006). "HLH proteins ABF-1 and Id2 inherently inhibit transcription factor E2A from mediating reprogramming of tumor B cells in Hodgkin' S lymphoma" (inductive inactivation factor E2A by HLH proteins ABF-1 and Id2 catalytic reprogramming of neurological B cells in Hodgkin lymphoma). Nat Immunol.7, 207-.

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Claims (23)

1. An isolated, synthetic or recombinant antibody or functional part thereof capable of specifically binding to respiratory syncytial virus F protein, which comprises:

heavy chain CDR1, which is the sequence NYIIN of SEQ ID NO. 1,

heavy chain CDR2, which is the sequence GIIPVLGTVHYAPKFQG of SEQ ID NO 2,

-a heavy chain CDR3, which is the sequence ETALVVSTTYLPHYFDN of SEQ ID NO 3,

a light chain CDR1, which is sequence QASQDIVNYLN of SEQ ID NO. 4,

a light chain CDR2 which is the sequence VASNLET of SEQ ID NO. 5, and

light chain CDR3, which is the sequence QQYDNLP of SEQ ID NO: 6.

2. An isolated, synthetic or recombinant antibody or functional part thereof capable of specifically binding to respiratory syncytial virus F protein, comprising:

a heavy chain having the amino acid sequence QVQLVQSGAEVKKPGSSVMVSCQASGGPLRNYIINWLRQAPGQGPEWMGGIIPVLGTVHYAPKFQGRVTITADESTDTAYIHLISLRSEDTAMYYCATETALVVSTTYLPHYFDN WGQGTLVTVSS of SEQ ID NO. 7; and

a light chain, the amino acid sequence of said light chain being DIQMTQSPSSLSAAVGDRVTITCQASQDIVNYLNWYQQKPGKAPKLLIYVASNLETGVPSRFSGSGSGTDFSLTISSLQPEDVATYYCQQYDNLPLTFGGGTKVEIKR.

3. The antibody or functional part thereof of claim 1 or 2, wherein the functional part is selected from the group consisting of a single domain antibody, a single chain variable fragment (scFv), a Fab fragment or a F (ab')₂And (3) fragment.

4. The antibody or functional part thereof according to claim 1 or 2, wherein the antibody is a monoclonal antibody.

5. An isolated, synthetic or recombinant antibody or functional part thereof capable of specifically binding to respiratory syncytial virus F protein, comprising:

a heavy chain having the amino acid sequence QVQLVQSGAEVKKPGSSVMVSCQASGGPLRNYIINWLRQAPGQGPEWMGGIIPVLGTVHYAPKFQGRVTITADESTDTAYIHLISLRSEDTAMYYCATETALVVSTTYLPHYFDN WGQGTLVTVSS of SEQ ID NO. 7; and

and a light chain having the amino acid sequence DIQMTQSPSSLSAAVGDRVTITCQASQDIVNYLNWYQQKPGKAPKLLIYVASNLETGVPSRFSGSGSGTDFSLTISSLQPEDVATYYCQQYDNLPLTFGGGTKVEIKRTV (SEQ ID NO: 8).

6. Such asThe antibody of claim 5, or a functional portion thereof, wherein the functional portion is selected from the group consisting of a single domain antibody, a single chain variable fragment (scFv), a Fab fragment, or a F (ab')₂And (3) fragment.

7. The antibody or functional part thereof according to claim 5, wherein the antibody is a monoclonal antibody.

8. An isolated, synthetic or recombinant nucleic acid sequence encoding the antibody or functional portion thereof of claim 1 or 2.

9. The nucleic acid sequence of claim 8, comprising:

a. a heavy chain nucleotide sequence with a nucleotide sequence of SEQ ID NO 9, 139 or 140;

b. a light chain nucleotide sequence with a nucleotide sequence of SEQ ID NO 10, 141 or 142; or

c. A heavy chain nucleotide sequence with a nucleotide sequence of SEQ ID NO 9, 139 or 140 and a light chain nucleotide sequence with a nucleotide sequence of SEQ ID NO 10, 141 or 142.

10. An isolated, synthetic or recombinant nucleic acid sequence encoding the antibody or functional portion thereof of claim 5.

11. An isolated mammalian cell capable of expressing the nucleic acid sequence of claim 8.

12. An isolated mammalian cell capable of expressing the nucleic acid sequence of claim 9.

13. An isolated mammalian cell capable of expressing the nucleic acid sequence of claim 10.

14. A method of producing an antibody or functional portion thereof, the method comprising:

-culturing the cell of claim 11 in vitro; and

-obtaining the antibody or functional part thereof produced by said cell.

15. A method of producing an antibody or functional portion thereof, the method comprising:

-culturing the cell of claim 12 in vitro; and

-obtaining the antibody or functional part thereof produced by said cell.

16. A method of producing an antibody or functional portion thereof, the method comprising:

-culturing the cell of claim 13 in vitro; and

-obtaining the antibody or functional part thereof produced by said cell.

17. A medicament comprising the antibody or functional fragment thereof of claim 1 or 2, and a pharmaceutically acceptable carrier, diluent or excipient.

18. A medicament comprising the antibody or functional fragment thereof of claim 5, and a pharmaceutically acceptable carrier, diluent, or excipient.

19. Use of an antibody or functional part thereof according to claim 1 or 2 for the manufacture of a medicament for the treatment or prevention of an RSV-associated disease, or for the prevention and/or combating of RSV infection and/or adverse effects of RSV infection in a human subject.

20. Use of an antibody or functional part thereof according to claim 5 for the manufacture of a medicament for the treatment or prevention of an RSV-associated disease, or for the prevention and/or combating RSV infection and/or adverse effects of RSV infection in a human subject.

21. Use of a nucleic acid sequence according to claim 8 for the manufacture of a medicament for the treatment or prevention of an RSV-associated disease, or for the prevention and/or combating of an RSV infection and/or an adverse effect of an RSV infection in a human subject.

22. Use of a nucleic acid sequence according to claim 9 for the manufacture of a medicament for the treatment or prevention of an RSV-associated disease, or for the prevention and/or combating of an RSV infection and/or an adverse effect of an RSV infection in a human subject.

23. Use of a nucleic acid sequence according to claim 10 for the manufacture of a medicament for the treatment or prevention of an RSV-associated disease, or for the prevention and/or combating of an RSV infection and/or an adverse effect of an RSV infection in a human subject.