Rsv-specific binding molecules and means for producing them

[0001] The invention relates to the field of biology and medicine.

[0002] A respiratory syncytial virus (RSV) is a common cold virus that belongs to the paramyxovirus family. RSV is virulent, easily transmissible and is the most common cause of lower respiratory tract disease in children under the age of 2 years. Up to 98% of children attending day care places will be infected in one RSV season. Between 0.5% and 3.2% of children with RSV infection require hospitalization. Annually, 90,000 admissions to the hospital and 4,500 deaths were recorded in the United States. The main risk factors for hospitalization due to RSV infection are pre-term labor, chronic lung disease, congenital heart disease, impaired immunity and age less than 6 weeks in children who are otherwise healthy. There is no effective treatment of RSV positive bronchiolitis other than supportive care in the form of proper nutrition and oxygen therapy. Antiviral therapies such as Ribavirin have not been shown to be effective in the treatment of RSV infection. One monoclonal antibody, Palivizumab (also called Synagis), is registered for prevention against RSV infection. Palivizumab is a genetically engineered (humanized) monoclonal antibody to the RSV fusion protein. However, Palivizumab is not always effective. Accordingly, there is a need in the art for alternative antibodies and therapies against RSV. Palivizumab (also called Synagis) is registered for prevention against RSV infection. Palivizumab is a genetically engineered (humanized) monoclonal antibody to the RSV fusion protein. However, Palivizumab is not always effective. Accordingly, there is a need in the art for alternative antibodies and therapies against RSV. Palivizumab (also called Synagis) is registered for prevention against RSV infection. Palivizumab is a genetically engineered (humanized) monoclonal antibody to the RSV fusion protein. However, Palivizumab is not always effective. Accordingly, there is a need in the art for alternative antibodies and therapies against RSV.

[0003] It is an object of the present invention to provide means and methods for combating and / or preventing an RSV related disease. It is a further object of the invention to provide alternative and / or improved anti-RSV antibodies and to provide stable cells capable of producing anti-RSV antibodies.

[0004] Thus, the present invention provides isolated antibodies according to claims 1-3, isolated nucleic acid sequences according to claims 4-5, a cell according to claim 6, a method according to claim 7, a composition according to claim 8 and antibodies, nucleic acids and compositions according to claims 9-11, for use according to claim 9-11.

[0005] The present invention provides antibodies that are capable of specific RSV binding. Such antibodies, also referred to herein as "anti-RSV antibodies" or "RSV specific antibodies", are capable of specifically binding at least one RSV component, such as, for example, an RSV protein epitope. Non-specific adhesion is not covered by the term "specific binding". The anti-RSV antibodies of the present invention are particularly suitable for counteracting and / or at least partially preventing RSV infection and / or side effects of RSV infection. One particularly preferred anti-RSV antibody of the present invention is an antibody labeled & quot; D25 & quot; that has a heavy chain region and a light chain region such as shown in Figures 11A-D. sequences

-2CDR D25, which in particular contribute to the antigen-binding properties of D25, are depicted in Figure HD. The D25 antibody appears to have superior properties compared to the Palivizumab anti-RSV antibody (Figure 8). For example, D25 has an IC50 value of about 0.4-1.5 ng / ml in an in vitro neutralization test, where HEp-2 cells are infected with RSV, whereas Palivizumab has an IC50 value of about 453 ng / ml.

[0006] A functional equivalent of an antibody is defined herein as a functional part, derivative or analog of an antibody.

[0007] A functional part of an antibody is defined as a part that has at least one property the same as that of an antibody in terms of its type, not necessarily in quantity. Said functional part is capable of binding to the same antigen as said antibody, although not necessarily to the same extent. The functional part of the antibody preferably includes a single domain antibody, single chain antibody, single chain variable fragment (scFv), Fab fragment or F (ab ') 2 fragment.

A functional derivative of the antibody is defined as an antibody that has been altered such that at least one property - preferably the antigen binding property of the resulting compound is the same in type, not necessarily in quantity. The derivative is provided in a number of ways, e.g. by substituting a conserved amino acid, thereby substituting the amino acid residue with another residue having typically similar properties (size, hydrophobicity, etc.) such that overall functioning is likely not to be seriously compromised.

[0009] A person skilled in the art is able to generate analogous compounds of the antibody. This is for example done by screening a peptide library or a phage display library. Such an analog has essentially at least one same property as said antibody in terms of type, not necessarily in amount.

[0010] As is well known to one skilled in the art, the heavy chain of an antibody is the larger of the two types of chains that form an immunoglobulin molecule. The heavy chain contains constant domains and a variable domain, wherein the variable domain participates in antigen binding. The light chain of the antibody is the smaller of the two types of chains that form the immunoglobulin molecule. The light chain contains a constant domain and a variable domain. The variable domain is, together with the heavy chain variable domain, involved in the binding of the antigen.

[0011] Complementarity Determining Regions (CDRs) are hypervariable regions present in the heavy chain variable domains and light chain variable domains. The CDRs of the heavy chain and the attached antibody light chain together form the antigen binding site.

[0012] Now that the present invention provides the observation that the CDR sequences shown in Figure 11 provide the desired RSV binding properties, the variants may comprise at least one altered CDR sequence. For example, a conservative amino acid substitution is used. A conservative amino acid substitution involves the substitution of one amino acid with another with generally similar properties

-3 (size, hydrophobicity, etc.), so that overall functioning is not likely to be severely affected.

[0013] It is also possible to change at least one CDR sequence shown in Figure 11 to generate a variant antibody, or its functional equivalent, with at least one property changed as compared to D25. An antibody or functional equivalent may comprise a CDR sequence that is at least 70% identical to the CDR sequence as shown in Figure 11, such that the preferred binding characteristics of D25 are at least partially maintained or even improved. The CDR sequence as shown in Figure 11 can be varied so that the resulting antibody or its functional equivalent contains at least one improved property such as, for example, improved binding affinity, selectivity and / or stability, as compared to D25. Various methods for altering the amino acid sequence are available in the art. For example, the heavy chain or light chain sequence is artificially synthesized with the desired CDR sequence. Preferably, the nucleic acid sequence encoding the CDR sequence is mutated, for example using random - or targeted - mutagenesis.

[0014] Herein disclosed is an isolated, synthetic or recombinant antibody or a functional equivalent thereof that is capable of specifically binding a Syncytial Respiratory Virus and which comprises:

- a heavy chain CDRI sequence comprising a sequence that is at least in

70% identical to the NYIIN sequence, and / or

- a heavy chain CDR2 sequence comprising a sequence that is at least in

75% identical to the GIIPVLGTVHYAPKFQG sequence, and / or

- a heavy chain CDR3 sequence comprising a sequence that is at least in

70% identical to the sequence ETAL WSTTYLPH YFD N, and / or

- a light chain CDRI sequence comprising a sequence that is at least w

85% identical to the QASQDIVNYLN sequence, and / or - a light chain CDR2 sequence comprising a sequence that is at least 70% identical to the VASNLET sequence.

[0015] Said antibody also comprises a light chain CDR3 sequence comprising a sequence that is at least 70% identical to the QQYDNLP sequence.

[0016] The antibody or functional equivalent may comprise a CDR sequence that is at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% identical to at least one of the sequences The CDRs depicted in Figure HD. Most preferably, the antibody or functional equivalent may comprise a CDR sequence that is at least 95% identical to at least one of the CDR sequences shown in Figure HD. A particularly preferred D25 antibody, described above, contains CDR sequences that consist of the CDR sequences shown in Figure HD. Particularly beneficial

The embodiment of the invention thereby provides an isolated, synthetic or recombinant antibody or a functional equivalent thereof that is capable of specifically binding the Syncytial Respiratory Virus and which comprises:

- a heavy chain CDRI sequence containing the NYIIN sequence,

- a heavy chain CDR2 sequence comprising a sequence

GIIPVLGTVHYAPKFQG, - heavy chain CDR3 sequence containing the ETALWSTTYLPHYFDN sequence,

- a light chain CDRI sequence containing the QAS QD IVNYLN sequence,

- a light chain CDR2 sequence comprising the VASNLET sequence and a light chain CDR3 sequence comprising the QQYDNLP sequence.

[0017] An antibody is disclosed herein or a functional equivalent is provided that comprises three heavy chain CDRs and three light chain CDRs as shown in Figure HD, or sequences that are at least 70%, preferably at least 80%, more preferably at least 85% identical to them. In addition, an isolated, synthetic or recombinant antibody or functional equivalent thereof is disclosed that comprises a heavy chain CDR1 sequence comprising a sequence that is at least 70% identical to the NYIIN sequence and a heavy chain CDR2 sequence comprising a sequence that is at least about 70% identical with the GIIPVLGTVHYAPKFQG sequence and the heavy chain CDR3 sequence containing the sequence, which is at least 70% identical to the ETALWSTTYLPHYFDN sequence and a light chain CDRI sequence comprising a sequence that is at least 70% identical to the QASQDIVNYLN sequence and a light chain CDR2 sequence comprising a sequence that is at least 70% identical to the VASNLET sequence and a light chain CDR3 sequence comprising a sequence that is at least 70% identical to the QQYDNLP sequence. Said antibody or functional equivalent preferably comprises CDR sequences that are at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% identical. with heavy chain CDR sequences and light chain CDR sequences as shown in Figure HD.

[0018] Antibodies or functional equivalents thereof comprising a variable heavy chain amino acid sequence that is at least 70% identical to the heavy chain sequence as shown in Figure 11 are also disclosed. Such heavy chain sequences provide desirable RSV binding properties, as evidenced by antibody D25. Furthermore, an antibody or a functional equivalent thereof is disclosed, having a heavy chain sequence comprising a sequence that is at least 70% identical to the sequence QVQLVQSGAEVKKPGSSVMVSCQASGGPLRNYIINWLRQAPGQGPEWMGGII

-5PVLGTVHYAPKFQGRVTITADESTDTAYIHLISLRSEDTAMYYCATETALWST

TYLPHYFDNWGQGTLVTVSS. In addition, variable light chain amino acid sequences that are at least 70% identical to the light chain sequences as shown in Figure 11 also provide desirable RSV binding properties, as evidenced by antibody D25. An antibody, or a functional equivalent thereof having a light chain sequence that is at least 70% identical to the sequence of DIQMTQSPSSLSAAVGDRVTITCQASQDIVNYLNWYQQKPGKAPKLLIYVASN

LETGVPSRFSGSGSGTDFSLTISSLQPEDVATYYCQQYDNLPLTFGGGTKVEIK RTV is therefore also disclosed. An antibody or functional part may comprise a heavy chain variable sequence and / or a light chain variable sequence that 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 shown in Figure 11. The higher the homology, the more specifically said antibody or functional portion resembles antibody D25. An antibody or functional part may comprise a heavy chain as well as a light chain that resemble the heavy and light chain of D25.

[0019] One embodiment provides an antibody comprising a heavy chain sequence consisting of a heavy chain sequence as shown in Figure 11, and a light chain sequence consisting of a light chain sequence as shown in Figure 11. Alternatively, as is well known to one skilled in the art, it is possible is to generate a truncated heavy chain or light chain sequence while retaining the ability of the binding property of interest. Preferably, such a truncated heavy chain or light chain is generated that has a shorter constant region compared to the original heavy or light chain. The variable domain is preferably maintained. For example, a Fab fragment or a F fragment (ab ') is produced

-6 shown in Figure HD. Preferably, said functional part may also comprise a sequence that is at least 70% identical to the light chain CDR3 sequence depicted in Figure HD. [0020] Another particularly preferred anti-RSV antibody of the present invention is the antibody designated "AM 14" which has a heavy chain region and a light chain region such as shown in Figure 14A. The AM14 CDR sequences that specifically contribute to AM14 antigen binding properties are also shown in Figure 14A.

[0021] Now that the present invention provides the observation that the CDR sequences shown in Figure 14A provide desirable RSV binding properties, the variants may comprise at least one altered CDR sequence. For example, a conservative amino acid substitution is used. A conservative amino acid substitution involves the substitution of one amino acid with another with generally similar properties (size, hydrophobicity, etc.), so that overall functioning is unlikely to be seriously compromised. It is also possible to change at least one CDR sequence shown in Figure 14A to generate a variant antibody, or a functional equivalent thereof, with at least one property changed compared to AM14. An antibody or functional equivalent may comprise a CDR sequence, which is at least 70% identical to the CDR sequence as shown in Figure 14A, such that the preferred binding characteristics of AM14 are at least partially maintained or even improved. The CDR sequence as shown in Figure 14A can be varied so that the resulting antibody or its functional equivalent contains at least one improved property such as, for example, improved binding affinity, selectivity and / or stability, as compared to AM14.

[0022] Various methods for altering the amino acid sequence are available in the art. For example, the heavy chain or light chain sequence is artificially synthesized with the desired CDR sequence. Preferably, the nucleic acid sequence encoding the CDR sequence is mutated, for example using random - or targeted - mutagenesis.

[0023] An isolated, synthetic or recombinant antibody or functional part thereof, derivative and / or analogue thereof that is capable of specifically binding a Syncytial Respiratory Virus is disclosed herein and which comprises:

- a heavy chain CDRI sequence comprising a sequence that is at least in

70% identical to the GFSFSHYA sequence, and / or

- a heavy chain CDR2 sequence comprising a sequence that is at least in

70% identical to the ISYDGENT sequence, and / or

- a heavy chain CDR3 sequence comprising a sequence that is at least in

70% identical to the sequence ARDRIVDDYYYYGMDV, and / or

- a light chain CDRI sequence comprising a sequence that is at least w

70% identical to the QDIKKY sequence, and / or - the light chain CDR2 sequence

Containing a sequence that is at least 70% identical to the DAS sequence and / or

- a light chain CDR3 sequence comprising a sequence that is at least 70% identical to the sequence of QQYD NLPPLT.

[0024] The antibody or functional equivalent may comprise a CDR sequence that is at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% identical to at least one of the sequences The CDRs depicted in Figure 14A. Most preferably, the antibody or functional equivalent may comprise a CDR sequence that is at least 95% identical to at least one of the CDR sequences shown in Figure 14A. A particularly preferred AM14 antibody, described above, contains CDR sequences that consist of the CDR sequences shown in Figure 14A. A particularly preferred embodiment of the invention thus provides an isolated, synthetic or recombinant antibody,

- a heavy chain CDRI sequence containing the GFSFSHYA sequence,

- a heavy chain CDR2 sequence containing the ISYDGENT sequence,

- a heavy chain CDR3 sequence comprising a sequence

ARDRIVDDYYYYGMDV,

- a light chain CDRI sequence containing the QDIKKY sequence,

- a light chain CDR2 sequence comprising the sequence DAS and

- a light chain CDR3 sequence comprising the QQYD sequence of NLPPLT.

[0025] An antibody or a functional equivalent is disclosed that comprises three heavy chain CDRs and three light chain CDRs as shown in Figure 14A, or sequences that are at least 70% identical to them. Further disclosed is an isolated, synthetic or recombinant antibody or a functional equivalent thereof that comprises a heavy chain CDRI sequence comprising a sequence that is at least 70% identical to the GFSFSHYA sequence and a heavy chain CDR2 sequence comprising a sequence that is at least 70% identical to the ISYDGENT sequence and heavy chain CDR3 sequence comprising a sequence that is at least 70% identical to the ARDRIVDDYYYYGMDV sequence and a light chain CDRI sequence comprising the sequence, which is at least 70% identical to the QDIKKY sequence and a light chain CDR2 sequence comprising a sequence that is at least 70% identical to the DAS sequence and a light chain CDR3 sequence comprising a sequence that is at least 70% identical to the QQYDNLPPLT sequence. Said antibody or functional equivalent preferably comprises CDR sequences that are at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% identical. with CDR sequences

Heavy chain and CDR sequences of the light chain as shown in Figure 14A. An antibody or functional equivalent comprising the aforementioned heavy chain CDRI, CDR2 and CDR3 sequences of Figure 14A as well as the aforementioned CDRI, CDR2 and CDR3 sequences of the light chain of Figure 14A are also disclosed.

[0026] Antibodies or functional equivalents thereof comprising a heavy chain amino acid sequence that is at least 70% identical to the heavy chain sequence as shown in Figure 14A are also disclosed. Such heavy chain sequences provide desirable RSV binding properties, as evidenced by the AM14 antibody. Further disclosed is an antibody or a functional equivalent thereof having a heavy chain sequence comprising a sequence that is at least 70% identical to the sequence EVQLVESGGGWQPGRSLRLSCAASGFSFSHYAMHWVRQAPGKGLEWVAVIS

YDGENTYYADSVKGRFSISRDNSKNTVSLQMNSLRPEDTALYYCARDRIVDD

YYYYGMDVWGQGATVTVSS. In addition, light chain amino acid sequences that are at least 70% identical to the light chain sequence as shown in Figure 14A also provide desirable RSV binding properties, as evidenced by AM14 antibody. An antibody, or a functional equivalent thereof having a light chain sequence that is at least 70% identical to the sequence DIQMTQS PS SLSASVGDRVT and TCQASQDIKKYLNWYHQKPGKVPELLMHDASNLETGVPSRF SGRGSGTDFTLTI SSLQPEDI GTYYCQQYDNLPPLTFGGGTKVEIKRTV, is therefore also disclosed. An antibody or functional part may preferably comprise a heavy chain variable sequence and / or a light chain variable sequence that 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 light chain sequence as shown in Figure 14A. The higher the homology, the more specifically said antibody or functional part is reminiscent of the AM14 antibody. An antibody or functional part may suitably include a heavy chain as well as a light chain that resemble the heavy and light chain AM14. In addition, an antibody or functional part comprising a heavy chain and light chain sequence is disclosed, 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 light chain sequence as shown in Figure 14A.

[0027] One embodiment provides an antibody comprising a heavy chain sequence consisting of a heavy chain sequence as shown in Figure 14A and a light chain sequence consisting of the light chain sequence as shown in Figure 14A. Alternatively, as is well known to one skilled in the art, it is possible to generate a truncated heavy chain or light chain sequence while maintaining the ability of the binding property of interest. Preferably, such a truncated heavy chain or light chain is generated that is shorter

-9region constant, compared to the original heavy or light chain. The variable domain is preferably maintained. For example, a Fab fragment or an F (ab ') 2 fragment based on the heavy chain sequence or light chain sequence shown in Figure 14A is produced. Accordingly, there is also disclosed a functional antibody equivalent comprising at least a functional part of the sequence as shown in Figure 14A. The functional part may be at least 20 amino acids long and may comprise a sequence that is at least 70% identical to at least one of the CDR sequences shown in Figure 14A.

Another particularly preferred anti-RSV antibody of the present invention is an antibody designated & quot; AM16 & quot; that has a heavy chain region and a light chain region as shown in Figure 14B. The AM16 CDR sequences that specifically contribute to the AM16 antigen binding properties are also shown in Figure 14B.

[0029] Now that the present invention provides the observation that the CDR sequences shown in Figure 14B provide desirable RSV binding properties, the variants may comprise at least one altered CDR sequence. For example, a conservative amino acid substitution is used. A conservative amino acid substitution involves the substitution of one amino acid with another with generally similar properties (size, hydrophobicity, etc.), so that overall functioning is unlikely to be seriously compromised.

[0030] It is also possible to change at least one CDR sequence shown in Figure 14B to generate a variant antibody, or a functional equivalent thereof, with at least one property changed as compared to AM16. An antibody or functional equivalent may comprise a CDR sequence that is at least 70% identical to the CDR sequence as shown in Figure 14B, such that the preferred binding characteristics of AM16 are at least partially maintained or even improved. The CDR sequence as shown in Figure 14B may be varied such that the resulting antibody or functional equivalent thereof contains at least one improved property such as, for example, improved binding affinity, selectivity and / or stability, as compared to AM16. Various methods for altering the amino acid sequence are available in the art. For example, the heavy chain or light chain sequence is artificially synthesized with the desired CDR sequence. Preferably, the nucleic acid sequence encoding the CDR sequence is mutated, for example using random - or targeted - mutagenesis.

[0031] An isolated, synthetic or recombinant antibody or functional part thereof, derivative and / or analogue thereof that is capable of specifically binding a Syncytial Respiratory Virus is disclosed herein and which comprises: a heavy chain CDRI sequence comprising a sequence that is at least 70 % identical to the GFTFSSYN sequence, and / or

A heavy chain CDR2 sequence comprising a sequence that is at least in

70% identical to the ISAGSSYI sequence, and / or

- a heavy chain CDR3 sequence comprising a sequence that is at least in

70% identical to the sequence AREDYGPGNYYSPNWFDP, and / or

- a light chain CDRI sequence comprising a sequence that is at least w

70% identical to the sequence SSNIGAGYD, and / or

- a light chain CDR2 sequence comprising a sequence that is at least 70% identical to the GNT sequence, and / or - a light chain CDR3 sequence comprising a sequence that is at least 70% identical to the HSYDRSLSG sequence.

[0032] The antibody or functional equivalent may comprise a CDR sequence that is at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% identical to at least one of the sequences The CDRs shown in Figure 14B. Most preferably, the antibody or functional equivalent may comprise a CDR sequence that is at least 95% identical to at least one of the CDR sequences shown in Figure 14B. A particularly preferred AM16 antibody, described above, contains CDR sequences that consist of the CDR sequences shown in Figure 14B. A particularly preferred embodiment of the invention thus provides an isolated, synthetic or recombinant antibody,

- a heavy chain CDR2 sequence containing the ISAGSSYI sequence,

- a heavy chain CDR3 sequence comprising a sequence

AREDYGPGNYYSPNWFDP,

- a light chain CDRI sequence containing the sequence SSNIGAGYD,

- a light chain CDR2 sequence comprising the sequence GNT and

- a light chain CDR3 sequence comprising the HSYDRSLSG sequence.

[0033] An antibody or a functional equivalent is disclosed that comprises three heavy chain CDR sequences and three light chain CDRs as shown in Figure 14B, or sequences that are at least 70% identical to them. Further disclosed is an isolated, synthetic or recombinant antibody or a functional equivalent thereof that comprises a heavy chain CDRI sequence comprising a sequence that is at least 70% identical to the GFTFSSYN sequence and a heavy chain CDR2 sequence comprising a sequence that is at least 70% identical to the ISAGSSYI sequence and heavy chain CDR3 sequence comprising a sequence that is at least 70% identical to the AREDYGPGNYYSPNWFDP sequence and a light chain CDRI sequence containing the sequence,

A light chain comprising a sequence that is at least 70% identical to the GNT sequence and a light chain CDR3 sequence comprising a sequence that is at least 70% identical to the HSYDRSLSG sequence. Said antibody or functional equivalent preferably comprises CDR sequences that are 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 aforementioned heavy chain CDR sequences and the aforementioned light chain CDR sequences as shown in Figure 14B. An antibody or functional equivalent comprising the aforementioned heavy chain CDRI, CDR2 and CDR3 sequences of Figure 14B as well as the aforementioned CDRI sequences,

[0034] Antibodies or functional equivalents thereof comprising a heavy chain amino acid sequence that is at least 70% identical to the heavy chain sequence as shown in Figure 14B are also disclosed. Such heavy chain sequences provide desirable RSV binding properties, as evidenced by the AM16 antibody. Further disclosed is an antibody or a functional equivalent thereof having a heavy chain sequence comprising a sequence that is at least 70% identical to the sequence EVQLVETGGGLAQPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSHISAGSS YIYYSD

SVKGRFTVSRDNVRNSVYLQMNSLRAADTAVYYCAREDYGPGNYYSPNWFDPWGQ GTLVTVS S. In addition, light chain amino acid sequences that are at least 70% identical to the light chain sequence as shown in Figure 14B also provide desirable RSV binding properties, as evidenced by AM16 antibody. An antibody, or a functional equivalent thereof having a light chain sequence that is at least 70% identical to the sequence QSWTQPPSVSGAPGQRVT and SCTGS SSNI GAGYDVHWYQQLPGTAPKLLIYGNTNRPSGVS D RFSGSKSGTSASLAI TGLQAEDEADYYCHSYDRSLSGSVFGGGTKLTV, is therefore also disclosed. The antibody or functional part may comprise a heavy chain variable sequence and / or a light chain variable sequence that 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 light chain sequence as shown in Figure 14B. The higher the homology, the more specifically said antibody or functional part is reminiscent of the AM16 antibody. An antibody or functional part may comprise a heavy chain as well as a light chain that resemble the heavy and light chain AM16. In addition, an antibody or functional part comprising a heavy chain and light chain sequence is disclosed, which are at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90 %

[0035] One embodiment provides an antibody comprising a heavy chain sequence consisting of a heavy chain sequence as shown in Figure 14B and a light chain sequence consisting of the light chain sequence as shown in Figure 14B. Alternatively, as is well known to one skilled in the art, it is possible to generate a truncated heavy chain or light chain sequence while maintaining the ability of the binding property of interest. Preferably, such a truncated heavy chain or light chain is generated that has a shorter constant region compared to the original heavy or light chain. The variable domain is preferably maintained. For example, a Fab fragment or a F fragment (ab ') is produced ) 2 based on the sequence of the heavy chain or light chain sequences shown in Figure 14B. Accordingly, there is also disclosed a functional antibody equivalent comprising at least a functional part of the sequence as shown in Figure 14B. The functional portion may be at least 20 amino acids long and may comprise a sequence that is at least 70% identical to at least one of the CDR sequences shown in Figure 14B.

[0036] Another particularly preferred anti-RSV antibody of the present invention is an antibody designated & quot; AM23 & quot; that has a heavy chain region and a light chain region as shown in Figure 14C. The AM23 CDR sequences that specifically contribute to AM23 antigen binding properties are also shown in Figure 14C.

[0037] Now that the present invention provides the observation that the CDR sequences shown in Figure 14C provide desirable RSV binding properties, the variants may comprise at least one altered CDR sequence. For example, a conservative amino acid substitution is used. A conservative amino acid substitution involves the substitution of one amino acid with another with generally similar properties (size, hydrophobicity, etc.), so that overall functioning is unlikely to be seriously compromised.

[0038] It is also possible to change at least one CDR sequence shown in Figure 14C to generate a variant antibody, or a functional equivalent thereof, with at least one property changed as compared to AM23. An antibody or functional equivalent may comprise a CDR sequence that is at least 70% identical to the CDR sequence as shown in Figure 14C, such that the preferred binding characteristics of AM23 are at least partially maintained or even improved. The CDR sequence as shown in Figure 14C can be changed such that the resulting antibody or its functional equivalent contains at least one improved property such as, for example, improved binding affinity, selectivity and / or stability, as compared to AM23. Various methods for altering the amino acid sequence are available in the art. For example, the heavy chain or light chain sequence is artificially synthesized with the desired CDR sequence. Preferably, the nucleic acid sequence encoding the CDR sequence is mutated, for example using random - or targeted - mutagenesis.

[0039] An isolated, synthetic or recombinant antibody or functional part thereof, derivative and / or analogue that is capable of specific binding is disclosed herein.

Syncytial Respiratory Virus and which contains:

- a heavy chain CDRI sequence comprising a sequence that is at least in

70% identical to the GFNFHNYG sequence, and / or

- a heavy chain CDR2 sequence comprising a sequence that is at least in

70% identical to the sequence VWYDGSKK, and / or

- a heavy chain CDR3 sequence comprising a sequence that is at least in

70% identical to the VRD KVGPTPYFD S sequence, and / or

- a light chain CDRI sequence comprising a sequence that is at least w

70% identical to the NIGSET sequence, and / or

- a light chain CDR2 sequence comprising a sequence that is at least 70% identical to the sequence of DDD, and / or - a light chain CDR3 sequence comprising a sequence that is at least 70% identical to the sequence of QVWDRSNYH QV.

[0040] The antibody or functional equivalent may comprise a CDR sequence that is at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% identical to at least one of the sequences The CDRs shown in Figure 14C. Most preferably, the antibody or functional equivalent may comprise a CDR sequence that is at least 95% identical to at least one of the CDR sequences shown in Figure 14C. A particularly preferred AM23 antibody, described above, contains CDR sequences that consist of the CDR sequences shown in Figure 14C. A particularly preferred embodiment of the invention thus provides an isolated, synthetic or recombinant antibody,

- a heavy chain CDR1 sequence comprising the GFNFHNYG sequence,

- a heavy chain CDR2 sequence comprising the VWYDGSKK sequence,

- a heavy chain CDR3 sequence comprising the VRDKVGPTPYFDS sequence,

- a light chain CDR1 sequence containing the NIGSET sequence,

- a light chain CDR2 sequence comprising the sequence DDD and

- a light chain CDR3 sequence comprising the QVWDRSNYH QV sequence.

[0041] An antibody or a functional equivalent is disclosed herein that comprises three heavy chain CDRs and three light chain CDRs as shown in Figure 14C. Furthermore, an isolated, synthetic or recombinant antibody or functional equivalent thereof is disclosed, which comprises a heavy chain CDRI sequence comprising a sequence that is at least in

-1470% identical to the GFNFHNYG sequence and a heavy chain CDR2 sequence comprising a sequence that is at least 70% identical to the VWYDGSKK sequence and a heavy chain CDR3 sequence comprising a sequence that is at least 70% identical to the VRDKVGPTPYFDS sequence and the light chain CDRI sequence comprising a sequence that is at least 70% identical to the NIGSET sequence and a light chain CDR2 sequence comprising a sequence that is at least 70% identical to the DDD sequence, and a light chain CDR3 sequence comprising a sequence that is at least about 70% identical with the sequence QVWDRSNYH QV. Said antibody or functional equivalent preferably comprises CDR sequences that are 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 aforementioned heavy chain CDR sequences and the above light chain CDR sequences as shown in Figure 14C. An antibody or functional equivalent comprising the aforementioned heavy chain CDRI, CDR2 and CDR3 sequences of Figure 14C as well as the aforementioned CDRI, CDR2 and CDR3 sequences of the light chain of Figure 14C are also disclosed.

[0042] Antibodies or functional equivalents thereof comprising a heavy chain amino acid sequence that is at least 70% identical to the heavy chain sequence as shown in Figure 14C are also disclosed. Such heavy chain sequences provide desirable RSV binding properties, as evidenced by the AM23 antibody. Further disclosed is an antibody or a functional equivalent thereof having a heavy chain sequence comprising a sequence which is at least 70% identical to the sequence EVQLVESGGNVVKPGTSLRLSCAATGFNFHNYGMNWVRQAPGKGLEWVAWWYDG SKKYYAD SVTGRFAI SRDNSKNTLYLQMNSLRVEDTAVYYCVRDKVGPTPYFDSWGQGTLVTVS S. In addition, light chain amino acid sequences that are at least 70% identical to the sequence chain light as depicted in Figure 14C also provide desirable RSV binding properties, as evidenced by the AM23 antibody. An antibody, or a functional equivalent thereof having a light chain sequence that is at least 70% identical to the sequence SYVLTQPPSVSLAPGGTAAI TCGRNNIGSETVHWYQQKPGQAPVLWYDDDDRPSGI PERFS GSNSGNTATLT AND SRVEAGDEADYYCQVWDRSNYHQVFGGGTKLTV, is therefore also disclosed. An antibody or functional part may comprise a heavy chain variable sequence and / or a light chain variable sequence that 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 shown in Figure 14C. The higher the homology, the more specifically said antibody or functional part is reminiscent of the AM23 antibody. An antibody or functional part may comprise a heavy chain as well as a light chain that resemble the heavy and light chain AM23. In addition, an antibody or functional part containing a chain sequence is therefore disclosed

And a light chain sequence that are at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least in

90%, most preferably at least 95% identical to the heavy chain sequence and light chain sequence as shown in Figure 14C.

[0043] One embodiment provides an antibody comprising a heavy chain sequence consisting of the heavy chain sequence as shown in Figure 14C and a light chain sequence consisting of the light chain sequence as shown in Figure 14C. Alternatively, as is well known to one skilled in the art, it is possible to generate a truncated heavy chain or light chain sequence while maintaining the ability of the binding property of interest. Preferably, such a truncated heavy chain or light chain is generated that has a shorter constant region compared to the original heavy or light chain. The variable domain is preferably maintained. For example, a Fab fragment or a F fragment (ab ') is produced ) 2 based on the sequence of the heavy chain or light chain sequences shown in Figure 14C. Accordingly, a functional equivalent of an antibody comprising at least a functional part of the sequence as shown in Figure 14C is also disclosed. The functional portion may be at least 20 amino acids long and may comprise a sequence that is at least 70% identical to at least one of the CDR sequences shown in Figure 14C.

[0044] The present invention provides RSV specific antibodies as defined in the claims with improved properties compared to antibodies of the prior art. The creators have managed to produce antibodies specific for RSV with low IC50 values. Such antibodies have particularly high or strong affinity for RSV and are therefore particularly suitable for counteracting and / or at least partially preventing RSV infection and / or side effects of RSV infection. One disclosure provides an antibody that has an IC5Q value of less than 10 ng / ml in an in vitro neutralization assay, wherein HEp-2 cells are infected with RSV, and a functional equivalent of said antibody. Said antibody or functional equivalent preferably has an IC 50 value of less than 5 ng / ml, more preferably less than 2 ng / ml.

[0045] The antibody of the present invention is preferably a human antibody. The use of human antibodies for human therapy reduces the risk of side effects due to an immune response in a human subject against non-human sequences. In another preferred embodiment, the antibody or functional part, derivative or analog of the invention is a chimeric antibody. In this way, sequences of interest such as, for example, a binding site of interest can be included in the antibody or functional equivalent of the invention.

[0046] The invention further provides an isolated, synthetic or recombinant nucleic acid sequence encoding an antibody of the invention. Such an acid

The nucleic acid is, for example, isolated from a B cell that is capable of producing the antibody of the invention as described in more detail below. The nucleic acid sequence disclosed herein may comprise a sequence that is at least 70% homologous to at least one portion of a functional nucleic acid sequence as shown in Figure 11, Figure 12, Figure 14A, Figure 14B and / or Figure 14B. Said nucleic acid sequence may comprise a sequence that 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 each other. least one portion of a functional nucleic acid sequence as shown in Figure 11, Figure 12, Figure 14A, Figure 14B and / or Figure 14B. Said functional part has a length of at least 30 nucleotides, preferably at least 50 nucleotides, more preferably at least 75 nucleotides. Said functional part may encode at least one nucleic acid sequence as shown in Figure HD, Figure 12, Figure 14A, Figure 14B and / or Figure 14B. Said sequence is preferably a CDR sequence.

[0047] The antibody of the invention is particularly suitable for use as a medicament or prophylactic agent. An antibody of the invention, for use as a medicament and / or prophylactic agent, is therefore also provided herein. In an especially preferred embodiment, said antibody comprises antibody D25, AM14, AM16 and / or AM23. Said medicament or prophylactic agent is preferably used to prevent or at least partially prevent RSV infection or to counter or at least partially prevent side effects of RSV infections. The use of antibody, functional part, a derivative or analog of the invention for the preparation of a medicament and / or prophylactic agent for at least partially treating and / or preventing an RSV related disorder therefore is also disclosed, as well as a method for at least partially treating or preventing an RSV related disorder, wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of an antibody or functional equivalent of the invention. Said antibody preferably comprises antibody D25, AM14, AM16 and / or AM23.

[0048] In order to counteract RSV, an antibody of the invention may be administered to a subject before RSV infection has taken place. Alternatively, the antibody of the invention may be administered when the subject is already infected by RSV. Said antibody may be administered to individuals at an increased risk for RSV-related disorders such as, for example, premature delivery children, individuals with chronic lung disease, congenital heart disease and / or immunodeficiency, and children younger than 6 weeks of age. Elderly people also have an increased risk of RSV-related disorders. Antibodies of the invention may be administered orally or by one or more injections. Dose ranges of antibodies of the invention for use in therapeutic applications as described herein above, are designed based on studies of increasing doses in the clinic in clinical trials for which stringent protocol requirements exist. Typical doses are

Between 0.1 and 10 mg per kg body weight. For therapeutic use, the antibodies of the invention are typically combined with a pharmaceutically acceptable carrier, adjuvant, diluent and / or excipient. Examples of suitable carriers include, for example, keyhole limpet hemocyanin (KLH), serum albumin (e.g., BSA or RSA) and ovalbumin. Many suitable oil-based and water-based adjuvants are known to those skilled in the art. In one embodiment, said adjuvant includes Specol. In another embodiment, said suitable carrier comprises a solution, such as, for example, physiological saline.

[0049] In yet another embodiment, a nucleic acid coding for the antibody of the invention is used. Following administration of such nucleic acid, antibodies or functional equivalents are produced by host systems. The antibodies or functional equivalents produced are capable of preventing and / or preventing RSV infection and / or side effects of RSV infection. The nucleic acid sequence of the invention for use as a medicament and / or prophylactic agent is therefore also provided herein. Said nucleic acid is preferably used to counteract RSV. Furthermore, the use of a nucleic acid sequence, a functional part, is disclosed

[0050] At least a functional part of a nucleic acid is understood to mean a portion of said nucleic acid, at least 30 base pairs long, preferably at least 50 base pairs long, more preferably at least 100 base pairs long, containing at least one characteristic expression (in the type, not necessarily in the amount) of the nucleic acid according to the invention. Said functional part at least encodes an amino acid sequence comprising a sequence that is at least 70% identical to the CDR sequence as shown in Figure HD, Figure 14A, Figure 14B and / or Figure 14C.

[0051] Furthermore, the invention provides an isolated cell producing an antibody capable of producing an antibody of the invention. Possible (but non-limiting) methods for obtaining such antibody-producing cells are described in detail in the examples. The inventors developed and used a new method to improve the stability of RSV-specific antibody-producing cells. Using this method, RSV specific antibody producing cells are generated that are stable for at least six months. A cell that produces an RSV-specific antibody of the invention that is stable for at least nine weeks, preferably for at least three months, more preferably for at least six months is also disclosed herein.

[0052] The present inventors have used their observation that the stability of the RSV-specific antibody-producing cell is affected by affecting the amount of the BCL6 and / or Blimp-1 expression product in said antibody-producing cell. The amount of the BCL6 and / or Blimp-1 expression product is influenced either directly or indirectly.

Preferably, both the amounts of BCL6 and Blimp-1 expression products in said antibody-producing cell are regulated, since both expression products are involved in the stability of the antibody-producing cell. The stability of the antibody-producing cell is defined as the ability of said antibody-producing cell to remain at a certain stage of development (preferably after said cell has been brought to said stage). Different stages of cell development include at least one other characteristic of said cell. For example, it is known that memory cell B differentiates after stimulation into plasma cells that secrete antibodies through a stage that some researchers call plasmoblast. Cell B memory, plasmoblast and plasma cell are different stages of B cell development, with B cell having different characteristics. Memory cell B shows low proliferation and antibody secretion. Plasmoblast shows both higher proliferation and higher levels of antibody secretion compared to memory B cells, whereas plasma cells secrete high levels of antibody but are unable to proliferate. Thanks to the methods disclosed herein, it has become possible to regulate the replication viability of the antibody-producing cell. The replication life of an antibody-producing cell is defined herein as the time period in which cell B and its daughter cells are capable of replicating, while maintaining their ability to produce the antibody and / or development into the cell that produces the antibody. Preferably, the replication life of the antibody-producing cell is prolonged, i.e. that said antibody-producing cell will not terminally differentiate - or only after a longer period compared to the same type of antibody producing cells that are currently used - and continues to proliferate in vitro. According to the inventors, it is possible to regulate the amount of the BCL6 and / or Blimp-1 expression product in the antibody-producing cell to the extent that the antibody-producing cell is brought to and / or maintained at a predetermined developmental stage in which the cells continue to proliferate. Thanks to the disclosed method of the inventors, it has become possible to increase the replication viability of the antibody-producing cell, because it is possible to maintain cell B at a particular developmental stage, where replication occurs. Reference is made to PCT / NL2006 / 000625, filed by the same applicant. Disclosed herein are agents and methods for producing a stable RSV specific antibody-producing cell.

[0053] An antibody producing cell is defined as a cell, which cell is capable of producing and / or secreting an antibody or a functional equivalent thereof and / or which cell is capable of developing into a cell that is capable of producing and / or secretion of an antibody or its functional equivalent. An RSV specific antibody-producing cell is defined herein as a cell capable of producing and / or secreting antibodies or functional equivalents thereof that are capable of specifically binding RSV and / or an RSV component such as, for example, an RSV F (fusion) epitope. , G protein (attachment) RSV or SH protein (small hydrophobic) RSV. Preferably, said cell producing the RSV specific antibody comprises a B cell and / or a plasma cell

The cell B is hereinafter referred to as the antibody-producing cell, even when the B cell is at a stage where antibody production is low or absent at all, such as a naive B cell or memory cell B, activated or absent. no, because such cells are able to grow into cells that produce an antibody such as plasmoblast and / or a plasma cell.

[0054] A cell that produces an RSV specific antibody preferably comprises mammalian cells. Non-limiting examples include antibody-producing cells from a human subject, rodent, rabbit, llama, pig, cow, goat, horse, monkey, gorilla. Preferably, said antibody-producing cell comprises a human cell, a mouse cell, a rabbit cell and / or a llama cell.

[0055] BCL6 encodes a transcriptional repressor that is required for the normal development of B cells and T cells and maturation and that is required for the creation of breeding centers. (Ye, 1997). BCL6 is highly expressed in B cells from reproduction centers, but is practically not expressed in plasma cells. BCL6 inhibits the differentiation of activated B cells into plasma cells. The B cell induced transcriptional repressor, maturation protein-1 (Blimp-1) is required for the development of B cells into plasma cells. The human variant of Blimp-1 is called Prdml. As used herein, any reference to Blimp-1 includes a reference to Prdml. Blimp-1 drives differentiation of plasma cells. BCL6 and Blimp-1 suppress their mutual expression; the same in a natural situation, when one achieves a higher level of expression than the other, the degree of differentiation is enforced. In the human body, the differentiation of plasma cells from the activation of naive B cells or memory involves down regulation of BCL6 and upregulation of Blimp-1. In cells from reproductive centers, the expression of BCL6 is high and Blimp-1 expression is low. In resting memory cells, the expression of BCL6 and Blimp-1 is low. Signals that cause differentiation cause upregulation of Blimp-1 and this Blimp-1 antibody of BCL6 expression. The stage at which both BCL6 and Blimp-1 are expressed is short-lived and is called plasmoblast. At gradually increasing levels of Blimp-1, the expression of BCL6 goes out, giving the plasma cell. the differentiation of plasma cells from the activation of naive B cells or memory involves down regulation of BCL6 and upregulation of Blimp-1. In cells from reproductive centers, the expression of BCL6 is high and expression of Blimp-1 is low. In resting memory cells, the expression of BCL6 and Blimp-1 is low. Signals that cause differentiation cause upregulation of Blimp-1 and this Blimp-1 antibody of BCL6 expression. The stage at which both BCL6 and Blimp-1 are expressed is short-lived and is called plasmoblast. At gradually increasing levels of Blimp-1, the expression of BCL6 goes out, giving the plasma cell. the differentiation of plasma cells from the activation of naive B cells or memory involves down regulation of BCL6 and upregulation of Blimp-1. In cells from reproductive centers, the expression of BCL6 is high and expression of Blimp-1 is low. In resting memory cells, the expression of BCL6 and Blimp-1 is low. Signals that cause differentiation cause upregulation of Blimp-1 and this Blimp-1 antibody of BCL6 expression. The stage at which both BCL6 and Blimp-1 are expressed is short-lived and is called plasmoblast. At gradually increasing levels of Blimp-1, the expression of BCL6 goes out, giving the plasma cell. which cause differentiation cause upregulation of Blimp-1, and this Blimp-1 antibody for BCL6 expression. The stage at which both BCL6 and Blimp-1 are expressed is short-lived and is called plasmoblast. At gradually increasing levels of Blimp-1, the expression of BCL6 goes out, giving the plasma cell. which cause differentiation cause upregulation of Blimp-1, and this Blimp-1 antibody for BCL6 expression. The stage at which both BCL6 and Blimp-1 are expressed is short-lived and is called plasmoblast. At gradually increasing levels of Blimp-1, the expression of BCL6 goes out, giving the plasma cell.

Co-expression of BCL6 and Blimp-1 in cell B results in stabilization of this B-cell at a plasmoblast-like stage. Plasmoblasts, like plasma cells, are capable of secretion of the antibody. However, plasmoblasts are still able to proliferate, whereas

The plasma cells have lost their ability to proliferate. Plasma cells are therefore not suitable for the growth of antibody-producing cell lines.

[0057] A cell producing an RSV specific antibody may comprise an exogenous nucleic acid sequence encoding BCL6 or a functional part, derivative and / or analog thereof. An exogenous nucleic acid is defined herein as a nucleic acid sequence that does not naturally belong to the cell genome. With such an exogenous nucleic acid molecule, it is possible to regulate the concentration of BCL6 in the antibody-producing cell regardless of the expression of endogenous BCL6. Hence, even if the expression of endogenous BCL6 is low or absent, e.g. caused by Blimp-1, the exogenous nucleic acid sequence encoding BCL6 or a functional part thereof, derivative and / or analogue is still able to produce a concentration of BCL6 that is sufficient to affect the stability of the antibody-producing cell. preferably,

[0058] Preferably, as described below in more detail, a cell that produces an RSV specific antibody may comprise an exogenous nucleic acid sequence encoding Bcl-xL or a functional part, derivative and / or analog thereof. If Bcl-xL or a functional part thereof, derivative and / or analog is present, it is possible to culture plasmoblasts under conditions of low cell density. Expression of said nucleic acid sequence encoding Bcl-xL or a functional part, derivative and / or analog thereof is preferably regulated by the exogenous inducer of the repressor, such that the expression amount of Bcl-xL is arbitrarily regulated. This discloses a cell that produces an RSV specific antibody comprising: - an exogenous nucleic acid sequence encoding BCL6 or a functional part thereof, a derivative and / or an analogue, and / or - an exogenous nucleic acid sequence encoding Bcl-xL or a functional part, derivative and / or analog thereof. Said cell that produces an RSV specific antibody preferably contains either an exogenous nucleic acid sequence encoding BCL6 or a functional part thereof, a derivative and / or an analogue - and an exogenous nucleic acid sequence encoding Bcl-xL - or a functional part, derivative and / or analog thereof. Preferably, the expression of said nucleic acid sequence encoding BCL6, BclxL or a functional part, derivative and / or analog of BCL6 or Bcl-xL is regulated by an activator and / or a repressor that is inducible by an exogenous compound. For example, an inducible promoter system such as Tet-on or Tet-off is used. Said cell that produces an RSV specific antibody preferably contains either an exogenous nucleic acid sequence encoding BCL6 or a functional part thereof, a derivative and / or an analogue - and an exogenous nucleic acid sequence encoding Bcl-xL - or a functional part, derivative and / or analog thereof. Preferably, the expression of said nucleic acid sequence encoding BCL6, BclxL or a functional part, derivative and / or analog of BCL6 or Bcl-xL is regulated by an activator and / or a repressor that is inducible by an exogenous compound. For example, an inducible promoter system such as Tet-on or Tet-off is used. Said cell that produces an RSV specific antibody preferably contains either an exogenous nucleic acid sequence encoding BCL6 or a functional part thereof, a derivative and / or an analogue - and an exogenous nucleic acid sequence encoding Bcl-xL - or a functional part, derivative and / or analog thereof. Preferably, the expression of said nucleic acid sequence encoding BCL6, BclxL or a functional part, derivative and / or analog of BCL6 or Bcl-xL is regulated by an activator and / or a repressor that is inducible by an exogenous compound. For example, an inducible promoter system such as Tet-on or Tet-off is used. a derivative and / or analog - and an exogenous nucleic acid sequence encoding Bcl-xL - or a functional part, derivative and / or analog thereof. Preferably, the expression of said nucleic acid sequence encoding BCL6, BclxL or a functional part, derivative and / or analog of BCL6 or Bcl-xL is regulated by an activator and / or a repressor that is inducible by an exogenous compound. For example, an inducible promoter system such as Tet-on or Tet-off is used. a derivative and / or analog - and an exogenous nucleic acid sequence encoding Bcl-xL - or a functional part, derivative and / or analog thereof. Preferably, the expression of said nucleic acid sequence encoding BCL6, BclxL or a functional part, derivative and / or analog of BCL6 or Bcl-xL is regulated by an activator and / or a repressor that is inducible by an exogenous compound. For example, an inducible promoter system such as Tet-on or Tet-off is used.

[0059] A stable cell producing an RSV specific antibody can be generated by co-expression of BCL6 and Blimp-1 in a cell that produces an RSV-specific antibody. A cell that produces an RSV specific antibody is preferably

-21 is kept from an individual who has been exposed to RSV. Methods for isolating antibody-producing cells are well known in the art. For example, compounds derived from RSV that are labeled with a tag and / or tag are incubated with a sample of a subject that has been exposed to RSV, which sample contains antibody-producing cells. Cells producing an RSV specific antibody that recognize tagged compounds derived from RSV are isolated, and unbound cells are washed away. The resulting RSV specific antibody-producing cells are then stabilized by co-expression of BCL6 as well as Blimp-1.

[0060] All cells producing antibodies from a donor exposed to RSV can first be stabilized, and then cells that recognize the tagged compound derived from RSV, isolate. Alternatively, the antibody-producing cells are equipped with a marker (fluorescent) downstream of their B cell receptor (BCR, a membrane-expressed form of the antibody) that signals when the antibody-producing cell binds non-labeled / unlabeled antigen by BCR. Antibody producing cells in which the marker is in turn are selected and then stabilized by co-expression of BCL6 as well as Blimp-1. If there are no compounds available from the antigen, but when tests are available to screen for unique antibodies, total / majority antibody-producing cells can be stabilized by co-expression of BCL6 as well as Blimp-1 and, optionally, also Bcl-XL. Accordingly, the cells are cultured at low densities, preferably between 10 and 100 cells per 96 wells, in the presence of L (mini bulk cultures, MBC) cells. Culture supernatants can be used directly in screening assays like ELISA, Western blot or functional assays like ELISPOT, neutralization tests or cell migration assays. And the MBC may be selected and, in order to obtain the monoclonal cell lines of the antibody producing cell of interest, cultures are made from limiting dilutions and, preferably 2-3 weeks later, the supernatants from these cultures and re-screened in a preferred test. also Bcl-XL. Accordingly, the cells are cultured at low densities, preferably between 10 and 100 cells per 96 wells, in the presence of L (mini bulk cultures, MBC) cells. Culture supernatants can be used directly in screening assays like ELISA, Western blot or functional assays like ELISPOT, neutralization tests or cell migration assays. And the MBC may be selected and, in order to obtain the monoclonal cell lines of the antibody producing cell of interest, cultures are made from limiting dilutions and, preferably 2-3 weeks later, the supernatants from these cultures and re-screened in a preferred test. also Bcl-XL. Accordingly, the cells are cultured at low densities, preferably between 10 and 100 cells per 96 wells, in the presence of L (mini bulk cultures, MBC) cells. Culture supernatants can be used directly in screening assays like ELISA, Western blot or functional assays like ELISPOT, neutralization tests or cell migration assays. And the MBC may be selected and, in order to obtain the monoclonal cell lines of the antibody producing cell of interest, cultures are made from limiting dilutions and, preferably 2-3 weeks later, the supernatants from these cultures and re-screened in a preferred test. Culture supernatants can be used directly in screening assays like ELISA, Western blot or functional assays like ELISPOT, neutralization tests or cell migration assays. And the MBC may be selected and, in order to obtain the monoclonal cell lines of the antibody producing cell of interest, cultures are made from limiting dilutions and, preferably 2-3 weeks later, the supernatants from these cultures and re-screened in a preferred test. Culture supernatants can be used directly in screening assays like ELISA, Western blot or functional assays like ELISPOT, neutralization tests or cell migration assays. And the MBC may be selected and, in order to obtain the monoclonal cell lines of the antibody producing cell of interest, cultures are made from limiting dilutions and, preferably 2-3 weeks later, the supernatants from these cultures and re-screened in a preferred test.

[0061] As is well known to one skilled in the art, many alternative methods are available in the art. The methods listed above are non-limiting.

[0062] Furthermore, a method of producing an antibody producing cell that is stable for at least three months and which is capable of producing RSV-specific antibodies or functional equivalents thereof is disclosed, the method comprising:

- increasing the level of Blimp-1 expression in a cell that is capable of producing antibodies specific for RSV or functional equivalents thereof; and

- increasing and / or maintaining the level of expression of BCL6 in said cell.

[0063] It became possible to convert the memory-specific memory cell B into a plasmoblast-like cell and to stabilize said cell, such that there is no rapid differentiation into the plasma cell. This is in contradiction with the natural development of plasma cells in which Blimp-1 expression in cell B memory

-22 causes rapid development in the plasma cell, thereby inhibiting BCL6 expression, so that the resulting plasma cell almost does not express BCL6. One embodiment of the disclosure thus includes co-expression of both BCL6 and Blimp-1 in an RSV specific cell, resulting in a cell that is capable of both proliferation and production of the antibody. The level of expression of BCL6 in said RSV specific cell B is preferably brought to, and maintained at, essentially at the same level or at a higher level as compared to plasmoblast. In this way, a stable culture of RSV-specific B cells is generated, which cells are still able to produce antibodies specific for RSV. These B cells specific for RSV, which co-express BCL6 and Blimp-1 are preferably further stabilized by the addition of the anti-apoptotic gene Bcl-xL. With the introduction of Bcl-xL, it is now possible to increase plasmoblasts under conditions of low cell density. Thus, the invention also provides a method for culturing plasmoblasts under low cell density conditions comprising generating a cell that produces an RSV specific antibody with expression levels of BCL6, Blimp-1 and Bcl-xL by any of the methods described herein.

[0064] The amount of the BCL6 expression product (preferably BCL6 proteins) in an RSV specific antibody-producing cell is regulated in a variety of ways.

[0065] The antibody-producing cell may be disclosed with a compound capable of directly or indirectly affecting the expression of BCL6. The antibody-producing cell is preferably disclosed with a compound capable of increasing BCL6 expression to counteract down-regulation of BCL6 during Blimp-1 expression. Such a compound preferably contains a STAT5 (Signal Transducer of Activation and Transcription 5) protein or a functional part thereof, a derivative and / or an analogue, and / or a nucleic acid sequence that encodes them. STAT5 is a transducer of a signal capable of increasing BCL6 expression. Two forms of STAT5, STAT5a and STAT5b are known which are encoded by two different genes, connected in tandem. Administration and / or activation of STAT5 results in increased levels of BCL6. Hence, down regulation of BCL6 by Blimp-1 is at least partially offset by upregulation of BCL6 expression by STAT5 or a functional part, derivative and / or analog thereof. Hence, STAT5 or a functional part thereof, derivative and / or analogue is able to directly affect the expression of BCL6. It is also possible to indirectly affect the expression of BCL6. This is for example done by regulating the amount of a compound that in turn is capable of directly or indirectly activating STAT5 and / or regulating STAT5 expression. Hence, the expression and / or activity of endogenous and / or exogenous STAT5 is increased. For example, it is possible to indirectly increase the expression of BCL6 by growing the antibody-producing cell in the presence of interleukin (IL) 2 and / or IL4 that are capable of activating STAT5.

[0066] A cell producing an RSV specific antibody may comprise a nucleic acid sequence encoding STAT5 or a functional part, derivative and / or analog thereof, wherein said nucleic acid sequence is constitutively active, i.e. that STAT5 is continuously expressed independently of the the presence of

-23 (endogenous) regulators. In the event that the endogenous expression of STAT5 is low or absent, the exogenous constitutively active nucleic acid sequence encoding STAT5 or a functional part thereof, derivative and / or analogue is preferably used to give a concentration of STAT5 or a functional part, derivative and / or analog which is sufficient to increase the expression of BCL6. Most preferably, the RSV-specific antibody-producing cell may comprise a nucleic acid sequence encoding a STAT5-containing compound or functional part thereof, derivative and / or analog, preferably a fusion protein whose activity is regulated by the exogenous repressor inducer, such that the amount of activation of the BCL6 expression is adjustable freely. Another system that allows the induction of BCL-6 is provided by the Tet-on system, wherein the addition of tetracycline and / or tetracycline derivatives induces transactivator activity which induced transcription of the BCL6 gene followed by the synthesis of the BCL protein. In one preferred embodiment of the disclosure, the antibody-producing cell may comprise the nucleic acid sequence encoding the estrogen receptor (ER) and STAT5 as the ER-STAT5 fusion protein. This fusion protein is inactive because it forms a complex with thermal shock proteins in the cytosol. In this way, STAT5 is unable to reach the nucleus and the expression of BCL6 is not amplified. After administration of the 4-hydroxy-tamoxifen exogenous inducer (4HT), the ER-STAT5 fusion protein dissociates from heat shock proteins such that STAT5 is able to enter the nucleus and activate BCL6 expression. which induced the transcription of the BCL6 gene, followed by the synthesis of the BCL protein. In one preferred embodiment of the disclosure, the antibody-producing cell may comprise the nucleic acid sequence encoding the estrogen receptor (ER) and STAT5 as the ER-STAT5 fusion protein. This fusion protein is inactive because it forms a complex with thermal shock proteins in the cytosol. In this way, STAT5 is unable to reach the nucleus and the expression of BCL6 is not amplified. After administration of the 4-hydroxy-tamoxifen exogenous inducer (4HT), the ER-STAT5 fusion protein dissociates from heat shock proteins such that STAT5 is able to enter the nucleus and activate BCL6 expression. which induced the transcription of the BCL6 gene, followed by the synthesis of the BCL protein. In one preferred embodiment of the disclosure, the antibody-producing cell may comprise the nucleic acid sequence encoding the estrogen receptor (ER) and STAT5 as the ER-STAT5 fusion protein. This fusion protein is inactive because it forms a complex with thermal shock proteins in the cytosol. In this way, STAT5 is unable to reach the nucleus and the expression of BCL6 is not amplified. After administration of the 4-hydroxy-tamoxifen exogenous inducer (4HT), the ER-STAT5 fusion protein dissociates from heat shock proteins such that STAT5 is able to enter the nucleus and activate BCL6 expression. the antibody-producing cell may comprise the nucleic acid sequence encoding the estrogen receptor (ER) and STAT5 as the ER-STAT5 fusion protein. This fusion protein is inactive because it forms a complex with thermal shock proteins in the cytosol. In this way, STAT5 is unable to reach the nucleus and the expression of BCL6 is not amplified. After administration of the 4-hydroxy-tamoxifen exogenous inducer (4HT), the ER-STAT5 fusion protein dissociates from heat shock proteins such that STAT5 is able to enter the nucleus and activate BCL6 expression. the antibody-producing cell may comprise the nucleic acid sequence encoding the estrogen receptor (ER) and STAT5 as the ER-STAT5 fusion protein. This fusion protein is inactive because it forms a complex with thermal shock proteins in the cytosol. In this way, STAT5 is unable to reach the nucleus and the expression of BCL6 is not amplified. After administration of the 4-hydroxy-tamoxifen exogenous inducer (4HT), the ER-STAT5 fusion protein dissociates from heat shock proteins such that STAT5 is able to enter the nucleus and activate BCL6 expression.

[0067] Additionally, or alternatively, the expression of BCL6 in an RSV-specific antibody-producing cell is increased by culturing said antibody-producing cell in the presence of a compound capable of directly or indirectly increasing BCL6 expression.

[0068] Disclosed herein is a method of producing a cell that produces an RSV specific antibody comprising:

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

- culturing a cell producing an antibody specific for RSV in the presence of a compound capable of directly or indirectly increasing BCL6 expression. Said compound capable of directly or indirectly enhancing BCL6 expression preferably comprises STAT5 or a functional part, derivative and / or analog thereof.

[0069] A method is thus disclosed comprising providing said cell producing an RSV specific antibody to STAT5 or a functional part thereof, derivative and / or analogue, or a STAT5 nucleic acid sequence or a functional part, derivative and / or analog thereof. Said antibody-producing cell may be cultured after introducing the nucleic acid sequence encoding STAT5 or a functional part thereof, a derivative and / or an analogue to said cell. Said nucleic acid sequence is introduced, for example, into said cell by means of transfection and / or virus-mediated gene transfer.

Many alternative methods are available in the art for introducing nucleic acid sequences into a cell that do not require further explanation here.

[0070] Due to the compound capable of directly or indirectly enhancing the expression of BCL6, it is possible to increase the expression of endogenous BCL6. However, the antibody-producing cell may comprise a nucleic acid sequence encoding the BCL6 or a functional part, derivative and / or analog thereof. As explained herein before, exogenous nucleic acid encoding BCL6 is advantageous because it allows the regulation of BCL6 concentration in the cell regardless of the expression of endogenous BCL6. Therefore, even if the expression of endogenous BCL6 is low or absent, for example caused by Blimp-1, the exogenous nucleic acid sequence encoding BCL6 or a functional part thereof, derivative and / or analogue is still able to produce a concentration of BCL6 that is sufficient to affect stability the antibody-producing cell. Also disclosed herein is a method comprising providing a cell that produces an RSV specific antibody with a nucleic acid sequence encoding BCL6 or a functional part, derivative and / or analog thereof. Preferably, said antibody-producing cell is provided with a constitutively active nucleic acid sequence encoding BCL6 or a functional part, derivative and / or analog thereof such that BCL6 expression is maintained even when the endogenous expression of BCL6 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 analog thereof is regulated by the exogenous repressor inducer such that the amount of expression of BCL6 is regulated arbitrarily. For example,

[0071] Also disclosed is a method wherein the amount of BCL6 is indirectly regulated by providing a cell that produces an RSV specific antibody with a nucleic acid sequence encoding E47 or a functional part, derivative and / or analog thereof. E47 encodes a transcription factor that belongs to the helix-loop-helix protein family called the E. protein. There are four E, E 12, E47, E2-2 and HEB proteins that are involved in the development of lymphocytes. E12 and E47 are encoded by one gene, named E2A, which is spliced ​​in a different way. Protein E can be inhibited by the protein E inhibitor Id2, and Id3, and by ABFI (Mathas S., 2006). Proteins E are described as tumor suppressors and their overexpression induces apoptosis. One of the specific goals of E47 are the Socsl and Socs3 genes. These Socs genes are known as negative STAT [delta] regulators and thus indirectly BCL6. In other words, the expression of E47 in cell B increases the expression of Blimp-1, which causes the differentiation of B cells towards the phenotype of antibody production (plasma cell).

[0072] The amount of Blimp-1 expression in the RSV-specific antibody-producing cell is also regulated in various ways. A cell producing an RSV specific antibody is provided with a compound capable of directly or indirectly affecting Blimp-1 expression. In addition, or alternatively, an antibody producing cell

Is grown in the presence of a compound capable of directly or indirectly affecting Blimp-1 expression. Furthermore, a method is disclosed that comprises providing a cell that produces an RSV specific antibody with a compound capable of directly or indirectly affecting Blimp-1 expression. In addition, a method comprising culturing said antibody-producing cell in the presence of a compound capable of directly or indirectly affecting Blimp1 expression is disclosed. A compound that is capable of increasing Blimp-1 expression may be used to counteract down-regulation of Blimp-1 during BCL6 expression. Said compound most preferably contains IL-21.

[0073] Said compound capable of directly or indirectly affecting Blimp-1 expression may comprise a STAT3 (Signal Transducer of Activation and Transcription 3) protein or a functional part thereof, a derivative and / or analogue, and / or a nucleic acid sequence that it codes them. STAT3 is a signal transducer that is involved in the development and differentiation of B. cells. STAT3 is able to up-regulate Blimp1 expression. Furthermore, a method is disclosed in which said compound capable of directly or indirectly affecting the expression of Blimp-1 comprises STAT3 or a functional part thereof, a derivative and / or an analogue or a nucleic acid sequence encoding STAT3 or a functional part thereof, a derivative and / or analog. Most preferably, the expression of said nucleic acid sequence encoding STAT3 or a functional part thereof, derivative and / or analogue is regulated by the exogenous inducer of the repressor, such that the expression amount of STAT3 is arbitrarily adjusted. For example, an inducible promoter system such as, for example, a Tet-on or Tet-off system is used. The fusion product comprising STAT3, derivative or analog, and ER can be introduced into said cell enabling regulation of STAT3 expression by hydroxy tamoxifen.

[0074] Because STAT3 is capable of affecting Blimp-1 expression, it is also possible to indirectly regulate Blimp-1 expression by administering a compound capable of directly or indirectly regulating STAT3 activity and / or expression. An antibody-producing cell with a compound that is capable of increasing STAT3 activity can be provided such that Blimp-1 expression is also indirectly increased. Furthermore, there is thus provided a method in which the antibody-producing cell is provided with a compound capable of directly or indirectly increasing STAT3 activity.

[0075] An antibody producing cell with a compound capable of directly or indirectly activating STAT3 can be provided to increase Blimp1 expression.

[0076] STAT3 is active in various ways. Preferably, STAT3 is activated by providing an antibody-producing cell with a cytokine. Cytokines that are naturally involved in the differentiation of B cells are very effective in the regulation of STAT proteins. Statins are very effective STAT3 activators of IL-21 and IL-6, but also IL-2, IL7, IL-IO, IL-15 and IL-27 are known to activate STAT3. In addition, Toll-like receptors (TLRs) that are involved in innate immunity are able to activate STAT3. One embodiment of this disclosure therefore provides a method in which said compound is capable

- directly or indirectly affecting the expression of Blimp-1 includes IL-21, IL-2, IL6, IL-7, IL-IO, IL-15 and / or IL-27. Most preferably, IL-21 is used because IL-21 is particularly suitable for affecting the stability of the antibody-producing cell. IL21 is able to up-regulate Blimp-1 expression even when the Blimp-1 expression counteracts BCL6.

[0077] In addition, or in addition, the mutant Janusa kinase (JAK) is used to activate STAT3. Naturally, JAK is able to phosphoryl STAT3 after being activated by at least one cytokine. The mutant Janse kinase capable of activating STAT3, regardless of the presence of cytokines, is particularly suitable in the method disclosed herein.

[0078] As previously explained, a compound capable of increasing Blimp-1 expression may comprise a nucleic acid sequence encoding STAT3 or a functional part, derivative and / or analog thereof. The presence of an exogenous nucleic acid sequence encoding STAT3 or a functional part, derivative and / or analog thereof permits the continued presence of STAT3 or a functional part, derivative and / or analog thereof even when the expression of endogenous STAT3 is very low or absent.

[0079] It is also possible to reduce the expression and / or activity of STAT5 to up-regulate Blimp-1. If the amount and / or activity of STAT5 is reduced, the activation of BCL6 expression is also reduced, resulting in a reduction in the amount of the BCL6 expression product. Because BCL6 and Blimp-1 counteract one another's expression, a reduced amount of the BCL6 expression product results in an increased amount of the Blimp-1 expression product. The compounds capable of down-regulating STAT5 activity can thus indirectly upregulate Blimp-1. Such compounds include, for example, members of the suppressor of cytokine signaling proteins (SOCS). The amount of the Blimp-1 expression product in the RSV-specific antibody-producing cell may therefore be up-regulated, providing such a cell with a SOCS protein and / or by activating the SOCS protein in said cell.

Expression and / or activity of STAT5 can be reduced when the cell producing the RSV specific antibody is provided with the nucleic acid sequence encoding E47 or a functional part, derivative and / or analog thereof. Expression of E47 within B cells expressing high levels of STAT [delta] b interferes with differentiation and proliferation, i.e. blocking STAT5 with E47 and SOCS results in reduced levels of BCL6 and then at increased levels of Blimp-1. The upregulated levels of Blimp-1 result in a reduction in cell proliferation and differentiation towards the antibody-producing cell. In other words, the expression of E47 in cell B increases the expression of Blimp-1, which results in the differentiation of the B cell towards the antibody producing phenotype (plasma cell).

[0081] At least a functional part of the STAT5 protein, STAT3, Bcl-xL and / or

BCL6 is understood to mean a protein molecule that has the same ability - in terms of its type, not necessarily in quantity - to influence the stability of the antibody-producing cell as compared to the STAT5 protein, STAT3, respectively,

-27Bcl-xL and / or BCL6. The functional part of the STAT5 protein or STAT3 protein is, for example, deprived of amino acids that are not, or only to a small extent, involved in such ability. A derivative of the STAT5 protein, STAT3 proteins, Bcl-xL and / or BCL6 is defined as a protein that has been altered so that the ability of this protein to affect the stability of the antibody-producing cell is basically the same in type, not necessarily in quantity. The derivative is provided in a number of ways, e.g. by conservative amino acid substitution, wherein one amino acid is substituted with another amino acid with generally similar properties (size, hydrophobicity, etc.), so that overall functioning is not likely to be seriously compromised. The derivative includes, for example, a fusion protein, for example in the form of a STAT5-ER or STAT3-ER fusion protein whose activity depends on the presence of 4-hydroxy tamoxifen (4HT). The STAT5 protein analogue, STAT3 proteins, Bcl-xL and / or BCL6 is defined as a molecule with the same ability to affect the stability of an antibody-producing cell in terms of type, not necessarily in quantity. Said analog does not have to be derived from said STAT5 protein, STAT3, Bcl-xL and / orBCL6.

[0082] Said RSV specific producing cell may be cultured in the presence of IL-21 before said antibody-producing cell is provided with the nucleic acid sequence encoding the BCL6 or a functional part, derivative and / or analog thereof. The culturing of an RSV-specific antibody-producing cell, preferably B-cell, in the presence of IL-21, before said cell is provided with the nucleic acid sequence encoding BCL6 or a functional part, derivative and / or analog thereof, is advantageous because stability is particularly well improved, proliferation and / or production of the antibody.

[0083] The disclosure provides a method of influencing the stability of an RSV specific antibody-producing cell as described herein, further comprising directly or indirectly increasing the amount of the Bcl-xL expression product in said antibody-producing cell. This is for example achieved by providing said antibody-producing cell with a nucleic acid sequence encoding Bcl-xL or a functional part thereof, derivative and / or analog or with sequences of nucleic acids coding for other anti-apoptotic genes including, but not limited to, Bcl- 2. In yet another embodiment of this disclosure, this is accomplished by providing said antibody-producing cell with a compound capable of directly or indirectly increasing Bcl-xL expression, preferably said compound comprises APRIL,

[0084] Bcl-xL is a member of the anti-apoptotic Bcl-2 family, Bcl2 proteins interact with and counteract the so-called members of the BH3 family (Bcl-2 homology domain 3) -only like Bax,

Bak, Bim and Bad, which induce the release of cytochrome c after specific death stimuli (Boise, LH, 1993). Thus, protecting the integrity of the mitochondrial membrane through proteins, like

Bcl-xL is critical for cell survival.

[0085] STAT5 activation has been shown to protect cells from cell death. STAT5 has been shown to regulate the expression of Bcl-xL, supporting the anti-apoptotic role for STAT5. STAT5 positively regulates the expression of Bcl-xL by STAT binding members in the BclxL promoter. In vivo, Bcl-xL expression is absent in bone marrow of STAT5A / B deficient mice. In addition, STAT5-mediated survival of erythroblasts is dependent on the activation of Bcl-xL. Bcl-xL transgenic overexpression has recently been demonstrated in mouse B cells to promote the survival of B cells and other non-malignant plasma cell foci.

[0086] The method of the present disclosure is particularly suitable for the production of a cell culture comprising cells producing an RSV specific antibody that are able to be used to obtain a B cell culture ex vivo. Said cell memory B is preferably human so that human antibodies are produced. Said cell B preferably originates from an individual, who has previously been exposed to a respiratory syncytial virus. RSV specific B cells can be isolated from a peripheral blood sample and / or tonsil sample using methods known in the art. Memory B cells are, for example, isolated by selection (magnetic bead sorting) for the B cell marker CD 19 and / or CD22 and (subsequent) selection for the IgG and / or CD27 cell surface and / or by negative selection for IgM, IgD and / or IgA. In B cells from reproductive centers, the expression of BCL6 is high, whereas the expression of Blimp-1 is low. The natural development of the antibody-secreting cell involves upregulation of Blimp-1 expression. Because Blimp-1 suppresses BCL6 expression, up-regulation of Blimp-1 results in down regulation of BCL6 in a natural situation. However, the Blimp-1 expression is up-regulated, while the expression of BCL6 is at least partially maintained. This gives the cell producing the antibody specific for RSV, whereas BCL6 and Blimp-1 are co-expressed. Said cell that produces an RSV specific antibody is capable of proliferating and secreting an anti-RSV antibody and is therefore suitable for use in ex vivo B cell culture. Said antibody-producing cell can be protected by apoptosis by Bcl-xL. A cell that produces an RSV-specific antibody has the advantage of being stable and not subject to terminal differentiation over an extended period of time. Said antibody-producing cell is stable for at least one week, preferably for at least one month, more preferably for at least three months, most preferably for at least six months. Cell B is preferably cultured in the presence of CD40L because the replication of most B cells is preferred by CD40L.

[0087] The expression of BCL6 can be maintained at substantially the same level or at a higher level as compared to cell B from proliferation centers, since significant expression of BCL6, together with Blimp-1 expression, results in an antibody-producing cell with favorable proliferation and production properties. antibodies and / or stability. Said BCL6 expression and / or Blimp-1 expression may be accompanied by the expression of Bcl-XL, resulting in even more favorable proliferation and antibody production properties and / or stability.

[0088] Disclosed herein is a method of producing an RSV specific production cell that is stable for at least one week, preferably for at least one month, more preferably for at least three months, more preferably for at least six months, at which method includes:

- provision of an RSV specific memory cell B; - increasing the level of Blimp-1 expression in said cell; and

- increasing and / or maintaining the level of expression of BCL6 in said cell. Also provided is an ex vivo method of producing an RSV-specific antibody-producing cell comprising increasing the expression level of Blimp1 in the B cell of RSV-specific memory and increasing and / or maintaining the level of expression of BCL6 in said cell. The above-mentioned expression levels of BCL6 and Blimp-1 are preferably brought to, and / or maintained at, substantially the same level or at a higher level as compared to plasmoblast. Said cell B may undergo transduction with BCL6 and Bcl-xL. Furthermore, a method is provided for producing a cell that produces an RSV specific antibody that is stable for at least three months, comprising:

- providing a B cell capable of producing antibodies specific for

RSV with BCL6, or a functional part thereof, derivative and / or analogue; and

- providing said B cell with Bcl-xL or a functional part, derivative and / or analog thereof; and - culturing said B cell.

[0089] Said cell B is preferably provided with a nucleic acid sequence encoding BCL6, or a functional part, derivative and / or analog thereof and with the nucleic acid sequence Bcl-xL or a functional part, derivative and / or analog thereof.

[0090] Said cell B is preferably cultured in the presence of a compound capable of increasing the expression of Blimp-1 such as, for example, IL-21, IL-2, IL-6, 11-7, IL-IO, IL15, IL-27, or the mutant Janus kinase. Preferably, IL-21 is used, because this cytokine is particularly useful for increasing Blimp-1 expression and stabilizing the antibody-producing cell by the method described herein. In addition, to increase transduction efficiency, said cell B is preferably cultivated in the presence of IL-21 before said cell B is a transduced nucleic acid sequence encoding BCL6 and / or Bcl-xL, or a functional part, derivative and / or analog thereof.

[0091] Said cell B is provided with a SOCS protein or a functional part thereof, a derivative and / or an analog or a nucleic acid encoding them, because the SOCS protein or functional part thereof, derivative and / or analog is capable of indirectly increasing Blimp-1 expression . In another alternative or additional embodiment of this disclosure, said cell B is provided with E47 or a functional part thereof, derivative and / or analog thereof, a nucleic acid encoding it. As indicated earlier, as a result of the increase in E47 or its functional part,

Of the derivative and / or analog, the function of the Socs protein is increased and indirectly the expression of Blimp-1 is increased.

[0092] In the Examples, particularly preferred embodiments according to the disclosure are shown. According to one particularly preferred embodiment of the disclosure, B-specific RSV cells are first cultured in the presence of IL-21. The B cells are then transduced with the nucleic acid coding for BCL6 and the nucleic acid coding for Bcl-xL. Eddy transduction is preferably used. Most preferably, B cells and a virus containing at least one nucleic acid of interest are mixed, whereafter the mixture is centrifuged to achieve high transduction efficiency. After transduction, B cells are grown in the absence of 11-21 and in the presence of 11-4 and L cells within 3-5 days to allow expression of BCL6. The B cells can then be transduced again using the nucleic acid encoding BCL6 and the nucleic acid encoding Bcl-xL. The B cells are then re-cultured in the absence of 11-21 and in the presence of 11-4 and L cells within 3-5 days to allow expression of BCL6. Then, cells expressing BCL6 and Bcl-xL are isolated. IL-21 is again administered to the culture to enhance replication and antibody production. Antibodies that are secreted by Bcl-6, Blimp 1 and Bcl-XL-expressing cells into the culture supernatant are preferably screened for neutralizing capacity / activity / in vitro to RSV. The antibody-producing cells that produce these antibodies are also preferably selected, for example, by culturing at limiting dilutions. Thus, stable RS-specific B cells are obtained, wherein BCL6 and Blimp-1 are co-expressed. Said B cells are capable of replication and antibody production in an in vitro culture for at least six months.

[0093] A method is disclosed herein further comprising selecting and / or isolating an RSV specific antibody or functional equivalent thereof. IgM producing cells and IgG producing cells can be selected and / or isolated. Preferably, the IgG producing cell is selected and / or isolated.

[0094] Cells producing an RSV specific antibody generated by the method described herein are suitable for the generation of anti-RSV antibodies. However, genes encoding heavy and / or light Ig chains are isolated from said cell and expressed in a second cell, such as, for example, Chinese hamster ovary (CHO) cell lines or 293 (T) cells. Said second cell, also referred to herein as the production cell, is preferably adapted for commercial production of antibodies. The proliferation of said production cell results in a producing cell line capable of producing antibodies specific for RSV. Preferably, said producer cell line is suitable for the preparation of compounds for human use.

[0095] The method disclosed herein is preferably used to produce an antibody-producing cell that is stable for at least one week, preferably at least one month, more preferably at least three months, more preferably at least six months, so that it has become possible production of commercial antibodies. Most preferably, a stable cell line capable of producing monoclonal antibodies is produced. This is preferably carried out using memory cells B, which, for example, were isolated from the sample by selection for CD 19 and / or CD22 (B cell marker) and cell surface IgG and / or CD27 (for memory cell determination) and / or by selection negative for IgM, IgD and / or IgA. In addition, the cell producing the RSV-specific antibody is, for example, being selected in an RSV binding assay or an RSV derived component such as, for example, F protein, G protein and / or RSV SH protein. Then, Blimp-1 and BCL6 can be co-expressed in said RSV-specific antibody-producing cell, resulting in a cell culture capable of specifically binding (component) RSV. In yet another preferred embodiment of the disclosure, said cell B is further provided with Bcl-xL or a functional part, derivative and / or analog thereof.

[0096] If only one memory cell is used, a cell line that produces monoclonal antibodies is obtained. It is also possible to generate a cell line that produces a monoclonal antibody with B cells capable of producing antibodies against RSV. After production of a stable B cell culture by a method described herein, a B cell capable of producing antibodies against a specific RSV antigen is isolated and preferably at least a functional part of the heavy chain and / or light chain Ig gene is expressed from said B cell in the other. cell line. Preferably at least the functional part of the gene encoding the heavy Ig chain and at least the functional part of the gene encoding the light chain Ig from said cell B are expressed in the second cell line.

[0097] The antibody-producing cell, preferably, but not necessarily, the memory cell B, which has been obtained from a subject that has been previously exposed to RSV, may be used in the method of the present disclosure. In this way, it became possible to produce human antibodies of interest in ex vivo.

[0098] Further disclosed herein is a method of producing antibodies that are capable of specifically binding and / or neutralizing a syncytial respiratory virus, the method comprising:

- generating a cell producing an antibody capable of producing antibodies specific for RSV according to the method of the invention; and

- obtaining antibodies produced by said antibody producing cell.

[0099] Also provided is an isolated or recombinant antibody as defined in the claims as well as a cell producing isolated or

A recombinant antibody as defined in the claims that may be obtained by a method of the invention as defined in the claims. Said antibody preferably comprises antibody D25, AM14, AM16 and / or AM23.

[0100] When a cell producing an RSV specific antibody of the invention as defined in the claims is obtained, the functional part of the gene encoding the heavy chain and the light chain Ig of said cell is preferably isolated and / or artificially generated. A nucleic acid sequence comprising at least a functional portion of the nucleic acid sequence as shown in Figure 11, Figure 12, Figure 14A, Figure 14B or Figure 14C is provided. Said functional part comprises at least one nucleic acid sequence as shown in Figure 11D, Figure 12, Figure 14A, Figure 14B and / or Figure 14C. Said functional part encodes a CDR as shown in Figure HD, Figure 12, Figure 14A, Figure 14B or Figure 14C.

[0101] Further disclosed is an isolated, synthetic or recombinant nucleic acid sequence comprising a heavy chain sequence that is at least 70%, preferably at least 80%, more preferably at least 90% homologous to at least a portion of the sequence

CAGGTGCAGCTGGTACAGTCTGGGGCTGAAGTGAAGAAGCCTGGGTCCTCGGTG ATGGTCTC CTGCCAGGCCTCTGGAGGCCCCCTCAGAA, ACTATATTATCAAC,

TGGCTACGACAGGCCCCTGGACAAGGCCCTGAGTGGATGGGA,

GGGATCATTCCTGTCTTGGGTACAGTACACTACGCACCGAAGTTCCAGGGC, AGAGTCACGATTACCGCGGACGAATCCACAGACACAGCCTACATCCATCTGATCA GCCTGAG ATCTGAGGACACGGCCATGTATTACTGTGCGACG,

GAAACAGCTCTGGTTGTATCTACTACCTACCTACCACACTACTTTACACAC, TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG, and / or

CAGGTGCAGCTGGTACAGTCTGGGGCTGAAGTGAAGAAGCCTGGGTCCTCGGTG

ATGGTCTC

CTGCCAGGCCTCTGGAGGCCCCCTCAGAAACTATATTATCAACTGGCTACGACAG

GCCCCTG

GACAAGGCCCTGAGTGGATGGGAGGGATCATTCCTGTCTTGGGTACAGTACACTA

CGCACCG

AAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACAGACACAGCCTAC

ATCCATCT

GATCAGCCTGAGATCTGAGGACACGGCCATGTATTACTGTGCGACGGAAACAGCT

CTGGTTG

TATCTACTACCTACCTACCACACTACTTTGACAACTGGGGCCAGGGAACCCTGGT CACCGTC TCCTCAG, said part having at least 15 nucleotides. Said heavy chain sequence is preferably derived from antibody D25. Said heavy chain sequence may preferably comprise a sequence that is at least 70%, preferably at least 80%, more preferably at least 90% homologous to the sequence as shown in Figure HD. An isolated, synthetic or recombinant nucleic acid sequence containing a chain sequence

A compound consisting of any of the aforementioned heavy chain sequences is also disclosed herein.

[0102] Also disclosed is an isolated, synthetic or recombinant nucleic acid sequence comprising a light chain sequence that is at least 70%, preferably at least 80%, more preferably at least 90% homologous to at least a portion of the GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCAGCTGTAGGAGAGACAGAG TCACCAT CACTTGC sequence , CAGGCGAGTCAGGACATTGTCAACTATTTAAAT, TGGTATCAACAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTAC,

GTTGCATCCAATTTGGAGACA,

GGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTAGTCTCACCA

TCAGCAG

CCTGCAGCCTGAAGATGTTGCAACATATTATTGT, CAACAATATGATAATCTCCCA, CTCACATTCGGCGGAGGGACCAAGGTTGAGATCAAAAGA and / or GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCAGCTGTAGGAGACAGAG TCACCAT

CACTTGCCAGGCGAGTCAGGACATTGTCAACTATTTAAATTGGTATCAACAGAAA

CCAGGGA

AAGCCCCTAAGCTCCTGATCTACGTTGCATCCAATTTGGAGACAGGGGTCCCATC

AAGGTTC

AGTGGAAGTGGATCTGGGACAGATTTTAGTCTCACCATCAGCAGCCTGCAGCCTG

AAGATGT

TGCAACATATTATTGTCAACAATATGATAATCTCCCACTCACATTCGGCGGAGGG ACCAAGG TTGAGATCAAAAGA, said part having at least 15 nucleotides. Said light chain sequence is preferably derived from antibody D25.

[0103] Said 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 as shown in Figure HD. An isolated, synthetic or recombinant nucleic acid sequence comprising a heavy chain sequence consisting of any of the aforementioned light chain sequences is also disclosed herein.

[0104] Further disclosed is an isolated, synthetic or recombinant nucleic acid sequence comprising a heavy chain sequence that is at least 70%, preferably at least 80% more preferably at least 90% homologous to at least a portion of the sequence GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGTCCCTG AGACTCTC CTGTGCGGCCTCT , GGATTCAGCTTCAGTCACTATGCC, ATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCAGTT,

ATATCTTATGATGGAGAAAATACA,

TATTACGCAGACTCCGTGAAGGGCCGATTCTCCATCTCCAGAGACAATTCCAAGA

-34ACACAGT

GTCTCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTCTATATTACTGT,

GCGAGAGACCGCATAGTGGACGACTACTACTACTACGGTATGGACGTC,

TGGGGCCAAGGGGCCACGGTCACCGTCTCCTCAG and / or

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTG

AGACTCTC

CTGTGCGGCCTCTGGATTCAGCTTCAGTCACTATGCCATGCACTGGGTCCGCCAG

GCTCCAG

GCAAGGGACTGGAGTGGGTGGCAGTTATATCTTATGATGGAGAAAATACATATTA

CGCAGAC

TCCGTGAAGGGCCGATTCTCCATCTCCAGAGACAATTCCAAGAACACAGTGTCTC

TGCAAAT

GAACAGCCTGAGACCTGAGGACACGGCTCTATATTACTGTGCGAGAGACCGCAT

AGTGGACG

ACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGGCCACGGTCACCGTCTC CTCA, said part having at least 15 nucleotides. Said heavy chain sequence is preferably derived from the AM14 antibody. An isolated, synthetic or recombinant nucleic acid sequence comprising a heavy chain sequence consisting of any of the aforementioned heavy chain sequences is also disclosed herein.

[0105] Also disclosed herein is an isolated, synthetic or recombinant nucleic acid sequence comprising a light chain sequence that is at least 70%, preferably at least 80% more preferably at least 90% homologous to at least a portion of the GACATCCAGATGACCCAGTCTCCATCTTCCCTGTCTGCATCTGTAGGAGACAGAG TCACCAT sequence CACTTGCCAGGCGAGT, CAGGACATTAAGAAGTAT, TTAAATTGGTATCATCAGAAACCAGGGAAAGTCCCTGAGCTCCTGATGCAC,

GATGCATCC,

AATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGCAGGGGATCTGGGACAGAT TTTACTCT CACCATTAGCAGCCTGCAGCCTGAAGATATTGGAACATATTACTGT, CAACAGTATGATAATCTGCCTCCGCTCACT,

TTCGGCGGAGGGACCAAGGTGGAGATCAAAC and / or

GACATCCAGATGACCCAGTCTCCATCTTCCCTGTCTGCATCTGTAGGAGACAGAG

TCACCAT

CACTTGCCAGGCGAGTCAGGACATTAAGAAGTATTTAAATTGGTATCATCAGAAA

CCAGGGA

AAGTCCCTGAGCTCCTGATGCACGATGCATCCAATTTGGAAACAGGGGTCCCATC

AAGGTTC

AGTGGCAGGGGATCTGGGACAGATTTTACTCTCACCATTAGCAGCCTGCAGCCTG

AAGATAT

TGGAACATATTACTGTCAACAGTATGATAATCTGCCTCCGCTCACTTTCGGCGGA

GGGACCA AGGTGGAGATCAAACGAACTGTG, wherein said part has co

At least 15 nucleotides. Said light chain sequence is preferably derived from the AM14 antibody.

[0106] An isolated, synthetic or recombinant nucleic acid sequence comprising a heavy chain sequence consisting of any of the aforementioned light chain sequences is also disclosed herein.

[0107] Further disclosed is an isolated, synthetic or recombinant nucleic acid sequence comprising a heavy chain sequence that is at least 70%, preferably at least 80%, more preferably at least 90% homologous to at least a portion of the sequence

GAGGTGCAGCTGGTGGAGACCGGGGGAGGCCTGGCCCAGCCTGGGGGGTCCCTG AGACTCTC CTGTGCAGCCTCT, GGATTCACATTCAGTAGTTACAC, ATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGTTGGGTCTCACAC, ATTAGTGCGGGTAGTAGTTACATA,

TACTACTCAGACTCAGTGAAGGGCCGATTCACCGTCTCCAGAGACAACGTCAGGA

ACTCAGT

ATATCTGCAAATGAACAGCCTGAGAGCCGCTGACACGGCTGTGTATTACTGT,

GCGAGAGAGGATTATGGTCCGGGAAATTATTATAGTCCTAACTGGTTCGACCCC, TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG and / or

GAGGTGCAGCTGGTGGAGACCGGGGGAGGCCTGGCCCAGCCTGGGGGGTCCCTG

AGACTCTC

CTGTGCAGCCTCTGGATTCACATTCAGTAGTTATAACATGAACTGGGTCCGCCAG

GCTCCAG

GGAAGGGGCTGGAGTGGGTCTCACACATTAGTGCGGGTAGTAGTTACATATACTA

CTCAGAC

TCAGTGAAGGGCCGATTCACCGTCTCCAGAGACAACGTCAGGAACTCAGTATATC

TGCAAAT

GAACAGCCTGAGAGCCGCTGACACGGCTGTGTATTACTGTGCGAGAGAGGATTAT

GGTCCGG

GAAATTATTATAGTCCTAACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCAC CGTCTCC TCA, said part having at least 15 nucleotides. Said heavy chain sequence is preferably derived from the AM16 antibody. An isolated, synthetic or recombinant nucleic acid sequence comprising a heavy chain sequence consisting of any of the aforementioned heavy chain sequences is also disclosed herein.

[0108] Also disclosed herein is an isolated, synthetic or recombinant nucleic acid sequence comprising a light chain sequence that is at least about 70%, preferably at least about 80%, more preferably at least about 90% homologous to at least a portion of the sequence

CAGTCTGTCGTGACGCAGCCGCCCTCAGTGTCTGGGCCCCCCAGGGCAGAGAGTCA CCATCTC CTGCACTGGGAGC, AGCTCCAACATCGGGGCAGGTTATGAT,

GTACACTGGTACCAGCAGCTTCCAGGAACAGCCCCCAAACTCCTCATCTAT, GGCA

-36ACACT,

AATCGGCCCTCAGGGGTCTCCGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAG CCTCCCT GGCCATCACTGGACTCCAGGCTGAGGATGAGGCTGATTATTACTGC, CACTCCTATGACAGAAGCCTGAGTGGT, TCAGTATTCGGCGGAGGGACCAAGCTG ACCGTCCTAG and / or CAGTCTGTCGTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGAGTCA CCATCTC

CTGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGATGTACACTGGTACCAG

CAGCTTC

CAGGAACAGCCCCCAAACTCCTCATCTATGGCAACACTAATCGGCCCTCAGGGGT

CTCCGAC

CGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGACTCCA

GGCTGA

GGATGAGGCTGATTATTACTGCCACTCCTATGACAGAAGCCTGAGTGGTTCAGTA TTCGGCG GAGGGACCAAGCTGACCGTC, said part having at least 15 nucleotides. Said light chain sequence is preferably derived from the AM16 antibody. An isolated, synthetic or recombinant nucleic acid sequence comprising a heavy chain sequence consisting of any of the aforementioned light chain sequences is also disclosed herein.

[0109] Further disclosed is an isolated, synthetic or recombinant nucleic acid sequence comprising a heavy chain sequence that is at least 70%, preferably at least 80%, more preferably at least 90% homologous to at least a portion of the CAGGTGCAACTGGTGGAGTCTGGGGGAAATGTGGTCAAGCCTGGGACGTCCCTG sequence

AGACTGTC CTGTGCAGCGACT, GGATTCAACTTCCATAACTACGGC,

ATGAACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCGGTT,

GTTTGGTATGATGGAAGTAAGAAA,

TACTATGCAGACTCCGTGACGGGCCGATTCGCCATCTCCAGAGACAATTCCAAGA

ACACTCT

GTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCTGTTTATTATTGT,

GTGAGAGATAAAGTGGGACCGACTCCCTACTTTGACTCC,

TGGGGCCAGGGAACCCTGGTCACCGTATCCTCAG and / or

GAGGTGCAGCTGGTGGAGTCTGGGGGAAATGTGGTCAAGCCTGGGACGTCCCTG

AGACTGTC

CTGTGCAGCGACTGGATTCAACTTCCATAACTACGGCATGAACTGGGTCCGCCAG

GCTCCAG

GCAAGGGGCTGGAGTGGGTGGCGGTTGTTTGGTATGATGGAAGTAAGAAATACT

ATGCAGAC

TCCGTGACGGGCCGATTCGCCATCTCCAGAGACAATTCCAAGAACACTCTGTATC

TGCAAAT

GAACAGCCTGAGAGTCGAGGACACGGCTGTTTATTATTGTGTGAGAGATAAAGTG

GGACCGA

-37CTCCCTACTTTGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT, said part having at least 15 nucleotides. Said heavy chain sequence is preferably derived from the AM23 antibody. An isolated, synthetic or recombinant nucleic acid sequence comprising a heavy chain sequence consisting of any of the aforementioned heavy chain sequences is also disclosed herein.

[0110] Also disclosed herein is an isolated, synthetic or recombinant nucleic acid sequence comprising a light chain sequence that is at least 70%, preferably at least 80%, more preferably at least 90% homologous to at least a portion of the sequence TCCTATGTGCTGACTCAGCCACCCTCGGTGTCACTGGCCCCAGGAGGGACGGCCG CGATCAC CTGTGGAAGAAAC, AACATTGGAAGTGAAACT,

GTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCGTCTAT, GAT

GATGAC,

GACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGG CCACCCT GACCATCAGCAGGGTCGAGGCCGGGGATGAGGCCGACTATTACTGT, CAGGTGTGGGATAGGAGTAATTATCATCAGGTA, TTCGGCGGAGGGACCAAGTTG ACCGTCCTAG and / or TCCTATGTGCTGACTCAGCCCCCCTCGGTGTCACTGGCCCCAGGAGGGACGGCCG CGATCAC

CTGTGGAAGAAACAACATTGGAAGTGAAACTGTGCACTGGTACCAGCAGAAGCC

AGGCCAGG

CCCCTGTGCTGGTCGTCTATGATGATGACGACCGGCCCTCAGGGATCCCTGAGCG

ATTCTCT

GGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAGGCCGGG

GATGAGGC

CGACTATTACTGTCAGGTGTGGGATAGGAGTAATTATCATCAGGTATTCGGCGGA GGGACCA AGCTGACCGTC, said part having at least 15 nucleotides. Said light chain sequence is preferably derived from the AM23 antibody. An isolated, synthetic or recombinant nucleic acid sequence comprising a heavy chain sequence consisting of any of the aforementioned heavy chain sequences is also disclosed herein.

[0111] Also disclosed is a nucleic acid sequence encoding an amino acid sequence that is at least 70%, preferably at least 80%, more preferably at least 90% identical to at least a functional part of the amino acid sequence as shown in Figure 11, Figure 14A, Figure 14B and / or Figure 14C, wherein said portion has at least 5 amino acid residues. Said nucleic acid sequence may encode an amino acid sequence that is at least 80% identical to the heavy chain 1, 2 and / or CDR sequence and / or light chain 1 or 2 CDR sequences shown in Figure 11D. Said nucleic acid sequence may encode an amino acid sequence which is at least 80% identical to at least one of the CDR sequences shown in Figure 14A,

-38Figura 14B and / or Figure 14C. Said nucleic acid sequence may encode an amino acid sequence that is at least 70% identical to the heavy chain sequence depicted in Figure 11A, to the heavy chain sequences shown in Figure 14A, to the heavy chain sequences shown in Figure 11B, to the heavy chain sequences shown in Figure 14C, to the light chain sequences shown in Figure 11A, to the light chain sequences shown in Figure 14A, to the light chain sequences shown in Figure 14B, and / or to the light chain sequences shown in Figure 14C.

[0112] Further disclosed is an isolated, synthetic or recombinant nucleic acid sequence comprising a sequence coding for an amino acid sequence that is at least 70%, preferably at least 80%, more preferably at least 85% identical to the amino acid sequence shown in Figure 11A-D. Said nucleic acid sequence may encode an amino acid sequence that is at least 80% identical to the heavy chain 1, 2 and / or CDR sequence and / or light chain 1 or 2 CDR sequences as shown in Figure 11A-D. One embodiment discloses an isolated, synthetic or recombinant nucleic acid sequence comprising a sequence coding for an amino acid sequence that is at least 70% identical to the NYIIN amino acid sequence,

PVLGTVHYAPKFQGRVTITADESTDTAYIHLISLRSEDTAMYYCATETALWST

TYLPHYFDN WGQGTLVTVSS, and / or at least 70% identical to the sequence DIQMTQSPSSLSAAVGDRVTITCQASQDIVNYLNWYQQKPGKAPKLLIYVASN LETGVPSRFSGSGSGTDFSLTISSLQPEDVATYYCQQYDNLPLTFGGGTKVEIK RTV. The nucleic acid sequence disclosed herein may be 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.

[0113] Further disclosed is an isolated, synthetic or recombinant nucleic acid sequence comprising a sequence coding for an amino acid sequence that is at least 70%, preferably at least 80%, more preferably at least 85% identical to the amino acid sequence as shown in Figure 14A-C. Said nucleic acid sequence may encode an amino acid sequence which is at least 70% identical to the CDR sequence as shown in Figure 14A, 14B and / or 14C. An isolated, synthetic or recombinant nucleic acid sequence may comprise a sequence coding for an amino acid sequence that is at least 70% identical to the amino acid sequence selected from the group consisting of: GFSFSHYA, ISYDGENT, ARDRIVDDYYYYMMDV, QDIKKY, DAS, QQYDNLPPLT, EVQLVESGGGWQPGRSLRLSCAASGFSFSHYAMHWVRQAPGKGLEWVAVIS

YDGENTYYADSVKGRFSISRDNSKNTVSLQMNSLRPEDTALYYCARDRIVDD

-39YYYYGMDVWGQGATVTVSS,

DIQMTQSPSSLSASVGDRVTITCQASQDIKKYLNWYHQKPGKVPELLMHDASNLETG VPSRF SGRGSGTDFTLTISSLQPEDIGTYYCQQYDNLPPLTFGGGTKVEIKRTV, GFTFSSYN, ISAGSSYI, AREDYGPGNYYSPNWFDP, SSNIGAGYD, GNT, HSYDRSLSG,

EVQLVETGGGLAQPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSHI sags SYIYYS D SVKGRFTVSRDNVRNSVYLQMNSLRAADTAVYYCAREDYGPGNYYSPNWFDPWGQ GTLVTVS s, QSVVTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNTNRPS GVSD RFSGSKSGTSASLAITGLQAEDEADYYCHSYDRSLSGSVFGGGTKLTV.

GFNFHNYG, VWYDGSKK, VRDKVGPTPYFDS, NIGSET, DDD, QVWDRSNYHQV, EVQ LVESGGNWKPGTSLRLSCAATGFNFHNY GMNWVRQAPGKGLEWVA

WWYDGSKKYYAD SVTGRFAI SRDNSKNTLYLQMNSLRVEDTAVYYCVRDKVGPTPYFDSWGQGTLVTVSS, and S YVLTQPPSVS LAPGGTAAI TCGRNNI GSETVHWYQQKPGQAPVL WYDDDDRPSGI PERFS GSNSGNTATLT AND SRVEAGDEADYYCQVWDRSNYHQVFGGGTKLTV. The nucleic acid sequence disclosed herein may be 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.

[0114] As already explained above, the nucleic acid sequences of the present invention as defined in the claims are particularly suitable for the expression of an antibody of the invention, preferably D25, AM14, AM16, AM23 in a nucleic acid expression system. The nucleic acid sequence of the present invention as defined in the claims is preferably expressed in a cell, more preferably in a production cell adapted to produce an antibody.

[0115] The invention is explained in more detail in the following examples. These examples do not limit the scope of the invention, but merely serve to explain the invention.

Examples

MATERIALS AND METHODS

Maintenance and isolation of human B cells [0116] Using standard procedures, CD19 positive human B cells were isolated from the top blood bank blood clot (other sources may be fresh blood with an anticoagulant factor, or an organ such as tonsil or spleen). Briefly, all peripheral blood mononuclear cells (PBMC) were isolated by Ficoll separation (Amersham, Buckinghamshire, UK). CD22 labeled beads were used for positive B cell selection using the MACS cell sorting technique as described by the manufacturer (Miltenyi, Utrecht, The Netherlands). The cells were then stained with the appropriate combinations of monoclonal antibodies (mAbs) to CD19, CD27, IgD, IgM and IgA (Becton Dickinson (BD), Franklin Lakes, NJ, USA). Memory B cells that are

-40 positive for CD19 and CD27 and negative for IgM, IgA and IgD, were then sorted using FACSAria (BD) (Figure 1). In addition, memory B cells, other subsets of B cells, such as naive, naive, vesicular, memory, producing antibodies, centroblasts, centrocytes, from proliferation centers, plasmoblasts, plasma cells, marginal zone, peri-lateral or transient B-cells (many of these subsets have been only determined in mice) can be isolated using appropriate markers.

Cell Culture [0117] Sorted cells were washed and grown in 24-well plates (1.5 to 2x10⁵cells / ml) on 80 Gray, irradiated L-cells expressing CD40L (5x10⁴cells / ml; provided by DR. J. Banchereau, Schering Plow France, Dardilly France), in complete medium (Iscove's Modified D Minimal Essential Medium containing 8% fetal calf serum (FCS) and Penicillin / Streptomycin). Unless otherwise indicated, these CD40L expressing L cells are always present in cultures, in combination with 8% FCS. To prepare cell B for retroviral transduction, cells were cultured for 36 hours in the presence of mouse IL-21 (50 ng / ml, R & D, Minneapolis, MN, USA). After transduction, the cells are preferably cultured in the presence of IL-21, however, the cells react on IL4, IL-15 and IL-10 (not excluding other cytokines). For example, IL-4 induced B-cell expansion is lower compared to IL-21 and lower levels of cell division may be necessary in some experiments.

Retroviral Constructs and Production of a Recombinant Retrovirus [0118] Constitutively active mutants of STAT5a and b have been described previously. DNA encoding these mutants and wild type STAT5b was obtained from T. Kitamura (IMSUT, Tokyo, Japan). Bcl-6 was identified in senescence rescue screenings in mouse fibroblasts as an anti-proliferative inhibitor of p19ARF-p53 signaling. Bcl-XL was identified as an anti-apoptotic agent that was provided courtesy of Dr. Korsmeyer (Howard Hughes Medical Institute, Boston, US). These DNAs were ligated into the LZRS-linker-IRESGFP vector (or IRES-YFP or IRES-NGFR), which has been described previously (Heemskerk et al., 1997; Heemskerk et al., 1999). Instead of the IRES-GFP marker (Green Fluorescent Protein, green fluorescent protein), IRES-YFP (Yellow Fluorescent Protein) was also used. yellow fluorescent protein) or IRES-NGFR (Nerve Growth Factor Receptor, nerve growth factor receptor). NGFR is an incompetent signaling NGFR mutant, provided courtesy of Dr. C. Bonini. Monoclonal antibodies against NGFR (Chromaprobe, Mountain View, CA, US or Miltenyi) were used to visualize cells expressing NGFR.

[0119] For the production of a recombinant retrovirus, retroviral plasmids were transfected into a virus-free amphotropic helper producing the Phoenix-A cell line derived from the human germline germ line 293 (Kinsella and Nolan, 1996) (courtesy Dr. G. Nolan, Stanford University , Palo Alto, CA), using Fugene-6 (Roche Diagnostics Netherlands, Almere, The Netherlands) according to the manufacturer's protocols. Two days later selection of transfected cells

Was started by adding 2 μg / ml puromycin (Becton Dickinson Clontech Laboratories, Palo Alto, CA). Ten to 14 days after transfection 6 x 10⁶ cells were plated on a 10 cm Petri dish (Becton Dickinson Discovery Labware, Bedford, MA) in 10 ml of complete medium without puromycin. The next day, the medium was refreshed and the retroviral supernatant was collected the next day, centrifuged and frozen in aliquots in cell-free 70 ° C. This approach gives reproducible rapid production, on a large scale and with high retroviral titers of more than 3 x 10⁶ infectious virus particles / ml.

Retroviral Transduction [0120] The procedure for the transduction of recombinant human fibronectin CH-296 fragments (RetroNectin ™, Takara, Otsu, Japan) was performed as previously described (Heemskerk et al., 1997; Heemskerk et al., 1999). 24-well plates not treated with tissue culture (Costar, Badhoevedorp, The Netherlands) were coated with 0.3 ml 30 μg / ml of the recombinant human fibronectin CH-296 fragment at room temperature for 2 hours or overnight at 4 ° C. When plates of different size without tissue culture were used, the reagents were used proportionally. The CH-296 solution was removed and then incubated with 2% human serum albumin (HSA) in phosphate buffered saline (PBS) for 30 min at room temperature followed by washing once with PBS. 5x10⁵ B cells that were prepared for retroviral transduction were seeded in 0.25 ml of RPMI without FCS and L cells and mixed with 0.25 ml of thawed retroviral supernatant. For Bcl-6 Bcl-XL double transduction, 125 μl Bcl-6-IRES-NGFR (or IRES-YFP) was mixed (Shvarts A. et al. Genes Dev., 2002) and 125 μl Bcl-XL-IRES-GFP (provided by S. Korsmeyer, Howard Hughes Medical Institute, Childrens Hospital, Boston, USA) and added to the cells. The culture was then centrifuged at 1800 rpm at 25 ° C for 60 minutes and incubated for 6 hours at 37 ° C. 0.25 ml of the supernatant was then removed and 0.25 ml of fresh retroviral supernatant was added. The culture was again centrifuged at 1800 rpm at 25 ° C for 60 minutes and incubated at 37 ° C overnight. The next morning, the cells were transferred to a 24-well tissue culture treated (Costar) plate and cultured for 3-5 days under normal conditions in the presence of human IL-4 (50 ng / ml) or mouse IL-21 (50 ng / ml, R & D, Minneapolis). , MN, USA). Transduction efficiency was determined by truncation with an antibody signaling an incompetent nerve growth factor receptor (ANCiFR, provided by C. Bonini, St. Raphael Hospital, Milan, Italy) or (co) expression of GFP and / or YFP. Then, cells containing the transgene (s) of interest were selected for further experiments. signaling an incompetent nerve growth factor receptor (ANCiFR, provided by C. Bonini, St. Raphael Hospital, Milan, Italy) or (co) expressing GFP and / or YFP. Then, cells containing the transgene (s) of interest were selected for further experiments. signaling an incompetent nerve growth factor receptor (ANCiFR, provided by C. Bonini, St. Raphael Hospital, Milan, Italy) or (co) expressing GFP and / or YFP. Then, cells containing the transgene (s) of interest were selected for further experiments.

Flow cytometry [0121] Antibodies against human IgD, IgG, CD3, CD19, CD20, CD27, CD38, CD40, CD45, CD56, CD70, CD80, CD86, HLA-DR (BD) molecules directly labeled with FITC were used for flow cytometric analysis. , PE, PERCP, PE-Cy5, APC or APC-Cy7 and IgM, kappa light chain, lambda light chain, CD138, directly labeled with PE (DAKO). Stained cells were analyzed using LSRII (BD) and FACS data were processed using FlowJo computer software (Tree Star, Inc).

Proliferation and memory B cells were isolated from fresh PBMCs on FACSAria: Naive B cells: CD19-Pe-Cy7 pos, CD27-APC neg, IgD-PE pos B memory cells: CD19-PeCy7 pos, CD27-APC pos, IgD-PE neg, IgA-FITC neg. Cells were washed with PBS and resuspended in 0.5 ml RPMI (37 ° C) without FCS. An equal amount of IMDM containing 2μΜ succinyl -limidyl ester of carboxyfluorescein (CFSE) was added to the cell mixture and incubated for 7 min at 37 ° C. Excessive cell labeling was stopped by washing the cells with cold FCS. The cells were resuspended in 500μl IMDM-8% FCS and cultured with L-cells and in the presence or absence of IL-21. Unlabeled cells were used as a control. After 36 hours (immediately prior to transduction), the cell proportion was analyzed for CFSE content. The remaining cells were transduced by centrifugation with Bcl-6-IRES-NGFR, grown for 3 days and analyzed for CFSE content by LSRII. Data was analyzed using FlowJo software (Treestar)

Isolation of Antigen-Specific Human B Cells by Fast Single Cell Sorting [0123] In addition to the method of isolating the memory B cells described above starting from MBC (i.e., 100 cell culture / well), human memory B cells can also be incubated with a fluorescent labeled antigen and sorted based on antigen recognition. An example is the isolation of B cells that bind tetanus toxoid (Tetanus Toxoid) labeled with phycoerythrin (PE) (provided by A. Radbruch, Berlin, Germany) (Figure 4). Cells were grown at 1 cells / well and checked for TT binding. Despite this, you can use any other labeled antigen.

Determination of B cell receptor expression (BCR) after prolonged culture of Bcl-6 and Bcl-XL transduced cells [0124] B cells that differentiate during in vitro culture are known to lose membrane expression of BCR, which is also observed in EBV transformed B cells. Therefore, B cells transduced with Bcl-6 and Bcl-XL and cultured in the presence of IL-21 were stained for GFP, NGFR, CD19, Kappa and / or Lambda or IgG or labeled tetanus toxoid. To show the usefulness of BCR expression, we sorted TT-PE binding cells (Radbruch) using FACSAria (BD) at 1 cells / well in 96 wells on which L cells were plated and IL-21 containing culture medium. After three weeks, binding of tetanus toxoid to growing clones using FACS Canto (BD) was checked.

Combined development of BCL6 and Bcl-XL with positive B cell lines that secrete antibodies [0125] Cell lines B that produce monoclonal antibodies and are 100% doubly positive Bcl-6 and Bcl-XL were created. First, this goal was achieved by inducing proliferation and differentiation using IL-21. At the same time, these cells

They are transduced with Bcl-6-IRES-NGFR and Bcl-XL-IRES-GFP retroviruses. The cells are kept on IL-4 for 3-4 days. Cells that are transduced with one or both retroviruses then express the transgene and thus express the NGFR or GFP protein. NGFR and / or GFP expression can be visualized using LSRII (BD). If necessary, the cells can be re-transduced to obtain higher cell numbers expressing both transgenes. Regardless of the second transduction, cells expressing both transgenes are classified using FACS Aria (BD) and grown at a cell density in the range of 10-500 cells / well in 96 well plates in the presence of IL-21 and 2500 to 5000 L cells / well. These MBC cultures (mini-bulkcultures) secrete relatively large amounts of antibodies in the culture supernatant already on day 5, which can then be used for screening purposes. Screening may be based on techniques available for the antigen of interest, e.g. ELISA / EIA / RIA, Western blot or direct functional assays, such as neutralization of the cytokine blocking experiment. After screening and selection of MBCs that recognize the antigen of interest (TT and RSV in our experiments), the cells were subcloned at 0.5-1 cells / well in 96 wells in the presence of IL-21. Subcloning usually takes 2-3 weeks and can be carried out by limiting dilution (LD) cultures or single-cell sorting using flow cytometry (FACSAria). Screening may be based on techniques available for the antigen of interest, e.g. ELISA / EIA / RIA, Western blot or direct functional assays, such as neutralization of the cytokine blocking experiment. After screening and selection of MBCs that recognize the antigen of interest (TT and RSV in our experiments), the cells were subcloned at 0.5-1 cells / well in 96 wells in the presence of IL-21. Subcloning usually takes 2-3 weeks and can be carried out by cultures at limiting dilution (LD) or single cell sorting using flow cytometry (FACSAria). Screening may be based on techniques available for the antigen of interest, e.g. ELISA / EIA / RIA, Western blot or direct functional assays, such as neutralization of the cytokine blocking experiment. After screening and selection of MBCs that recognize the antigen of interest (TT and RSV in our experiments), the cells were subcloned at 0.5-1 cells / well in 96 wells in the presence of IL-21. Subcloning usually takes 2-3 weeks and can be carried out by limiting dilution (LD) cultures or single-cell sorting using flow cytometry (FACSAria). which recognize the antigen of interest (TT and RSV in our experiments), the cells were subcloned at 0.5-1 cells / well in 96 wells in the presence of IL-21. Subcloning usually takes 2-3 weeks and can be carried out by limiting dilution (LD) cultures or single-cell sorting using flow cytometry (FACSAria). which recognize the antigen of interest (TT and RSV in our experiments), the cells were subcloned at 0.5-1 cells / well in 96 wells in the presence of IL-21. Subcloning usually takes 2-3 weeks and can be carried out by limiting dilution (LD) cultures or single-cell sorting using flow cytometry (FACSAria).

RSV A-2 virus resource and HEp2 cell line [0126] RSV A-2 virus (courtesy of G. van Bleek, WKZ, Utrecht) and the HEp2 cell line (Clinical Laboratory, AMC, Amsterdam), grown in large quantities and frozen in liquid nitrogen.

[0127] The adjacent HEp2 cell line was grown in normal medium in Falcon T175 bottles, after which the aliquots were frozen.

[0128] To obtain a high RSV titer resource, HEp2 cells were seeded and grown to reach 50-60% confluence. The original RSV stock was added (1/20 total dilution volume 5 ml) for 45 min at RT on HEp2 cells. 15 ml of fresh medium was added and the cells were left overnight at 37 ° C, 5% CO 2 with the lid open. The next morning, the culture supernatant was carefully removed and 15 ml of medium containing 1% FCS was added. The cells were left for 24 to 36 hours at 37 ° C, 5% CO 2 with the lid closed. If

RSV induced syncytia were clearly visible and most of the syncytes were intact, the medium was collected, filtered (0.22 μm) and centrifuged at 1450 rpm at RT, after which the samples were immediately frozen and stored in liquid nitrogen. The second harvest can be obtained by immediately adding new medium containing 1% FCS and freezing this batch 4-6 hours later.

RSV lysate for ELISA [0129] HEp2 cells that were infected with RSV A-2 to obtain virus stocks were used to isolate RSV proteins. First, the cells were carefully washed with PBS and styzsized. Trypsin (Gibco) was washed away and the cell pellet was lysed in 1%

-4-polytyl glucose (the cell pellet from one T175 flask was treated with 2 ml of octyl glucoside). The suspension was homogenised using a syringe and needle (10 times up and down), incubated for 1 hour on ice and then dialyzed against 2L of buffer

TBS pH 7.4, overnight at 4 ° C. The supernatant was obtained after centrifugation of cellular debris.

The protein content was determined at 3.6 mg / ml and was used at 20 μg / ml (50 μΐ) in ELISA. Determination of TCID50 and PFU of RSV resources [0130] To determine TCID50, 10⁴ HEp2 was seeded in 96-well plates and infected with 2 or 10 step serial dilutions of RSV virus in a 4-well. 2-3 days later the supernatant was removed and the cells fixed with 80% acetone for 10 min at RT. After removing the acetone, the layer of fixed cells was dried and kept at 4 ° C or frozen at -20 ° C. To stain the RSV HEp2 cells, the plates were first blocked with 5% powdered milk in PBS 0.1% Tween 20. The plates were then washed 3 times and then incubated for 3-5 hours at 37 ° C with a polyclonal goat anti-RSV-HRP (1 : 500, Biodesign, Saco, ME, US) and washed extensively. The wells were then incubated with the substrate on AEC for 30 min at RT. Infected foci are colored red and can be visually inspected with an optical microscope and can be counted. The standard Excel program was used to determine the TCID50.

[0131] To determine the number of plaque-forming units (PFU) of the virus, 1x10⁵/ ml HEp-2 cells in 24-well plates were incubated with 10-fold serial dilutions (10^-3 - 10^-7) of RSV virus in 1% FCS medium at 37 ° C for 45 (200 μΐ), after which cells and virus were covered with 0.5 ml of 0.25% SeaPlaque agar (Biozyme) at body temperature. The agarose layer prevents the virus from spreading to uninfected cells through the culture medium. In this way, the virus can infect only neighboring cells that are finally killed by the virus to form plaques in a single layer of HEp2 cells. Plaques can best be visualized by staining the fixed cells (96% ethanol - 100% acetic acid - 10% formalin 6: 2: 1) with 1% crystal violet solution. Plaques are counted (by at least two different people) and the PFU value can be determined.

Selection of Neutralizing Antibodies Syncytial Respiratory Virus (RSV) [0132] To obtain B cell clones of anti-respiratory respiratory virus (RSV), peripheral blood cells (PBMCs) from two donors were isolated from the upper blood bank blood clot (donor B62 and B63 ). Before sorting CD19 cells^posIgM^negIgD^negIgA^negCD27^pos using FACSAria (BD) (Figure 1), CD22 + cells were isolated using beads and MACS columns (Miltenyi). Only, when mentioned differently, the cells were cultured with L cells. Cells were grown for 36 hours in the presence of IL-21, before transduction with Bcl-6-IRES-NGFR alone. After 12 h, the cells were harvested and cultured for 3 days in the presence of IL-4, after which NGFR expressing cells were sorted using MACS beads (Miltenyi) and immediately transduced with Bcl-XL-IRES-GFP. B cells that do not bind to MACS beads were washed and transduced with Bcl-6 and Bcl-XL at the same time. After 12 h, the cells were harvested, combined and cultured

For 3 days in the presence of IL-4, and then sorted for expression of GFP and NGFR on FACSAria.

The cells were washed and grown at 100 cells / well in 96 well plates (Costar) in the presence of IL-21.

[0133] Double transduced B cell cultures of Bcl-6 and Bcl-XL were screened for RSV binding using ELISA on an RSV infected cell lysate of HEp2 and tested in parallel using RSV microneutralization experiments. In short, 10⁴ HEp2 cells are plated in 96-well flat-bottom plates (Costar) in complete medium. The next day, the medium is replaced for 1 h at RT with a RSV virus mixture and a cell culture supernatant that is pre-incubated for 30 min at 37 ° C. The total volume is 25 μΐ and the final concentration of RSV is 0.1 MOI. After 1h, the virus supernatant mixture is diluted 9 times with PBS and replaced with 100 μΐ IMDM / 5% FCS. After 2 days, the cells are fixed with 80% acetone and polyclonal anti-RSV-HRP colors (Biodesign). Due to H2O2 and AEC, red color appears in cells infected with RSV. By means of light microscopy, infected cells can be observed and counted as necessary. As RSV neutralization control, polyclonal goat anti-RSV (Abcam, Cambridge, MA) is used. RT-PCR and cloning of VH and VL regions [0134] Total RNA was isolated from ~ 5x10⁵ B cells using the RNeasy® mini kit (Qiagen, Venlo, The Netherlands). 250 ng of total RNA was reverse-transcribed in a volume of 20 μl containing 1x first strand buffer, 500 μΜ dNTP, 250 ng random hexamers, 5 mM DTT, 40 U RNasin (Promega) and 200 U SuperScript III RT (Invitrogen). cDNA was diluted 10X in Ultrapure water and 2.5 μΐ cDNA was PCR-tested in 50 μΐ of a solution containing 20 mM Tris-HCL, 50 mM KCL, 2.5 mM MgCl2, 250 μΜ dNTP, 1 U AmpliTaq Gold DNA polymerase (Applied Biosystems Inc.) and 25 pmol each starter. The PCR conditions were: 8 min denaturation step at 96 ° C, then 35 cycles at 96 ° C, 30 s at 60 ° C, 1 min at 72 ° C, and finally at 10 min at 72 ° C.

[0135] The PCR products were analyzed in agarose gels, purified and cloned into the cloning vector pCR2.1 TA according to manufacturers' recommendations. Sequence analysis was performed using BigDye Terminator chemistry (Applied Biosystems Inc.) and Vector-NTI software (Invitrogen).

[0136] To exclude DNA reverse transcriptase and / or DNA polymerase mutations, several independent cDNA and PCR reactions were performed and the sequence was individually cloned and analyzed. Consensus sequences were determined using the Vector-NTI Contig Express software.

[0137] For expression of recombinant protein antibodies in 293T cells, full-length heavy and light chain constructs were generated in pCDNA3.1 (+) Zeo (Invitrogen). The heavy chain expression vector was constructed by PCR amplification of the heavy chain leader sequence and the VH region of clone D25 introducing the 5'-NheI site and the 3'-XhoI site. The IgG1 constant region (CH1-hinge-CH2-CH3) was amplified from the same cDNA while introducing the 5'-XhoI and 3'-NotI site. The full-length heavy chain expression vector was obtained by ligation of three points to digested

-46Nhel / Notl pCDNA3.1 (+) Zeo. The full-light light chain expression construct was generated by PCR amplification of the light chain leader sequence, the VL region and the light chain constant region with the 5'-NheI and 3'-NotI primers. The latter product was cloned into NheI / NotI digested pCDNA3.1 (+) Zeo to obtain the full-length light chain expression vector.

[0138] Sequence analysis was performed to confirm the correctness of the expression constructs.

[0139] Transient dual transfection (Fugene-6, Roche, Germany or Lipofectamine LTX, Invitrogen) of 293T cells with expression vectors of both the heavy and light chain was performed to generate a recombinant monoclonal antibody. FACS staining with the obtained culture supernatant (48 hours) on RSV-infected Hep2 cells was performed to show the functional binding of the antibody to the RSV F protein.

[0140] The oligonucleotides used for PCR amplification were:

VH regions:

VH1-For

VH1B-For

VH2A-For

VH2B-For

VH3-For

VH3B-For

VH4-For

VH5-For

VH6- For

Cgamma-Rev

AAATCGATACCACCATGGACTGGACCTGGAGG 5'-3 '

AAATCGATACCACCATGGACTGGACCTGGAGM 5'-3 '

AAATCGATACCACCATGGACACACTTTGCTMCAC 5'-3 '

AAATCGATACCACCATGGACATACTTTGTTCCAAC 5'-3 '

AAATCGATACCACCATGGAGTTTGGGCTGAGC 5'-3 '

AAATCGATACCACCATGGARYTKKGRCTBHGC 5'-3 '

AAATCGATACCACCATGAAACACCTGTGGTTCTT 5'-3 '

AAATCGATACCACCATGGGGTCAACCGCCATC 5'-3 '

AAATCGATACCACCATGTCTGTCTCCTTCCTC 5'-3 '

GGGTCTAGACAGGCAGCCCAGGGCCGCTGTGC 5'-3 '

Vkappa regions:

VK1-For

Vk1B-For

Vk2-For

Vk3-For

VK4-For

Ck-Rev

AAATCGATACCACCATGGACATGAGGGTCCCY 5'-3 '

AAATCGATACCACCATGGACATGAGRGTCCYY 5'-3 '

AAATCGATACCACCATGAGGCTCCCTGCTCAG 5'-3 '

AAATCGATACCACCATGGAARCCCCAGCGCA 5'-3 '

AAATCGATACCACCATGGTGTTGCAGACCCAG 5'-3 '

GATCGCGGCCGCTTATCAACACTCTCCCCTGTTGAAGCTCTT 5'-3 '

Vlambda regions:

-47Vllaecb 5'-AAATCGATACCACCATGGCCTGGTCCCCTCTCCTCC-3 '

Vl1g 5'-AAATCGATACCACCATGGCCGGCTTCCCTCTCCTCC-3 '

Vl2 / 10 5'-AAATCGATACCACCATGGCCTGGGCTCTGCTCCTCC-3 '

Vl3jpah 5'-AAATCGATACCACCATGGCCTGGACCGCTCTCCTGC-3 '

V15 / 7 5'-AAATCGATACCACCATGGCCTGGTCTCCTCTCCTTC-3 '

V16 / 9 5'-AAATCGATACCACCATGGCCTGGCTCTCCTCTCCTTC-3 '

Vl3rm 5'-AAATCGATACCACCATGGCCTGGATCCCTCTCCTCC-3 '

Vl3l 5'-AAATCGATACCACCATGGCCTGGACCCCTCTCTGGC-3 '

Vl3e 5'-AAATCGATACCACCATGGCCTGGGCCACACTCCTGC-3 '

Vl4c 5'-AAATCGATACCACCATGGCCTGGGTCTCCTTCTACC-3 '

Vl8a 5'-AAATCGATACCACCATGGCCTGGATGATGCTTCTCC-3 '

C12 / 7 5'-GATCGCGGCCGCTTATCAWGARCATTCTGYAGGGGCCACTG-3 '[0141] The oligonucleotides used to construct the expression vector were as follows:

Expressive vector heavy chain:

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

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

CHfw-XhoI: 5'-CGCGCTCGAGTGCCTCCACCAAGGGCCCATCGGTC-3 'CHrev-NotI: 5'-GATCGCGGCCGCTTATCATTTACCCGGRGACAGGGAGAGGC-3'

Light chain expressive vector:

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

CK-NotI: 5'-GATCGCGGCCGCTTATCAACACTCTCCCCTGTTGAAGCTCTT-3 '

EBV RT-PCR [0142] To test whether a strong proliferative response was associated with the presence of EBV, EBV RT-PCR was performed. The RT procedure is described above. The PCR conditions were as follows: a 7-minute denaturation step at 94 ° C, followed by 30 cycles of 30s at 94 ° C, 30s at 62 ° C (HPRT1), 52 ° C (LMP-1) and 58 ° C (EBNA1 / 2) ) and 30s at 72 ° C, and finally a 7-minute extension at 72 ° C. The oligonucleotides used for RT-PCR were as follows: HPRT1 forward (5'-TATGGACAGGACTGAACGTCTTGC-3 ') GACACAAACATGATTCAAATCCCTGA-3'); GCGACTCTGCTGGAAATGAT-3 ') and GACATGGTAATGCCTAGAAG-3');

AGCAAGAAGAGGAGGTGGTAAG-3 ')

GGCTCAAAGTGGTCTCTAATGC-3 ').

and HPRT1 LMP-1

LMP-1

EBNA1 / 2 and EBNA1 / 2 reverse front:

Inverse reverse (5 '(5') (5 '(5') (5'-48) In addition to RT-PCR, we perform PCR directly on the cell pellet and DNA supernatant, which were isolated using QIAmp isolation kit (Qiagen) ).

EXAMPLE 1

RESULTS

B cell phenotype [0144] The use of human memory B cells as a platform for the isolation of therapeutic drugs is based on the ability to grow and test these cells over a relatively long period of time. Human B cells can be cultured and maintained in laboratory conditions, but not long enough to expand, select and clone single B cell lines against the antigen of interest. We have developed immortalization methods based on the genetic modification of human B cells. We studied the later targets of STAT5. One goal, apart from others, is Bcl-6. Bcl-6 inhibits the differentiation of B cells into plasma cells that are arrested in proliferation. Overexpression of Bcl-6 maintains the balance of BLIMP1, a transcription factor, whose expression is strongly increased by stimulation of B cells with IL-21 (works by STAT3). BLIMP1 is necessary to induce the development of Ig-producing cells (CD20-CD38 +), whereas Bcl-6 can prevent this (cells maintain the expression of CD20, the so-called breeding center phenotype).

[0145] To investigate possible warping of some cell populations in the B cell compartment, CFSE labeling before stimulation of fresh human B naive cells and memory showed that all cells start dividing and that all B cell populations are equally transduced (Figure 2). B memory cells transduced with Bcl-6 and cultured in the presence of IL-21 and IL-4 are shown. Naive B cells were transduced at a lower level and the division rates were lower after 36 hours, but identical to memory B cells after a further 3 days of culture (data not shown).

[0146] Next, we show that Bcl-6, together with Bcl-XL (anti-apoptotic, downstream STAT5 target), CD40L signaling and in the presence of IL-21, maintain human IgG memory B cells in the CD20 + CD38dull phenotype over a long period of time (> 3 months) (Figure 3). In addition, Bcl-6 Bcl-XL B cells have a phenotype corresponding to activated B cells (see Table 1, for an example of FACS staining of 3 B + TTT clones), because these cells have high expression of CD80, CD86 and HLA-DR. determined on three different Bcl-6 Bcl-XL B cell clones cultured with IL-21 and CD40L signaling

<td>coloration</td><td>score</td><td>coloration</td><td>score</td>

<td>CD2</td><td>neg</td><td>CD69</td><td>neg</td>

<td>CD5</td><td>neg</td><td>CD70</td><td>item</td>

<td>CD7</td><td>neg</td><td>CD71</td><td>item</td>

<td>CD10</td><td>item</td><td>CD73</td><td>neg</td>

<td>coloration</td><td>score</td><td>coloration</td><td>score</td>

<td>CD20</td><td>item</td><td>CD80</td><td>h / High</td>

<td>CD21</td><td>item</td><td>CD86</td><td>item</td>

<td>CD22</td><td>item</td><td>CD95</td><td>h / High</td>

<td>CD23</td><td>neg / 5% item</td><td>CD126</td><td>neg</td>

<td>CD24</td><td>neg</td><td>CD132 (typically gamma)</td><td>item</td>

<td>CD25</td><td>item</td><td>CD138</td><td>neg / 2% pos</td>

<td>CD27</td><td>neg / low</td><td>CD154 (CD40L)</td><td>8% pos</td>

<td>CD28</td><td>neg</td><td>ICOSL</td><td>item</td>

<td>CD30</td><td>pos (56-74%)</td><td>IgM</td><td>neg</td>

<td>CD38</td><td>pos / intermediate</td><td>IgG</td><td>item</td>

<td>CD40</td><td>item</td><td>HLA-DR</td><td>pos (high)</td>

<td>CD44</td><td>item</td><td>Kappa</td><td>pos / neg</td>

<td>CD45</td><td>item</td><td>lambda</td><td>pos / neg</td>

<td>CD45RA</td><td>h / High</td><td>IL21-R</td><td>item</td>

Antibody Expression of Antibodies [0147] Transduced with Bcl-6 Bcl-XL, EBV negative cells retained positive BCR expression as determined by antigen binding or Kappa and Lambda staining (Figure 3 and 4). Accordingly, such cells are particularly suitable for isolation and / or screening after a long period of culture for the desired specificity, e.g. using a labeled antigen, as such cells will bind said tagged antigen to their BCR. This was confirmed by single-cell sorting of B-double-trimoped Bcl-6 and Bcl-XL cells that bind PE-labeled TT using FACSAria. After three weeks, single-cell sorted clones were stained with the appropriate markers and TT-PE in 96-well plates and measured for binding in FACS Canto (BD) (Figure 4). In summary, in the case

Cell division and growth curves [0148] The Bcl-6 Bcl-XL transduced B cells are on average 0.6 times a day.

The partition rates vary between donors and cell density of the culture (Figure 5a).

The D25 anti-RSV clone had a division rate of 0.47 times a day (Figure 5b). Cells can be grown at densities below 1 cell / 96 well for cloning purposes.

Isolation of antibodies from Bcl-6 Bcl-XL B cells

[0149] Bcl-6 Bcl-XL transduced B cells secrete an average of one μg / ml of antibodies, which is sufficient for culturing the amounts necessary for preclinical studies (Figure 6).

Surprisingly, the D25 anti-RSV clone produced three times as many antibodies as compared to the other cell lines tested.

Determination of ABV content [0150] EBV RT-PCR on mRNA of Bcl-6 Bcl-XL cell lines that were cultured with IL21 and CD40L signaling. In the cell lines obtained by this immortalization technique, no EBV gene transcript was ever detected (data not shown). Selection procedure [0151] Due to the stability of BCR growth and expression, these cells are well-suited for isolating antigen-specific B cells. This gave us the opportunity to use several different selection and cloning procedures. One is to immediately obtain antigen-specific cells after introducing Bcl-6 and Bcl-XL by FACS or sorting into Magnetic Bead using the labeled antigen of interest, thereby increasing the likelihood of generating multiple clones of antigen-specific B cells. Another option is the growth of purified, pooled B memory cells (or any other) transduced with Bcl-6 Bcl-XL at low cell densities (e.g., 100 cells / well). The supernatants from these 100c / w cultures can be collected and tested for their specificity. 100 cell / well cultures that have been shown to be positive for antigen recognition are then subcloned by limiting dilution cultures to generate monoclonal cell line antibodies. With both methods we could isolate over 40 clones of B cells recognizing tetanus toxoid (TT). Therefore, these clones were either selected for binding TT to BCR on FACSAria or were screened by screening the ELISA series of cultures until a single monoclonal anti-TT cell line was isolated (not shown). cumulative B memory cells (or any other) transduced with Bcl-6 Bcl-XL at low cell densities (e.g., 100 cells / well). The supernatants from these 100c / w cultures can be collected and tested for their specificity. 100 cell / well cultures that have been shown to be positive for antigen recognition are then subcloned by limiting dilution cultures to generate monoclonal cell line antibodies. With both methods we could isolate over 40 clones of B cells recognizing tetanus toxoid (TT). Therefore, these clones were either selected for binding TT to BCR on FACSAria or were screened by screening the ELISA series of cultures until a single monoclonal anti-TT cell line was isolated (not shown). cumulative B memory cells (or any other) transduced with Bcl-6 Bcl-XL at low cell densities (e.g., 100 cells / well). The supernatants from these 100c / w cultures can be collected and tested for their specificity. 100 cell / well cultures that have been shown to be positive for antigen recognition are then subcloned by limiting dilution cultures to generate monoclonal cell line antibodies. With both methods we could isolate over 40 clones of B cells recognizing tetanus toxoid (TT). Therefore, these clones were either selected for binding TT to BCR on FACSAria or were screened by screening the ELISA series of cultures until a single monoclonal anti-TT cell line was isolated (not shown).

Selection of RSV neutralizing antibodies [0152] From donor B63, a culture of 100 cells / well completely blocked infection and RSV replication. D10, one of the neutralizing cultures of 100 cells / well produced a strong anti-RSV antibody that was cloned by culture by limiting dilutions. One of the monoclonal antibodies, D25, was used for further studies. D25, a monoclonal antibody with an IgG1 heavy chain as determined by commercial ELISA (Sanquin, Amsterdam, not shown) and the Kappa light chain (Figure 7), very effectively blocked RSV infection with an IC50 between 0.5 and 1.5 ng / ml (± 10pM), while the IC50 of the standard anti-RSV antibody used in the clinic (Palivizumab developed by MedImmune) is 0.453 μg / ml (3.02nM) (H. Wu et al. 2005 J.Mol.Biol. and A. Mejias et al. 2005 Antimicrob. Agents Chemother. ) (Figure 8). Antigen recognition

[0153] In addition to neutralization experiments, D25 binding to RSV infected HEp2 cells was determined. HEp2 cells were infected using a regular virus production protocol. HEp2 cells infected with RSV were styzsynized and incubated with 25-50 μΐ culture supernatant. The cells were washed and stained with mouse-anti-human IgG-PE (BD or Jackson) to detect the binding of D25 antibodies to infected cells. The r-Biopharm ELISA control antibody was used as an internal control. Figure 9a shows binding of D25 to intact RSV-infected HEp2 cells.

[0154] Since the RSV envelope membrane proteins are present in two proteins, namely the G and F proteins, D25 binding has been tested on VSV virus infected cells with either pseudotyped or neither RSV or G RSV F protein (supplied courtesy of John K Rose). As shown in Figure 9b, D25 was strongly bound to EL-4 cells infected with the VSV-F protein. In an attempt to examine the epitope recognized by D25 versus palivizumab, VSV-F protein-infected EL-4 cells were incubated with an increasing amount of D25 and palivizumab. The cells were washed and stained with a mixture of 3 mouse anti-RSV-F antibodies (Dako). In contrast to Palivizumab, which competed for binding to infected VSV-F cells with a mouse anti-RSV-F antibody, D25 binding was unaffected (data not shown).

[0155] Figure 9c shows the binding of Palivizumab (Synagis) and D25 in a concentration-dependent manner to infected HEp 2 cells. Since both antibodies bind 1 to 1 to their target protein, there is no difference in binding to infected HEp 2 cells.

Incidence of antigen-binding RSV binding clones We calculated that the incidence of antigen-specific memory B cells that bind RSV was 17%, and the frequency of antigen-specific cells that neutralize RSV was 6% as determined for the B63 donor. . D25 binds to a conformational epitope which is different from the one recognized by palivizumab. This is illustrated in Figure 10, where D25 does not bind to denatured, linear epitopes presented by the lysate of lysed RSV infected cells coated on ELISA plates, whereas palivizumab binds to denatured protein (F).

Isolation and purification of antibody fragments [0157] From several B cell lines, including the RSV D25 highly neutralizing clone, we were able to grow quantities such as 500 ml. These culture supernatants contain at least 2 μLg / ml, so we should be able to get a sufficiently purified antibody to perform preclinical studies (on animals). Purification is carried out using a Montage Antigen Purification Kit (Millipore, Billerica, MA, USA) and HiTrap Protein A HP columns (GE Healthcare, Diegem, Belgium).

[0158] In addition, 293T cells were transfected with a heavy and light D25 chain that was subcloned in protein expression vectors pCDA3.1 using lipofectamine.

LTX (Invitrogen). The amount of IgG that was present in the supernatant was about 22 μLg / ml (total volume 50 ml). This is an antibody derived from a cloned sequence

The 52-nucleotide antibody expressed by the B cell line D25 also recognized infected HEp2 cells (a dish not shown).

Antibody sequence [0159] Figure 11a shows the nucleotide and amino acid sequence of the heavy and light chain of the B63D10-D25 clone. Using standard RT-PCR primers and antibody-specific primers, the heavy (Vh1-69) and light chain (VkI O8 / 018) sequences were determined. The entire antibody sequence was cloned using TOPO vectors and after sequence control, it was subcloned into the mammalian expression protein pCDNA3.1 (Invitrogen). Figures 11b and 11c show the VH and VL4 clone chains, Stars indicate mutations compared to the Vh1-69 germline sequences that must take place during affinity maturation and further selection of B cells.

[0160] In summary, we show here the isolation, characterization and long-term culture of human memory B cells using the Bcl-6 and Bcl-XL transgene. They give us the tools necessary to isolate antibodies with unique properties, such as anti-RSV B63D10-B25 monoclonal antibody. Because B cells are of human origin, they can be easily used as a therapeutic drug.

EXAMPLE 2 [0161] The heavy and light chain of D25 was cloned into a standard expression vector as previously described (p44 'antibody sequence'). To create an expression construct that allows expression of the protein at the maximum level, the D25 heavy and light chain sequences have been codon optimized using GENEART (Regensburg, Germany). Additional restriction sites have been created in this procedure to simplify cloning procedures in the future, but most importantly, the nucleotide codons that are translated into the amino acid sequences have been optimized for maximum translation into the protein. In this way, the nucleotide sequence was optimized, but the amino acid sequence remained unchanged. EXAMPLE 4 shows the ability to neutralize purified D25 derived from B cell supernatant, recombinant D25 and D25 optimized using GENEART. All effectively neutralize RSV.

[0162] GENEART modifications compared to the original D25 sequence are shown in Figure 12.

EXAMPLE 3 [0163] In addition to RSV in vitro neutralization experiments, we tested monoclonal antibody D25 in in vivo models. The models that have been described in relation to in vivo anti-RSV tests are BALB / ci cotton (Sigmodon hispidus) mice (Mejias A et al., Antimicrobial Agents and chemotherapy 2004; p1811, Johnson S et al., JID 1997; p1215 and Wu H et al., JMB 2007: p652). The BALB / c mouse model is clearly the weakest model, but because cottoncaps are difficult to obtain and maintain, we first set the D25 tests on BALB / c mice.

-53 Protocol: Antibodies specific to RSV in BALB / c, Day 5 [0164] Experimental design:

Day 1. IP injection of 100μΐ of antibodies

Day 0. Infection with 1x10⁷ pfu RSV A2 in 50μΐ

Day 1 to 5, check the general well-being and weight of the mouse

Day 5, autopsy, collect BAL, blood and lungs

Get blood via venipuncture

Collect 2.0ml BAL through the tracheal cannula

Collect lungs

Immediately start TCID50 on BAL material (1 ml)

Freeze 1ml of BAL material (ELISA cytokine / RT-PCR) -80C. Perform TCID50 on the prepared longer material (1ml)

Freeze 1 ml of long material (ELISA cytokine / RT-PCR) -80C. Collect / centrate blood for hIgG ELISA serum and store at -80C [0165] The results are shown in Figure 13:

(A) One day before RSV challenge (1x10⁷ RSV-A2 particles) via a nasal spray, animals were injected with different amounts of Synagis (MedImmune), purified by D25 or IgG1 control antibody (Eureka) (Table 3). (Figure 13B) Human IgG levels were determined in mouse sera from day 5 and decrease in serum antibody concentration after 5 days; Table 4 provides an overview of half-life values. Figure 13D shows viral loads found in lung lavage (BAL) on day 5 in treated and untreated animals, while Figure 13E shows the number of T and B cells in the peripheral blood of treated and untreated mice. Figure 13F shows lung and bronchial histology with infiltration (usually mainly eosinophils) of treated and untreated animals.

Conclusion / Result:

[0166] The estimate of half-life of D25 is 5 to 9 days based on (linear) calculation that 60 and 30Pg of antibody were injected on day 0 (2 and 1 mg / kg respectively) and on day 5 33 or 16Pg was detected (total volume of mouse 1 5). When we started with 0.5mg / kg of injections per animal at d0, then Ig levels drop from 15μg to 1 g at day 5, which could indicate a 9-day half-life (Table 4).

Table 4 mg / kg in total given d0 Xg) detected in d5 Xg) half-life (days)

2.0

5.6

-541.0 30 16 5.4

0.5. 9 9.4. [0167] The virus titer determined in the TCID50 assay shows that 1x10 10 can be detected in control animals.⁴ PFU, while the virus was not detected in animals treated with Synagis (2mg / kg) or D25 (2, 1 and 0.5mg / kg).

[0168] Animals treated with Synagis or D25 maintain higher% of peripheral CD4 T cells and B220 B cells. Synagis treated animals (2mg / kg) have fewer CD4 T cells compared to those treated with D25. Although this may not be significant, it is important to remember that animals treated with a low dose of D25 (1 and 0.5 mg / kg) maintain a high level of B and T lymphocytes relative to the animals treated with the control.

[0169] Although the histological data (Figure 13F) are not quantitative, it is evident that Synagis and D25 reduce the influx of immune cells to the lungs and around the bronchi compared to the control. When D25 and Synagis are compared, animals treated with D25 appear to have less cellular infiltration on the lungs and around the bronchi.

[0170] To test D25 in cotton boxes, experiments are set to compare pre-treated Synagis and D25 animals prior to challenge with RSV-X virus in NVI (Bilthoven, The Netherlands).

EXAMPLE 4 [0171] In addition to B63-D10-D25, we isolated three new strong RSV neutralizing antibodies (AM14, AM16 and AM23) from the same donor (B63). 100 cells per well of bulk B cell culture, which were originally selected for neutralization of RSV virus and frozen and stored in liquid nitrogen, thawed and the supernatant tested for binding to RSV infected HEp2 cells. We tested for binding to infected Hep2 cells because it is a marker for the recognition of antibodies of native RSV membrane oligomeric proteins like F and G proteins and can serve as a good prognostic factor for neutralization. When binding was detected, the cells were grown as single cells and screened for binding to obtain clones. All three antibodies were cloned into the GENEART vector, which was originally built for D25. In addition, as D25 they all recognize the RSV F protein (not shown). After cloning and expression in 293T cells, the recombinant protein was purified (the nucleotide and amino acid sequences are shown in Figure 14A, B and C). Antibodies were tested for neutralization against several primary RSV isolates on Vero and HEp2 cells (Figure 15). All three antibodies are of the IgG1 isotype. AM14 has a Kappa light chain, whereas AM16 and AM23 have a Lambda light chain. All three antibodies, like D25, contain somatic hypermutations of their antibody variable domains, suggesting that in vivo they have undergone affinity maturation during the reaction at breeding sites, in a process that creates unique antibody sequences. the recombinant protein was purified (nucleotide and amino acid sequences are shown in Figure 14A, B and C). Antibodies were tested for neutralization against several primary RSV isolates on Vero and HEp2 cells (Figure 15). All three antibodies are of the IgG1 isotype. AM14 has a Kappa light chain, whereas AM16 and AM23 have a Lambda light chain. All three antibodies, like D25, contain somatic hypermutations of their antibody variable domains, suggesting that in vivo they have undergone affinity maturation during the reaction at breeding sites, in a process that creates unique antibody sequences. the recombinant protein was purified (nucleotide and amino acid sequences are shown in Figure 14A, B and C). Antibodies were tested for neutralization against several primary RSV isolates on Vero and HEp2 cells (Figure 15). All three antibodies are of the IgG1 isotype. AM14 has a Kappa light chain, whereas AM16 and AM23 have a Lambda light chain. All three antibodies, like D25, contain somatic hypermutations of their antibody variable domains, suggesting that in vivo they have undergone affinity maturation during the reaction at breeding sites, in a process that creates unique antibody sequences. All three antibodies are of the IgG1 isotype. AM14 has a Kappa light chain, whereas AM16 and AM23 have a Lambda light chain. All three antibodies, like D25, contain somatic hypermutations of their antibody variable domains, suggesting that in vivo they have undergone affinity maturation during the reaction at breeding sites, in a process that creates unique antibody sequences. All three antibodies are of the IgG1 isotype. AM14 has a Kappa light chain, whereas AM16 and AM23 have a Lambda light chain. All three antibodies, like D25, contain somatic hypermutations of their antibody variable domains, suggesting that in vivo they have undergone affinity maturation during the reaction at breeding sites, in a process that creates unique antibody sequences.

[0172] The results are shown in Figures 15-I and 15-II: RS virus neutralization test with purified supernatant of cell line B D25 (sD25),

-55-recombinant purified D25 (rD25), recombinant-optimized for codon using GENEART D25 (rD25 GA), AM14, AM16, AM23 (all being purified recombinant protein) and Synagis. Viral antibody neutralization was tested on two different cell cell lines (Figure 15-I). Vero and (Figure 15-II) Hep2 using different antibodies: A2 (A), X (B) and 2006/1 (C) are subtype A RSV, while the viruses Z (D) and 2007-2 (E) are subtype B. 100TCID50 of each virus was added to serial dilutions of antibodies in DMEM / 1% FCS and incubated for 1 hour at 37 degrees, after which 100ul Vero or HEp2 cells were added ( 1x10⁶/ Ml). The virus antibody mixtures were not washed. After three days, the supernatant was removed and the cells were fixed with 80% acetone for 10 minutes at RT. After removal of acetone, the fixed cell layer was dried and kept at 4 ° C or frozen at -20 ° C. For staining of RSV-infected HEp2 cells, the plates were first blocked with 5% powdered milk in PBS 0.1% Tween 20, then the plates were washed 3 times and then incubated for 3-5 hours at 37 ° C of the goat polyclonal anti-RSV-HRP (1: 500, Biodesign, Saco, ME, US) and washed extensively. All wells were then incubated with the AEC substrate for 30 min at RT. Infected foci are colored red and can be visually observed with an optical microscope and can be counted.

Result / conclusion [0173] All antibodies neutralize RSV strains A and B (Table 5). In general, different D25 antibodies neutralize RSV viruses effectively, although small differences between experiments can be seen. AM14 is as strong as D25, while AM ​​16 is just as strong as Synagis. Although AM23 neutralizes RSV strains very effectively, it is, however, less potent with respect to the neutralization of RSV strains B, although still comparable with Synagis.

Table 5 IC50 values ​​(ng / ml)

<td>Cell line used</td><td>subtype RSV</td><td>SD25</td><td>RD25</td><td>RD25 GA</td><td>AM14</td><td>ÄM16</td><td>AM23</td>

<td>vero</td><td>AND</td><td>3.4</td><td>1.6</td><td>3.2</td><td>15.2</td><td>304.3</td><td>19.4</td>

<td>vero</td><td>B</td><td>9.0</td><td>0.3</td><td>1.2</td><td>1.1</td><td>126.4</td><td>168.8</td>

<td>HepG2</td><td>AND</td><td>3.3</td><td>2.1</td><td>5.3</td><td>21.5</td><td>285.6</td><td>25.0</td>

<td>HepG2</td><td>B</td><td>14.3</td><td>1.9</td><td>1.3</td><td>6.7</td><td>124.8</td><td>190.7</td>

The IC50 value for each antibody of the RS virus subtype on Vero or HEp2 cells was calculated as the average of 50% neutralization from the three virus strains (A2, X and 2006-1). The IC 50 value for each antibody on the RS virus subtype B on Vero or HEp2 cells was calculated as the average 50% neutralization on the two virus strains (2007-2 and Z). Each of the neutralization tests was performed in triplicate and repeated twice (also shown in Figure 15A and B).

sD25 = purified supernatant of a culture derived from B cells

-25RD25 = purified recombinant D25 rD25 GA = supernatant of 293T cells with recombinant codon optimized using GENEART D25 EXAMPLE 5

Synergistic and blocking effects of anti-RSV antibodies.

[0174] To analyze whether D25, Synagis or a new set of AM antibodies mutually interfere with the recognition of RSV F protein, we pre-incubated RSV infected HEp2 cells with increasing concentrations of unlabeled antibodies until they reached the maximum binding plateau. We determine for each antibody the plateau phase in which no binding increase was detected when the Ig amount was increased. (Not shown). After washing, the samples were incubated with a standard dose (3 pmol) of labeled PE D25 or APC labeled with Synagis. This dose also gives the maximum binding.

Result [0175] As shown in Figure 16, tagged Synagis and D25 show reduced binding to RSV infected HEp2 cells when these cells are preincubated with unlabeled Synagis or D25. Synagis also shows a slight decrease in the binding of induced AM16. Binding of D25 is strongly blocked by AM23, but in contrast, D25 binding is strongly elevated after pre-incubation with AM14. This indicates that the epitope recognized by D25 is usually not even fully exposed, but the exposure increases after binding of AM14 to its native epitope. This shows that these two antibodies can cooperate with each other and increase neutralization.

Brief description of the drawings [0176]

Figure 1.

Isolation of human, IgG positive, memory B cells. PBMCs isolated from the upper blood clot by Ficoll separation (Amersham) were incubated with anti-CD22 magnetic beads and then isolated using MACS (Miltenyi) columns. CD22 positive cells were then incubated with antibodies against human CD19, CD27, IgM, IgD and IgA (BD). Negative cells for IgM, IgD and IgA and positive for CD19 and CD27 were sorted by rapid single cell sorting (FACSAria, BD).

Figure 2

CFSE staining. Fresh human memory B cells were isolated, labeled with CSFE and stimulated for 36h with IL-21, and transduced with Bcl-6-IRES-NGFR. The cells were kept for an additional 3 days on IL-21, and the CFSE content was determined. The CFSE dye is diluted with each cell division.

-57 Figure 3

An example of human B cells transduced with Bcl-6 and Bcl-XL or with Bcl-XL alone. Cells were maintained on irradiated L cells expressing CD40L and the cytokine IL-21. On the left, the BCR expression determined by kappa and lambda staining is shown (93% positive kappa lambda cells are of the IgG isotype, not shown). On the right side, CD38 expression is shown on the X-axis and CD20 expression on the Y-axes. CD38 staining^dullCD20 + indicates memory B cells or from breeding centers; CD38 staining⁺CD20^- indicates plasmoblasts.

Figure 4

Isolation of immortalized, antigen-specific human B cells. Human memory B cells were isolated as described in Figure 1 and then transduced with Bcl-6-IRES-NGFR and Bcl-XL-IRES-GFP. Cells expressing NGFR, GFP and binding to PE tagged toxin were isolated using FACSAria. The cells were cultured in single-celled flat-bottomed 96-well plates in the presence of irradiated L and IL-21 cells, and were subjected to selection based on TT-PE binding using FACS Canto (BD).

Figure 5

Accumulated cell growth and rate of division of B 6XL 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.

Figure 6

Fresh cultures were started from 200,000 cells / 24 wells in 1.0 ml IMDM with 8% FCS and pen / strep. The FCS used was either normal (HyClone) or FCS with ultra-low bovine IgG Ultralow Bovine IgG FCS (Gibco). After 3 days, the culture of the supernatant was replaced and the cell numbers were adjusted to 200,000 cells / ml. Average production of IgG after 3 days is measured at 3 consecutive time points, this difference was not statistically significant (p value 0.2).

Figure 7

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

Figure 8

From donor B63, cultures of 100 cells / well were grown using Bcl-6 Bcl-XL positive human memory B cells. One of these cultures, D10 showed strong neutralization. Monoclonal cell lines derived from LD were prepared, one D25 neutralizing RSV A-2 virus efficiently. D25 is shown here compared to palivizumab (synagis) and polyclonal goat anti-RSV. They are not shown

-Important culture supernatants of Bcl6 Bcl-XL culture of transduced B cell clones cultured with IL-21 and CD40L signaling, which produce high levels of antibodies, but do not block RSV infection. Clone D25 was used for further characterization.

Figure 9

In Figure 9a: HEp2 cells were plated at 10-12e6 cells per T175 (Nunc) flask in IMDM / 5% FCS. The next day, the medium was replaced with 5ml medium with RSV virus (1.0 MOI) and incubated for 45 minutes at RT, after which 20 ml of fresh medium was added and the cells were grown overnight at 37 ° C. The next day, the medium was replaced with IMDM / 1% FCS and grown overnight with the lid closed at 37 ° C. The next day, the cells were washed with PBS and treated with trypsin. For the coloration of infected cells, the primary incubation was carried out with the culture supernatant. Secondary incubation was done with anti-human IgG-PE (BD). The cells were analyzed using LSRII (BD). As a positive control, a positive ELISA KIT commercial control from r-Biopharm was used.

In Figure 9b: EL-4 cells were infected with VSV virus by pseudotyped F or G RSV protein (provided by courtesy of John Rose) and incubated with the culture supernatant D25. The cells were washed and incubated with anti-human-IgG-PE (Jackson) to detect binding of D25 to infected cells. Only binding of D25 to cells infected with the VSV virus by pseudotyped RSV F protein was detected. Figure 9c shows the binding of Palivizumab (Synagis) and D25 in a concentration-dependent manner to infected HEp 2 cells. Average Fluorescence Intensity (MFI) is shown.

Figure 10

Polyclonal goat anti-RSV binding (positive control), palivizumab (Synagis) and D25 to coated cell lysate of HEp2 infected cells.

Figure 11

Analysis of the sequence of clone D25. 11a shows the nucleotide sequence and predicted amino acid sequence of the heavy and light chain variable domains. 11b / c show the sequence of the heavy and light chain D25 in comparison to the expected germline. Asterisks are mutations that probably occurred during selection and maturation of the B-cell clone in vivo.

Figure 12

Cloning and expression of recombinant human antibodies from BCL6 BCL-xL transfected BCL cell lines. This has already been described for antibody D25 (Figure 11). The GENEART nucleotide modifications are shown in relation to the original D25 sequence, note that these mutations do not affect the amino acid composition of the D25 antibody.

-59 Figure 13

Provocation of BALB / c mice with purified, derived from B cell supernatant D25 and Synagis. (A) One day before RSV challenge (1x10⁷ RSV-A2 particles) by nasal spray, animals were injected IP with different amounts of Synagis (MedImmune), purified D25 or IgG1 control antibody (Eureka) (Table 3). (B) levels of human IgG were measured in mouse sera from day 5 and decrease in serum antibody levels after 5 days (C); Table 4 provides an overview of half-life values. Figure 13D shows viral loads found in lung lavage (BAL) on day 5 in treated and untreated animals, while Figure 13E indicates the numbers of T and B cells in the peripheral blood of treated and untreated mice. (F) shows lung histology with bronchi and infiltration (usually mainly eosinophils) of treated and untreated animals.

Figure 14

Nucleotide and amino acid sequences of the three new strong RSV neutralizing antibodies (A) AM14, (B) AM16 and (C) AM23.

Figure 15

RS virus neutralization test with purified supernatant B D25 cell line (sD25), recombinant purified D25 (rD25), recombinant codon optimized with GENEART D25 (rD25 GA), AM14, AM16, AM23 (all being purified recombinant protein) and Synagis. Viral antibody neutralization was tested on two different cell lines (Figure 15-I). Vero and (Figure 15-II) Hep2 using different antibodies A2 (A), X (B) and 2006/1 (C) are subtype A RSV, while the viruses Z (D) and 2007-2 (E) are subtype B. 100TCID50 of each virus was added to serial dilutions of antibodies in DMEM / 1% FCS and incubated for 1 hour at 37 degrees, after which 100ul Vero or HEp2 cells were added (1 x 10⁶/ Ml). Figure 16

Relative binding of a fixed amount (3pmol) of APC-labeled Synagis and PE-labeled rD25 to HEV2 cells infected with RSV that had previously been incubated with increasing concentrations of indicated unlabelled antibodies.

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