U.S. patent application number 14/026182 was filed with the patent office on 2014-12-25 for rsv-specific binding molecules and means for producing them.
This patent application is currently assigned to MedImmune Limited. The applicant listed for this patent is MedImmune Limited. Invention is credited to Tim Beaumont, Mark Jeroen Kwakkenbos, Hergen Spits, Etsuko Yasuda.
Application Number | 20140377279 14/026182 |
Document ID | / |
Family ID | 38578633 |
Filed Date | 2014-12-25 |
United States Patent
Application |
20140377279 |
Kind Code |
A9 |
Spits; Hergen ; et
al. |
December 25, 2014 |
RSV-SPECIFIC BINDING MOLECULES AND MEANS FOR PRODUCING THEM
Abstract
The invention provides antibodies and functional equivalents
thereof which are capable of specifically binding RSV, and means
and methods for producing them.
Inventors: |
Spits; Hergen; (Amsterdam,
NL) ; Beaumont; Tim; (Ouderkerk aan de Amstel,
NL) ; Kwakkenbos; Mark Jeroen; (Amsterdam, NL)
; Yasuda; Etsuko; (Amsterdam, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MedImmune Limited |
Cambridgeshire |
|
GB |
|
|
Assignee: |
MedImmune Limited
Cambridgeshire
GB
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20140072575 A1 |
March 13, 2014 |
|
|
Family ID: |
38578633 |
Appl. No.: |
14/026182 |
Filed: |
September 13, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12600950 |
May 6, 2010 |
8562996 |
|
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PCT/NL08/50333 |
May 30, 2008 |
|
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14026182 |
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Current U.S.
Class: |
424/159.1 ;
435/339; 435/455; 435/69.6; 530/389.4; 536/23.53 |
Current CPC
Class: |
C07K 16/1027 20130101;
A61P 31/14 20180101; A61P 9/10 20180101; C07K 2317/56 20130101;
A61P 37/04 20180101; A61P 31/12 20180101; C07K 2317/565 20130101;
A61P 31/18 20180101; A61P 31/16 20180101; C07K 2317/76 20130101;
A61P 9/00 20180101; A61P 11/00 20180101; A61K 2039/505
20130101 |
Class at
Publication: |
424/159.1 ;
530/389.4; 536/23.53; 435/339; 435/455; 435/69.6 |
International
Class: |
C07K 16/10 20060101
C07K016/10 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 1, 2007 |
EP |
07109472.6 |
Claims
1-3. (canceled)
4. An isolated, synthetic or recombinant antibody or a functional
part, derivative and/or analogue thereof which is capable of
specifically binding Respiratory Syncytial Virus and which
comprises: a heavy chain CDR1 sequence comprising a sequence which
is at least 80% identical to the sequence GFSFSHYA (SEQ ID NO:73),
and a heavy chain CDR2 sequence comprising a sequence which is at
least 80% identical to the sequence ISYDGENT (SEQ ID NO:74), and/or
a heavy chain CDR3 sequence comprising a sequence which is at least
80% identical to the sequence ARDRIVDDYYYYGMDV (SEQ ID NO:75), and
a light chain CDRI sequence comprising a sequence which is at least
80% identical to the sequence QDIKKY (SEQ ID NO:76), and a light
chain CDR2 sequence comprising a sequence which is at least 80%
identical to the sequence DAS, and a light chain CDR3 sequence
comprising a sequence which is at least 80% identical to the
sequence QQYDNLPPLT (SEQ ID NO:77).
5. An antibody, functional part, derivative or analogue according
to claim 4, having a heavy chain sequence comprising a sequence
which is at least 70% identical to the sequence
EVQLVESGGGVVQPGRSLRLSCAASGFSFSHYAMHWVRQAPGKGLEWVAVISYDGEN
TYYADSVKGRFSISRDNSKNTVSLQMNSLRPEDTALYYCARDRIVDDYYYYGMDVWG QGATVTVSS
(SEQ ID NO:78), and having a light chain sequence which is at least
70% identical to the sequence TABLE-US-00006 (SEQ ID NO: 79)
DIQMTQSPSSLSASVGDRVTITCQASQDIKKYLNWYHQKPGKVPELLMHD
ASNLETGVPSRFSGRGSGTDFTLTISSLQPEDIGTYYCQQYDNLPPLTFG GGTKVEIKRTV.
6. An isolated, synthetic or recombinant antibody or a functional
part, derivative and/or analogue thereof which is capable of
specifically binding Respiratory Syncytial Virus and which
comprises: a heavy chain CDR1 sequence comprising a sequence which
is at least 80% identical to the sequence GFTFSSYN (SEQ ID NO:80),
and a heavy chain CDR2 sequence comprising a sequence which is at
least 80% identical to the sequence ISAGSSYI (SEQ ID NO:81), and a
heavy chain CDR3 sequence comprising a sequence which is at least
80% identical to the sequence AREDYGPGNYYSPNWFDP (SEQ ID NO:82),
and a light chain CDR1 sequence comprising a sequence which is at
least 80% identical to the sequence SSNIGAGYD (SEQ ID NO:83), and a
light chain CDR2 sequence comprising a sequence which is at least
80% identical to the sequence GNT, and a light chain CDR3 sequence
comprising a sequence which is at least 80% identical to the
sequence HSYDRSLSG (SEQ ID NO:84).
7. An antibody, functional part, derivative or analogue according
to claim 6, having a heavy chain sequence comprising a sequence
which is at least 70% identical to the sequence
EVQLVETGGGLAQPGGSLRLSCAASGFTFSSYNMNWvRQAPGKGLEWVSHISAGSSYIY
YSDSVKGRFTVSRDNVRNSVYLQMNSLRAADTAVYYCAREDYGPGNYYSPNWFDPW GQGTLVTVSS
(SEQ ID NO:85), and having a light chain sequence which is at least
70% identical to the sequence TABLE-US-00007 (SEQ ID NO: 86)
QSVVTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLI
YGNTNRPSGVSDRFSGSKSGTSASLAITGLQAEDEADYYCHSYDRSLSGS VFGGGTKLTV.
8. An isolated, synthetic or recombinant antibody or a functional
part, derivative and/or analogue thereof which is capable of
specifically binding Respiratory Syncytial Virus and which
comprises: a heavy chain CDR1 sequence comprising a sequence which
is at least 80% identical to the sequence GFNFHNYG (SEQ ID NO:87),
and a heavy chain CDR2 sequence comprising a sequence which is at
least 80% identical to the sequence VWYDGSKK (SEQ ID NO:88), and a
heavy chain CDR3 sequence comprising a sequence which is at least
80% identical to the sequence VRDKVGPTPYFDS (SEQ ID NO:89), and a
light chain CDR1 sequence comprising a sequence which is at least
80% identical to the sequence NIGSET (SEQ ID NO:90), and a light
chain CDR2 sequence comprising a sequence which is at least 80%
identical to the sequence DDD, and a light chain CDR3 sequence
comprising a sequence which is at least 80% identical to the
sequence QVWDRSNYHQV (SEQ ID NO:91).
9. An antibody, functional part, derivative or analogue according
to claim 8, having a heavy chain sequence comprising a sequence
which is at least 70% identical to the sequence
EVQLVESGGNVVKPGTSLRLSCAATGFNFHNYGMNWvRQAPGKGLEvNAVVWYDGSK
KYYADSVTGRFAISRDNSKNTLYLQMNSLRVEDTAVYYCVRDKVGPTPYFDSWGQGT LVTVSS
(SEQ ID NO:92), and/or having a light chain sequence which is at
least 70% identical to the sequence TABLE-US-00008 (SEQ ID NO: 93)
SYVLTQPPSVSLAPGGTAAITCGRNNIGSETVHWYQQKPGQAPVLVVYDDD
DRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDRSNYHQVFGGG TKLTV.
10. (canceled)
11. An isolated, synthetic or recombinant nucleic acid sequence, or
a functional part, derivative or analogue thereof, encoding an
antibody, functional part, derivative or analogue according to
claim 4.
12. An antibody, functional part, derivative or analogue according
to claim 4, for use as a medicament and/or prophylactic agent.
13-15. (canceled)
16. An isolated antibody producing cell capable of producing an
antibody, functional part, derivative or analogue according to
claim 4.
17-18. (canceled)
19. An antibody producing cell according to claim 16, comprising:
an exogenous nucleic acid sequence encoding BCL6 or a functional
part, derivative and analogue thereof, and/or an exogenous nucleic
acid sequence encoding Bcl-xL or a functional part, derivative
and/or analogue thereof.
20. (canceled)
21. A method for producing an antibody producing cell, which is
stable for at least three months and which is capable of producing
RSV-specific antibodies, the method comprising: increasing an
expression level of Blimp 1 in a B cell which is capable of
producing RSV-specific antibodies; and increasing and/or
maintaining a BCL6 expression level in said B cell.
22-29. (canceled)
30. A method for producing antibodies which are capable of
specifically binding Respiratory Syncytial Virus, the method
comprising: producing an antibody producing cell capable of
producing RSV-specific antibodies with a method according to claim
21; and obtaining antibodies produced by said antibody producing
cell.
31. An isolated antibody obtainable by a method according to claim
30, or a functional part, derivative and/or analogue of said
antibody.
32-34. (canceled)
35. A method for at least in part treating or preventing an
RSV-related disorder, the method comprising administering to an
individual in need thereof a therapeutically effective amount of an
antibody or functional part, derivative or analogue according to
claim 4.
Description
[0001] The invention relates to the fields of biology and
medicine.
[0002] Respiratory Syncytial Virus (RSV) is a common cold virus
belonging to the family of paramyxovirus. RSV is virulent, easily
transmissible and the most common cause of lower respiratory tract
disease in children of less than 2 years of age. Up to 98% of
children attending day care will be infected in a single RSV
season. Between 0.5% and 3.2% of children with RSV infection
require hospitalization. Approximately 90,000 hospital admissions
and 4500 deaths per year were reported in United States. Major risk
factors for hospitalization due to RSV are premature birth, chronic
lung disease, congenital heart disease, compromised immunity, and
age younger than 6 weeks in otherwise healthy children. No
effective treatment of RSV positive bronchiolitis beside supportive
care in the form of adequate nutrition and oxygen therapy is
available. Antiviral therapies such as Ribavirin have not been
proven to be effective in RSV infection. One monoclonal antibody,
Palivizumab (also called Synagis), is registered for prophylaxis
against RSV infection. Palivizumab is a genetically engineered
(humanized) monoclonal antibody to the fusion protein of RSV.
However, Palivizumab is not always effective. Therefore, 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 counteracting and/or preventing an RSV-related
disease. It is a further object of the invention to provide
alternative and/or improved antibodies against RSV, or functional
equivalents of such antibodies, and to provide stable cells capable
of producing antibodies--or functional equivalents thereof--against
RSV.
[0004] The present invention provides antibodies and functional
equivalents thereof which are capable of specifically binding RSV.
Such antibodies and/or functional equivalents, also called herein
"anti-RSV antibodies" or "RSV-specific antibodies", are capable of
specifically binding at least one component of RSV, such as for
instance an epitope of an RSV protein, Non-specific sticking is not
encompassed by the term "specifically binding". Anti-RSV antibodies
and functional equivalents according to the present invention are
particularly suitable for counteracting and/or at least in part
preventing an RSV-infection and/or adverse effects of an RSV
infection. One particularly preferred anti-RSV antibody according
to the present invention is the antibody designated "D25", which
has a heavy chain region and a light chain region as depicted in
FIGS. 11A-C. The CDR sequences of D25, which in particular
contribute to the antigen-binding properties of D25, are depicted
in FIGS. 11B and 11C. Antibody D25 appears to have superior
characteristics as compared to the registered anti-RSV antibody
Palivizumab (FIG. 8). For instance, D25 has an IC50 value of about
0.4-1.5 ng/ml in an in vitro neutralization assay wherein HEp-2
cells are infected with RSV, whereas Palivizumab has an IC50 value
of about 453 ng/ml.
[0005] A functional equivalent of an antibody is defined herein as
a functional part, derivative or analogue of an antibody.
[0006] A functional part of an antibody is defined as a part which
has at least one same property as said antibody in kind, not
necessarily in amount. Said functional part is capable of binding
the same antigen as said antibody, albeit not necessarily to the
same extent. A functional part of an antibody preferably comprises
a single domain antibody, a single chain antibody, a single chain
variable fragment (scFv), a Fab fragment or a F(ab').sub.2
fragment.
[0007] A functional derivative of an antibody is defined as an
antibody which has been altered such that at least one
property--preferably an antigen-binding property--of the resulting
compound is essentially the same in kind, not necessarily in
amount. A derivative is provided in many ways, for instance through
conservative amino acid substitution, whereby an amino acid residue
is substituted by another residue with generally similar properties
(size, hydrophobicity, etc), such that the overall functioning is
likely not to be seriously affected.
[0008] A person skilled in the art is well able to generate
analogous compounds of an antibody. This is for instance done
through screening of a peptide library or phage display library.
Such an analogue has essentially at least one same property as said
antibody in kind, not necessarily in amount.
[0009] As is well known by the skilled person, a heavy chain of an
antibody is the larger of the two types of chains making up an
immunoglobulin molecule. A heavy chain comprises constant domains
and a variable domain, which variable domain is involved in antigen
binding. A light chain of an antibody is the smaller of the two
types of chains making up an immunoglobulin molecule. A light chain
comprises a constant domain and a variable domain. The variable
domain is, together with the variable domain of the heavy chain,
involved in antigen binding.
[0010] Complementary-determining regions (CDRs) are the
hypervariable regions present in heavy chain variable domains and
light chain variable domains. The CDRs of a heavy chain and the
connected light chain of an antibody together form the
antigen-binding site.
[0011] Now that the present invention provides the insight that the
CDR sequences depicted in FIG. 11 provide desired RSV-binding
characteristics, a skilled person is well capable of generating
variants comprising at least one altered CDR sequence. For
instance, conservative amino acid substitution is applied.
Conservative amino acid substitution involves substitution of one
amino acid with another with generally similar properties (size,
hydrophobicity, etc), such that the overall functioning is likely
not to be seriously affected,
[0012] It is also possible to change at least one CDR sequence
depicted in FIG. 11 in order to generate a variant antibody, or a
functional equivalent thereof, with at least one altered property
as compared to D25. Preferably, an antibody or functional
equivalent is provided comprising a CDR sequence which is at least
70% identical to a CDR sequence as depicted in FIG. 11, so that the
favorable binding characteristics of D25 are at least in part
maintained or even improved. A CDR sequence as depicted in FIG. 11
is preferably altered such that the resulting antibody or
functional equivalent comprises at least one improved property,
such as for instance an improved binding affinity, selectivity
and/or stability, as compared to D25. Variant antibodies or
functional equivalents thereof comprising an amino acid sequence
which is at least 70% identical to a CDR sequence as depicted in
FIG. 11 are therefore within the scope of the present invention.
Various methods are available in the art for altering an amino acid
sequence. For instance, a heavy chain or light chain sequence with
a desired CDR sequence is artificially synthesized. Preferably, a
nucleic acid sequence encoding a CDR sequence is mutated, for
instance using random- or site-directed-mutagenesis.
[0013] In a first aspect the invention thus provides an isolated,
synthetic or recombinant antibody or a functional equivalent
thereof which is capable of specifically binding Respiratory
Syncytial Virus and which comprises:
[0014] a heavy chain CDR1 sequence comprising a sequence which is
at least 70% identical to the sequence NYIIN (SEQ ID NO: 1),
and/or
[0015] a heavy chain CDR2 sequence comprising a sequence which is
at least 75% identical to the sequence GIIPVLGTVHYAPKFQG (SEQ ID
NO: 2), and/or
[0016] a heavy chain CDR3 sequence comprising a sequence which is
at least 70% identical to the sequence ETALVVSTTYLPHYFDN (SEQ ID
NO: 3), and/or
[0017] a light chain CDR1 sequence comprising a sequence which is
at least 85% identical to the sequence QASQDIVNYLN (SEQ ID NO: 4),
and/or
[0018] a light chain CDR2 sequence comprising a sequence which is
at least 70% identical to the sequence VASNLET (SEQ ID NO: 5).
[0019] Preferably, said antibody also comprises a light chain CDR3
sequence comprising a sequence which is at least 70% identical to
the sequence QQYDNLP (SEQ ID NO: 6).
[0020] Preferably, an antibody or a functional equivalent according
to the invention comprises a CDR sequence which is at least 75%,
more preferably at least 80%, more preferably at least 85%, more
preferably at least 90% identical to at least one of the CDR
sequences depicted in FIG. 11A. Most preferably, an antibody or a
functional equivalent according to the invention comprises a CDR
sequence which is at least 95% identical to at least one of the CDR
sequences depicted in FIG. 11A. The particularly preferred antibody
D25, described above, comprises CDR sequences which consist of the
CDR sequences depicted in FIG. 11A. A particularly preferred
embodiment according to the invention thus provides an isolated,
synthetic or recombinant antibody or a functional equivalent
thereof which is capable of specifically binding Respiratory
Syncytial Virus and which comprises:
[0021] a heavy chain CDR1 sequence comprising the sequence NYIIN
(SEQ ID NO: 1), and/or
[0022] a heavy chain CDR2 sequence comprising the sequence
GIIPVLGTVHYAPKFQG (SEQ ID NO: 2), and/or
[0023] a heavy chain CDR3 sequence comprising the sequence
ETALVVSTTYLPHYFDN (SEQ ID NO: 3), and/or a light chain CDR1
sequence comprising the sequence QASQDIVNYLN (SEQ ID NO: 4),
and/or
[0024] a light chain CDR2 sequence comprising the sequence VASNLET
(SEQ ID NO: 5).
[0025] Preferably, said antibody also comprises a light chain CDR3
sequence comprising the sequence QQYDNLP (SEQ ID NO: 6).
[0026] In one embodiment an antibody or functional equivalent is
provided which comprises the three heavy chain CDR sequences and
the three light chain CDR sequences as depicted in FIGS. 11B and
11C, or sequences that are at least 70%, preferably at least 80%,
more preferably at least 85% identical thereto. Further provided is
therefore an isolated, synthetic or recombinant antibody or a
functional equivalent thereof which comprises a heavy chain CDR1
sequence comprising a sequence which is at least 70% identical to
the sequence NYIIN (SEQ ID NO: 1) and a heavy chain CDR2 sequence
comprising a sequence which is at least 70% identical to the
sequence GIIPVLGTVHYAPKFQG (SEQ ID NO: 2) and a heavy chain CDR3
sequence comprising a sequence which is at least 70% identical to
the sequence ETALVVSTTYLPHYFDN (SEQ ID NO: 3) and a light chain
CDR1 sequence comprising a sequence which is at least 70% identical
to the sequence QASQDIVNYLN (SEQ ID NO: 4) and a light chain CDR2
sequence comprising a sequence which is at least 70% identical to
the sequence VASNLET (SEQ ID NO: 5), and a light chain CDR3
sequence comprising a sequence which is at least 70% identical to
the sequence QQYDNLP (SEQ ID NO: 6). Said antibody or functional
equivalent preferably comprises CDR sequences which 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 heavy chain CDR sequences and the light chain CDR
sequences as depicted in FIGS. 11B and 11C. An antibody or
functional equivalent comprising the above mentioned heavy chain
CDR1, CDR2 and CDR3 sequences as well as the above mentioned light
chain CDR1, CDR2 and CDR3 sequences is also provided.
[0027] Antibodies or functional equivalents thereof comprising a
variable heavy chain amino acid sequence which is at least 70%
identical to the heavy chain sequence as depicted in FIG. 11 is
also provided. Such heavy chain sequences provide desired
RSV-binding properties, as evidenced by antibody D25. Further
provided is therefore an antibody or a functional equivalent
thereof, having a heavy chain sequence comprising a sequence which
is at least 70% identical to the sequence
QVQLVQSGAEVKKPGSSVMVSCQASGGPLRNYIINWLRQAPGQGPEWMGGII
PVLGTVHYAPKFQGRVTITADESTDTAYIHLISLRSEDTAMYYCATETALVVST
TYLPHYFDNWGQGTLVTVSS (SEQ ID NO: 7). Moreover, variable light chain
amino acid sequences which are at least 70% identical to the light
chain sequence as depicted in FIG. 11 also provide desired
RSV-binding properties, as evidenced by antibody D25. An antibody,
or a functional equivalent thereof having a light chain sequence
which is at least 70% identical to the sequence
DIQMTQSPSSLSAAVGDRVTITCQASQDIVNYLNWYQQKPGKAPKLLIYVASNLETG
VPSRFSGSGSGTDFSLTISSLQPEDVATYYCQQYDNLPLTFGGGTKVEIKRTV (SEQ ID NO:
8) is therefore also provided. An antibody or functional part
according to the invention preferably comprises a variable heavy
chain sequence and/or a variable light chain sequence which is at
least 75%, more preferably at least 80%, more preferably at least
85%, more preferably at least 90%, most preferably at least 95%
identical to the heavy chain sequence and/or the light chain
sequence as depicted in FIG. 11. The higher the homology, the more
closely said antibody or functional part resembles antibody D25. An
antibody or functional part according to the invention preferably
comprises a heavy chain as well as a light chain which resemble the
heavy and light chain of D25. Further provided is therefore an
antibody or functional part comprising a heavy chain sequence and a
light chain sequence which are at least 70%, more preferably at
least 80%, more preferably at least 85%, more preferably at least
90%, most preferably at least 95% identical to the heavy chain
sequence and the light chain sequence as depicted in FIG. 11.
[0028] One embodiment provides an antibody or functional equivalent
thereof comprising a heavy chain sequence consisting of the heavy
chain sequence as depicted in FIG. 11, and a light chain sequence
consisting of the light chain sequence as depicted in FIG. 11.
Alternatively, as is well known by the skilled person, it is
possible to generate a shortened heavy chain or light chain
sequence while maintaining a binding property of interest.
Preferably, such a shortened heavy chain or light chain is
generated which has a shorter constant region, as compared to the
original heavy or light chain. The variable domain is preferably
maintained. For instance, a Fab fragment or F(ab).sub.2 fragment
based on a heavy chain sequence or light chain sequence depicted in
FIG. 11 is produced. A functional equivalent of an antibody
comprising at least a functional part of a sequence as depicted in
FIG. 11 is therefore also provided. Said functional part has a
length of at least 20 amino acids and comprises a sequence which is
at least 70% identical to the heavy chain CDR1 sequence depicted in
FIGS. 11B and 11C, and/or a sequence which is at least 75%
identical to the heavy chain CDR2 sequence depicted in FIGS. 11B
and 11C, and/or a sequence which is at least 70% identical to the
heavy chain CDR3 sequence depicted in FIGS. 11B and 11C, and/or a
sequence which is at least 85% identical to the light chain CDR1
sequence depicted in FIGS. 11B and 11C, and/or a sequence which is
at least 70% identical to the light chain CDR2 sequence depicted in
FIGS. 11B and 11C. Preferably, said functional part also comprises
a sequence which is at least 70% identical to the light chain CDR3
sequence depicted in FIGS. 11B and 11C.
[0029] Another particularly preferred anti-RSV antibody according
to the present invention is the antibody designated "AM14", which
has a heavy chain region and a light chain region as depicted in
FIG. 14A. The CDR sequences of AM14, which in particular contribute
to the antigen-binding properties of AM14, are also depicted in
FIG. 14A.
[0030] Now that the present invention provides the insight that the
CDR sequences depicted in FIG. 14A provide desired RSV-binding
characteristics, a skilled person is well capable of generating
variants comprising at least one altered CDR sequence. For
instance, conservative amino acid substitution is applied.
Conservative amino acid substitution involves substitution of one
amino acid with another with generally similar properties (size,
hydrophobicity, etc), such that the overall functioning is likely
not to be seriously affected.
[0031] It is also possible to change at least one CDR sequence
depicted in FIG. 14A in order to generate a variant antibody, or a
functional equivalent thereof, with at least one altered property
as compared to AM14. Preferably, an antibody or functional
equivalent is provided comprising a CDR sequence which is at least
70% identical to a CDR sequence as depicted in FIG. 14A, so that
the favorable binding characteristics of AM14 are at least in part
maintained or even improved. A CDR sequence as depicted in FIG. 14A
is preferably altered such that the resulting antibody or
functional equivalent comprises at least one improved property,
such as for instance an improved binding affinity, selectivity
and/or stability, as compared to AM14. Variant antibodies or
functional equivalents thereof comprising an amino acid sequence
which is at least 70% identical to a CDR sequence as depicted in
FIG. 14A are therefore within the scope of the present invention.
Various methods are available in the art for altering an amino acid
sequence. For instance, a heavy chain or light chain sequence with
a desired CDR sequence is artificially synthesized. Preferably, a
nucleic acid sequence encoding a CDR sequence is mutated, for
instance using random- or site-directed-mutagenesis.
[0032] In one aspect the invention thus provides an isolated,
synthetic or recombinant antibody or a functional part, derivative
and/or analogue thereof which is capable of specifically binding
Respiratory Syncytial Virus and which comprises:
[0033] a heavy chain CDR1 sequence comprising a sequence which is
at least 70% identical to the sequence GFSFSHYA (SEQ ID NO: 73),
and/or
[0034] a heavy chain CDR2 sequence comprising a sequence which is
at least 70% identical to the sequence ISYDGENT (SEQ ID NO: 74),
and/or
[0035] a heavy chain CDR3 sequence comprising a sequence which is
at least 70% identical to the sequence ARDRIVDDYYYYGMDV (SEQ ID NO:
75), and/or
[0036] a light chain CDR1 sequence comprising a sequence which is
at least 70% identical to the sequence QDIKKY(SEQ ID NO: 76),
and/or
[0037] a light chain CDR2 sequence comprising a sequence which is
at least 70% identical to the sequence DAS, and/or
[0038] a light chain CDR3 sequence comprising a sequence which is
at least 70% identical to the sequence QQYDNLPPLT (SEQ ID NO;
77).
[0039] Preferably, an antibody or a functional equivalent according
to the invention comprises a CDR sequence which is at least 75%,
more preferably at least 80%, more preferably at least 85%, more
preferably at least 90% identical to at least one of the CDR
sequences depicted in FIG. 14A. Most preferably, an antibody or a
functional equivalent according to the invention comprises a CDR
sequence which is at least 95% identical to at least one of the CDR
sequences depicted in FIG. 14A. The particularly preferred antibody
AM14, described above, comprises CDR sequences which consist of the
CDR sequences depicted in FIG. 14A. A particularly preferred
embodiment according to the invention thus provides an isolated,
synthetic or recombinant antibody or a functional equivalent
thereof which is capable of specifically binding Respiratory
Syncytial Virus and which comprises:
[0040] a heavy chain CDR1 sequence comprising the sequence GFSFSHYA
(SEQ ID NO: 73), and/or
[0041] a heavy chain CDR2 sequence comprising the sequence ISYDGENT
(SEQ ID NO: 74), and/or
[0042] a heavy chain CDR3 sequence comprising the sequence
ARDRIVDDYYYYGMDV (SEQ ID NO: 75), and/or
[0043] a light chain CDR1 sequence comprising the sequence QDIKKY
(SEQ ID NO: 76), and/or
[0044] a light chain CDR2 sequence comprising the sequence DAS,
and/or
[0045] a light chain CDR3 sequence comprising the sequence
QQYDNLPPLT (SEQ ID NO: 77).
[0046] In one embodiment an antibody or functional equivalent is
provided which comprises the three heavy chain CDR sequences and
the three light chain CDR sequences as depicted in FIG. 14A, or
sequences that are at least 70% identical thereto. Further provided
is therefore an isolated, synthetic or recombinant antibody or a
functional equivalent thereof which comprises a heavy chain CDR1
sequence comprising a sequence which is at least 70% identical to
the sequence GFSFSHYA (SEQ ID NO: 73) and a heavy chain CDR2
sequence comprising a sequence which is at least 70% identical to
the sequence ISYDGENT (SEQ ID NO: 74) and a heavy chain CDR3
sequence comprising a sequence which is at least 70% identical to
the sequence ARDRIVDDYYYYGMDV (SEQ ID NO: 75) and a light chain
CDR1 sequence comprising a sequence which is at least 70% identical
to the sequence QDIKKY (SEQ ID NO 76) and a light chain CDR2
sequence comprising a sequence which is at least 70% identical to
the sequence. DAS, and a light chain CDR3 sequence comprising a
sequence which is at least 70% identical to the sequence QQYDNLPPLT
(SEQ ID NO: 77). Said antibody or functional equivalent preferably
comprises CDR sequences which 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 heavy chain CDR
sequences and the light chain CDR sequences as depicted in FIG.
14A. An antibody or functional equivalent comprising the above
mentioned heavy chain CDR1, CDR2 and CDR3 sequences of FIG. 14A as
well as the above mentioned light chain CDR1, CDR2 and CDR3
sequences of FIG. 14A is also provided.
[0047] Antibodies or functional equivalents thereof comprising a
heavy chain amino acid sequence which is at least 70% identical to
a heavy chain sequence as depicted in FIG. 14A is also provided.
Such heavy chain sequences provide desired RSV-binding properties,
as evidenced by antibody AM14. Further provided is therefore an
antibody or a functional equivalent thereof, having a heavy chain
sequence comprising a sequence which is at least 70% identical to
the sequence EVQLVESGGGVVQPGRSLRLSCAASGFSFSHYAMHWVRQAPGKGLEWVAVIS
YDGENTYYADSVKGRFSISRDNSKNTVSLQMNSLRPEDTALYYCARDRIVDD
YYYYGMDVWGQGATVTVSS (SEQ ID NO: 78). Moreover, light chain amino
acid sequences which are at least 70% identical to a light chain
sequence as depicted in FIG. 14A also provide desired RSV-binding
properties, as evidenced by antibody AM14. An antibody, or a
functional equivalent thereof having a light chain sequence which
is at least 70% identical to the sequence
DIQMTQSPSSLSASVGDRVTITCQASQDIKKYLNWYHQKPGKVPELLMHDASNLETG
VPSRFSGRGSGTDFTLTISSLQPEDIGTYYCQQYDNLPFLTFGGGTKVEIKRTV (SEQ ID NO:
79) is therefore also provided. An antibody or functional part
according to the invention preferably comprises a variable heavy
chain sequence and/or a variable light chain sequence which is at
least 75%, more preferably at least 80%, more preferably at least
85%, more preferably at least 90%, most preferably at least 95%
identical to a heavy chain sequence and/or a light chain sequence
as depicted in FIG. 14A. The higher the homology, the more closely
said antibody or functional part resembles antibody AM14. An
antibody or functional part according to the invention preferably
comprises a heavy chain as well as a light chain which resemble the
heavy and light chain of AM14. Further provided is therefore an
antibody or functional part comprising a heavy chain sequence and a
light chain sequence which are at least 70%, more preferably at
least 80%, more preferably at least 85%, more preferably at least
90%, most preferably at least 95% identical to the heavy chain
sequence and the light chain sequence as depicted in FIG. 14A.
[0048] One embodiment provides an antibody or functional equivalent
thereof comprising a heavy chain sequence consisting of the heavy
chain sequence as depicted in FIG. 14A, and a light chain sequence
consisting of the light chain sequence as depicted in FIG. 14A.
Alternatively, as is well known by the skilled person, it is
possible to generate a shortened heavy chain or light chain
sequence while maintaining a binding property of interest.
Preferably, such a shortened heavy chain or light chain is
generated which has a shorter constant region, as compared to the
original heavy or light chain. The variable domain is preferably
maintained. For instance, a Fab fragment or F(ab').sub.2 fragment
based on a heavy chain sequence or light chain sequence depicted in
FIG. 14A is produced. A functional equivalent of an antibody
comprising at least a functional part of a sequence as depicted in
FIG. 14A is therefore also provided. Said functional part has a
length of at least 20 amino acids and comprises a sequence which is
at least 70% identical to at least one of the CDR sequences
depicted in FIG. 14A.
[0049] Another particularly preferred anti-RSV antibody according
to the present invention is the antibody designated "AM16", which
has a heavy chain region and a light chain region as depicted in
FIG. 14B. The CDR sequences of AM16, which in particular contribute
to the antigen-binding properties of AM16, are also depicted in
FIG. 14B.
[0050] Now that the present invention provides the insight that the
CDR sequences depicted in FIG. 14B provide desired RSV-binding
characteristics, a skilled person is well capable of generating
variants comprising at least one altered CDR sequence. For
instance, conservative amino acid substitution is applied.
Conservative amino acid substitution involves substitution of one
amino acid with another with generally similar properties (size,
hydrophobicity, etc), such that the overall functioning is likely
not to be seriously affected.
[0051] It is also possible to change at least one CDR sequence
depicted in FIG. 14B in order to generate a variant antibody, or a
functional equivalent thereof, with at least one altered property
as compared to AM16. Preferably, an antibody or functional
equivalent is provided comprising a CDR sequence which is at least
70% identical to a CDR sequence as depicted in FIG. 14B, so that
the favorable binding characteristics of AM 16 are at least in part
maintained or even improved. A CDR sequence as depicted in FIG. 14B
is preferably altered such that the resulting antibody or
functional equivalent comprises at least one improved property,
such as for instance an improved binding affinity, selectivity
and/or stability, as compared to AM16. Variant antibodies or
functional equivalents thereof comprising an amino acid sequence
which is at least 70% identical to a CDR sequence as depicted in
FIG. 14B are therefore within the scope of the present invention.
Various methods are available in the art for altering an amino acid
sequence. For instance, a heavy chain or light chain sequence with
a desired CDR sequence is artificially synthesized. Preferably, a
nucleic acid sequence encoding a CDR sequence is mutated, for
instance using random- or site-directed-mutagenesis.
[0052] In one aspect the invention thus provides an isolated,
synthetic or recombinant antibody or a functional part, derivative
and/or analogue thereof which is capable of specifically binding
Respiratory Syncytial Virus and which comprises:
[0053] a heavy chain CDR1 sequence comprising a sequence which is
at least 70% identical to the sequence GFTFSSYN (SEQ ID NO: 80),
and/or
[0054] a heavy chain CDR2 sequence comprising a sequence which is
at least 70% identical to the sequence ISAGSSYI (SEQ ID NO: 81),
and/or
[0055] a heavy chain CDR3 sequence comprising a sequence which is
at least 70% identical to the sequence AREDYGPGNYYSPNWFDP (SEQ ID
NO: 82), and/or
[0056] a light chain CDR1 sequence comprising a sequence which is
at least 70% identical to the sequence SSNIGAGYD (SEQ ID NO: 83),
and/or
[0057] a light chain CDR2 sequence comprising a sequence which is
at least 70% identical to the sequence GNT, and/or
[0058] a light chain CDR3 sequence comprising a sequence which is
at least 70% identical to the sequence HSYDRSLSG (SEQ ID NO:
84).
[0059] Preferably, an antibody or a functional equivalent according
to the invention comprises a CDR sequence which is at least 75%,
more preferably at least 80%, more preferably at least 85%, more
preferably at least 90% identical to at least one of the CDR
sequences depicted in FIG. 14B. Most preferably, an antibody or a
functional equivalent according to the invention comprises a CDR
sequence which is at least 95% identical to at least one of the CDR
sequences depicted in FIG. 14B. The particularly preferred antibody
AM16, described above, comprises CDR sequences which consist of the
CDR sequences depicted in FIG. 14B. A particularly preferred
embodiment according to the invention thus provides an isolated,
synthetic or recombinant antibody or a functional equivalent
thereof which is capable of specifically binding Respiratory
Syncytial Virus and which comprises:
[0060] a heavy chain CDR1 sequence comprising the sequence GFTFSSYN
(SEQ ID NO: 80), and/or
[0061] a heavy chain CDR2 sequence comprising the sequence ISAGSSYI
(SEQ ID NO: 81), and/or
[0062] a heavy chain CDR3 sequence comprising the sequence
AREDYGPGNYYSPNWFDP (SEQ ID NO: 82), and/or
[0063] a light chain CDR1 sequence comprising the sequence
SSNIGAGYD (SEQ ID NO: 83), and/or
[0064] a light chain CDR2 sequence comprising the sequence GNT,
and/or
[0065] a light chain CDR3 sequence comprising the sequence
HSYDRSLSG (SEQ ID NO: 84).
[0066] In one embodiment an antibody or functional equivalent is
provided which comprises the three heavy chain CDR sequences and
the three light chain CDR sequences as depicted in FIG. 14B, or
sequences that are at least 70% identical thereto. Further provided
is therefore an isolated, synthetic or recombinant antibody or a
functional equivalent thereof which comprises a heavy chain CDR1
sequence comprising a sequence which is at least 70% identical to
the sequence GFTFSSYN (SEQ ID NO: 80) and a heavy chain CDR2
sequence comprising a sequence which is at least 70% identical to
the sequence ISAGSSYI (SEQ ID NO: 81) and a heavy chain CDR3
sequence comprising a sequence which is at least 70% identical to
the sequence AREDYGPGNYYSPNWFDP (SEQ ID NO: 82) and a light chain
CDR1 sequence comprising a sequence which is at least 70% identical
to the sequence SSNIGAGYD (SEQ ID NO: 83) and a light chain CDR2
sequence comprising a sequence which is at least 70% identical to
the sequence GNT, and a light chain CDR3 sequence comprising a
sequence which is at least 70% identical to the sequence HSYDRSLSG
(SEQ ID NO: 84). Said antibody or functional equivalent preferably
comprises CDR sequences which 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 above mentioned
heavy chain CDR sequences and the above mentioned light chain CDR
sequences as depicted in FIG. 14B. An antibody or functional
equivalent comprising the above mentioned heavy chain CDR1, CDR2
and CDR3 sequences of FIG. 14B as well as the above mentioned light
chain CDR1, CDR2 and CDR3 sequences of FIG. 14B is also
provided.
[0067] Antibodies or functional equivalents thereof comprising a
heavy chain amino acid sequence which is at least 70% identical to
a heavy chain sequence as depicted in FIG. 14B is also provided.
Such heavy chain sequences provide desired RSV-binding properties,
as evidenced by antibody AM16. Further provided is therefore an
antibody or a functional equivalent thereof, having a heavy chain
sequence comprising a sequence which is at least 70% identical to
the sequence
EVQLVETGGGLAQPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSHISAGSS
YIYYSDSVKGRFTVSRDNVRNSVYLQMNSLRAADTAV YYCAREDYGPGNYYSPNW
FDPWGQGTLVTVSS (SEQ ID NO: 85). Moreover, light chain amino acid
sequences which are at least 70% identical to a light chain
sequence as depicted in FIG. 14B also provide desired RSV-binding
properties, as evidenced by antibody AM16. An antibody, or a
functional equivalent thereof having a light chain sequence which
is at least 70% identical to the sequence
QSVVTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNTNRPS
GVSDRFSGSKSGTSASLAITGLQAEDEADYYCHSYDRSLSGSVFGGGTKLTV (SEQ ID NO:
86) is therefore also provided. An antibody or functional part
according to the invention preferably comprises a variable heavy
chain sequence and/or a variable light chain sequence which is at
least 75%, more preferably at least 80%, more preferably at least
85%, more preferably at least 90%, most preferably at least 95%
identical to the heavy chain sequence and/or the light chain
sequence as depicted in FIG. 14B. The higher the homology, the more
closely said antibody or functional part resembles antibody AM16.
An antibody or functional part according to the invention
preferably comprises a heavy chain as well as a light chain which
resemble the heavy and light chain of AM 16. Further provided is
therefore an antibody or functional part comprising a heavy chain
sequence and a light chain sequence which are at least 70%, more
preferably at least 80%, more preferably at least 85%, more
preferably at least 90%, most preferably at least 95% identical to
the heavy chain sequence and the light chain sequence as depicted
in FIG. 14B.
[0068] One embodiment provides an antibody or functional equivalent
thereof comprising a heavy chain sequence consisting of the heavy
chain sequence as depicted in FIG. 14B, and a light chain sequence
consisting of the light chain sequence as depicted in FIG. 14B.
Alternatively, as is well known by the skilled person, it is
possible to generate a shortened heavy chain or light chain
sequence while maintaining a binding property of interest.
Preferably, such a shortened heavy chain or light chain is
generated which has a shorter constant region, as compared to the
original heavy or light chain. The variable domain is preferably
maintained. For instance, a Fab fragment or F(ab').sub.2 fragment
based on a heavy chain sequence or light chain sequence depicted in
FIG. 14B is produced. A functional equivalent of an antibody
comprising at least a functional part of a sequence as depicted in
FIG. 14B is therefore also provided. Said functional part has a
length of at least 20 amino acids and comprises a sequence which is
at least 70% identical to at least one of the CDR sequences
depicted in FIG. 14B,
[0069] Another particularly preferred anti-RSV antibody according
to the present invention is the antibody designated "AM23", which
has a heavy chain region and a light chain region as depicted in
FIG. 14C. The CDR sequences of AM23, which in particular contribute
to the antigen-binding properties of AM23, are also depicted in
FIG. 14C.
[0070] Now that the present invention provides the insight that the
CDR sequences depicted in FIG. 14C provide desired RSV-binding
characteristics, a skilled person is well capable of generating
variants comprising at least one altered CDR sequence. For
instance, conservative amino acid substitution is applied.
Conservative amino acid substitution involves substitution of one
amino acid with another with generally similar properties (size,
hydrophobicity, etc), such that the overall functioning is likely
not to be seriously affected.
[0071] It is also possible to change at least one CDR sequence
depicted in FIG. 14C in order to generate a variant antibody, or a
functional equivalent thereof, with at least one altered property
as compared to AM23. Preferably, an antibody or functional
equivalent is provided comprising a CDR sequence which is at least
70% identical to a CDR sequence as depicted in FIG. 14C, so that
the favorable binding characteristics of AM23 are at least in part
maintained or even improved. A CDR sequence as depicted in FIG. 14C
is preferably altered such that the resulting antibody or
functional equivalent comprises at least one improved property,
such as for instance an improved binding affinity, selectivity
and/or stability, as compared to AM23. Variant antibodies or
functional equivalents thereof comprising an amino acid sequence
which is at least 70% identical to a CDR sequence as depicted in
FIG. 14C are therefore within the scope of the present invention.
Various methods are available in the art for altering an amino acid
sequence. For instance, a heavy chain or light chain sequence with
a desired CDR sequence is artificially synthesized. Preferably, a
nucleic acid sequence encoding a CDR sequence is mutated, for
instance using random- or site-directed-mutagenesis.
[0072] In one aspect the invention thus provides an isolated,
synthetic or recombinant antibody or a functional part, derivative
and/or analogue thereof which is capable of specifically binding
Respiratory Syncytial Virus and which comprises:
[0073] a heavy chain CDR1 sequence comprising a sequence which is
at least 70% identical to the sequence GFNFHNYG (SEQ ID NO: 87),
and/or
[0074] a heavy chain CDR2 sequence comprising a sequence which is
at least 70% identical to the sequence VWYDGSKK (SEQ ID NO: 88),
and/or
[0075] a heavy chain CDR3 sequence comprising a sequence which is
at least 70% identical to the sequence VRDKVGPTPYFDS (SEQ ID NO:
89), and/or
[0076] a light chain CDR1 sequence comprising a sequence which is
at least 70% identical to the sequence NIGSET (SEQ ID NO: 90),
and/or
[0077] a light chain CDR2 sequence comprising a sequence which is
at least 70% identical to the sequence DDD, and/or
[0078] a light chain CDR3 sequence comprising a sequence which is
at least 70% identical to the sequence QVWDRSNYHQV (SEQ ID NO:
91).
[0079] Preferably, an antibody or a functional equivalent according
to the invention comprises a CDR sequence which is at least 75%,
more preferably at least 80%, more preferably at least 85%, more
preferably at least 90% identical to at least one of the CDR
sequences depicted in FIG. 14C. Most preferably, an antibody or a
functional equivalent according to the invention comprises a CDR
sequence which is at least 95% identical to at least one of the CDR
sequences depicted in FIG. 14C. The particularly preferred antibody
AM23, described above, comprises CDR sequences which consist of the
CDR sequences depicted in FIG. 14C. A particularly preferred
embodiment according to the invention thus provides an isolated,
synthetic or recombinant antibody or a functional equivalent
thereof which is capable of specifically binding Respiratory
Syncytial Virus and which comprises:
[0080] a heavy chain CDR1 sequence comprising the sequence GFNFHNYG
(SEQ ID NO: 87), and/or
[0081] a heavy chain CDR2 sequence comprising the sequence VWYDGSKK
(SEQ ID NO: 88), and/or
[0082] a heavy chain CDR3 sequence comprising the sequence
VRDKVGPTPYFDS (SEQ ID NO 89), and/or
[0083] a light chain CDR1 sequence comprising the sequence NIGSET
(SEQ ID NO: 90), and/or
[0084] a light chain CDR2 sequence comprising the sequence DDD,
and/or
[0085] a light chain CDR3 sequence comprising the sequence
QVWDRSNYHQV (SEQ ID NO: 91).
[0086] In one embodiment an antibody or functional equivalent is
provided which comprises the three heavy chain CDR sequences and
the three light chain CDR sequences as depicted in FIG. 14C, or
sequences that are at least 70% identical thereto. Further provided
is therefore an isolated, synthetic or recombinant antibody or a
functional equivalent thereof which comprises a heavy chain CDR1
sequence comprising a sequence which is at least 70% identical to
the sequence GFNFHNYG (SEQ ID NO: 87) and a heavy chain CDR2
sequence comprising a sequence which is at least 70% identical to
the sequence VWYDGSKK (SEQ ID NO: 88) and a heavy chain CDR3
sequence comprising a sequence which is at least 70% identical to
the sequence VRDKVGPTPYFDS (SEQ ID NO: 89) and a light chain CDR1
sequence comprising a sequence which is at least 70% identical to
the sequence NIGSET (SEQ ID NO: 90) and a light chain CDR2 sequence
comprising a sequence which is at least 70% identical to the
sequence DDD, and a light chain CDR3 sequence comprising a sequence
which is at least 70% identical to the sequence QVWDRSNYHQV (SEQ ID
NO: 91). Said antibody or functional equivalent preferably
comprises CDR sequences which 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 above mentioned
heavy chain CDR sequences and the above mentioned light chain CDR
sequences as depicted in FIG. 14C. An antibody or functional
equivalent comprising the above mentioned heavy chain CDR1, CDR2
and CDR3 sequences of FIG. 14C as well as the above mentioned light
chain CDR1, CDR2 and CDR3 sequences of FIG. 14C is also
provided.
[0087] Antibodies or functional equivalents thereof comprising a
heavy chain amino acid sequence which is at least 70% identical to
a heavy chain sequence as depicted in FIG. 14C is also provided.
Such heavy chain sequences provide desired RSV-binding properties,
as evidenced by antibody AM23. Further provided is therefore an
antibody or a functional equivalent thereof, having a heavy chain
sequence comprising a sequence which is at least 70% identical to
the sequence EVQLVESGGNVVKPGTSLRLSCAATGFNFHNYGMNWVRQAPGKGLEWVAVVWYD
GSKKYYADSVTGRFAISRDNSKNTLYLQMNSLRVEDTAVYYCVRDKVGPTPYFDSW GQGTLVTVSS
(SEQ ID NO: 92). Moreover, light chain amino acid sequences which
are at least 70% identical to a light chain sequence as depicted in
FIG. 14C also provide desired RSV-binding properties, as evidenced
by antibody AM23. An antibody, or a functional equivalent thereof
having a light chain sequence which is at least 70% identical to
the sequence
SYVLTQPPSVSLAPGGTAAITCGRNNIGSETVHWYQQKPGQAPVLWYDDDDRPSGIP
ERFSGSNSGNTATLTISRVEAGDEADYYCQVWDRSNYHQVFGGGTKLTV (SEQ ID NO: 93)
is therefore also provided. An antibody or functional part
according to the invention preferably comprises a variable heavy
chain sequence and/or a variable light chain sequence which is at
least 75%, more preferably at least 80%, more preferably at least
85%, more preferably at least 90%, most preferably at least 95%
identical to the heavy chain sequence and/or the light chain
sequence as depicted in FIG. 14C. The higher the homology, the more
closely said antibody or functional part resembles antibody AM23.
An antibody or functional part according to the invention
preferably comprises a heavy chain as well as a light chain which
resemble the heavy and light chain of AM23. Further provided is
therefore an antibody or functional part comprising a heavy chain
sequence and a light chain sequence which are at least 70%, more
preferably at least 80%, more preferably at least 85%, more
preferably at least 90%, most preferably at least 95% identical to
the heavy chain sequence and the light chain sequence as depicted
in FIG. 14C.
[0088] One embodiment provides an antibody or functional equivalent
thereof comprising a heavy chain sequence consisting of the heavy
chain sequence as depicted in FIG. 14C, and a light chain sequence
consisting of the light chain sequence as depicted in FIG. 14C.
Alternatively, as is well known by the skilled person, it is
possible to generate a shortened heavy chain or light chain
sequence while maintaining a binding property of interest.
Preferably, such a shortened heavy chain or light chain is
generated which has a shorter constant region, as compared to the
original heavy or light chain. The variable domain is preferably
maintained. For instance, a Fab fragment or F(ab').sub.2, fragment
based on a heavy chain sequence or light chain sequence depicted in
FIG. 14C is produced. A functional equivalent of an antibody
comprising at least a functional part of a sequence as depicted in
FIG. 14C is therefore also provided. Said functional part has a
length of at least 20 amino acids and comprises a sequence which is
at least 70% identical to at least one of the CDR sequences
depicted in FIG. 14C.
[0089] The present invention provides RSV-specific antibodies or
functional equivalents thereof having improved properties as
compared to prior art antibodies. The inventors have succeeded in
generating RSV-specific antibodies with low IC.sub.50 values. Such
antibodies have a particular high or strong affinity for RSV and
are therefore particularly suitable for counteracting and/or at
least in part preventing an RSV-infection and/or adverse effects of
an RSV infection. One embodiment provides an antibody which has an
IC.sub.50 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.sub.50 value of less than 5 ng/ml, more
preferably less than 2 ng/ml. The preferred antibody D25 has an
IC.sub.50 value of about 0.5-1.5 ng/ml in the in vitro
neutralization assay described in the examples (see FIG. 8).
[0090] An antibody according to the invention is preferably a human
antibody. The use of human antibodies for human therapy diminishes
the chance of side-effects due to an immunological reaction in a
human individual against non-human sequences. In another preferred
embodiment an antibody or functional part, derivative or analogue
according to the invention is a chimeric antibody. This way,
sequences of interest, such as for instance a binding site of
interest, can be included into an antibody or functional equivalent
according to the invention.
[0091] The invention further provides an isolated, synthetic or
recombinant nucleic acid sequence, or a functional part, derivative
or analogue thereof, encoding an antibody or functional equivalent
according to the invention. Such nucleic acid is for instance
isolated from a B-cell which is capable of producing an antibody
according to the invention, as outlined in more detail below. A
preferred embodiment provides a nucleic acid sequence comprising a
sequence which is at least 70% homologous to at least a functional
part of a nucleic acid sequence as depicted in FIG. 11, FIG. 12,
FIG. 14A, FIG. 14B and/or FIG. 14B. Said nucleic acid sequence
preferably comprises a sequence which is at least 75%, more
preferably at least 80%, more preferably at least 85%, more
preferably at least 90%, most preferably at least 95% homologous to
at least a functional part of a nucleic acid sequence as depicted
in FIG. 11, FIG. 12, FIG. 14A, FIG. 14B and/or FIG. 14B. Said
functional part has a length of at least 30 nucleotides, preferably
at least 50 nucleotides, more preferably at least 75 nucleotides.
Preferably, said functional part encodes at least one nucleic acid
sequence as depicted in FIG. 11A, FIG. 12, FIG. 14A, FIG. 14B
and/or FIG. 14B. Said sequence is preferably a CDR sequence.
[0092] An antibody or functional equivalent according to the
invention is particularly suitable for use as a medicine or
prophylactic agent. An antibody according to the invention, or a
functional part, derivative or analogue thereof, for use as a
medicament and/or prophylactic agent is therefore also herewith
provided. In a particularly preferred embodiment said antibody
comprises antibody D25, AM14, AM16 and/or AM23, or a functional
part, derivative or analogue thereof. Said medicament or
prophylactic agent is preferably used for counteracting or at least
in part preventing an RSV-infection or for counteracting or at
least in part preventing adverse effects of an RSV-infection. A use
of an antibody, functional part, derivative or analogue according
to the invention for the preparation of a medicament and/or
prophylactic agent for at least in part treating and/or preventing
a RSV-related disorder is therefore also provided, as well as a
method for at least in part treating or preventing an RSV-related
disorder, the method comprising administering to an individual in
need thereof a therapeutically effective amount of an antibody or
functional equivalent according to the invention. Said antibody
preferably comprises antibody D25, AM14, AM16 and/or AM23, or a
functional part, derivative or analogue thereof.
[0093] In order to counteract RSV, an antibody or functional
equivalent according to the invention is preferably administered to
an individual before an RSV-infection has taken place.
Alternatively, an antibody or functional equivalent according to
the invention is administered when an individual is already
infected by RSV. Said antibody or functional equivalent is
preferably administered to individuals with an increased risk of
RSV-related disorders, such as for instance children with premature
birth, individuals with chronic lung disease, congenital heart
disease and/or compromised immunity, and children with an age
younger than 6 weeks. Also elderly people have an increased risk of
RSV-related disorders. Antibodies or functional equivalents
according to the invention are preferably administered orally or
via one or more injections. Dose ranges of antibodies and/or
functional equivalents according to the invention to be used in the
therapeutic applications as described herein before are designed on
the basis of rising dose studies in the clinic in clinical trials
for which rigorous protocol requirements exist. Typical doses are
between 0.1 and 10 mg per kg body weight. For therapeutic
application, antibodies or functional equivalents according to the
invention are typically combined with a pharmaceutically acceptable
carrier, adjuvant, diluent and/or excipient. Examples of suitable
carriers for instance comprise keyhole limpet haemocyanin (KLH),
serum albumin (e.g. BSA or RSA) and ovalbumin. Many suitable
adjuvants, oil-based and water-based, are known to a person skilled
in the art. In one embodiment said adjuvant comprises Specol. In
another embodiment, said suitable carrier comprises a solution like
for example saline.
[0094] In yet another embodiment a nucleic acid encoding an
antibody or functional part according to the invention is used.
Upon administration of such nucleic acid, antibodies or functional
equivalents are produced by the host's machinery. Produced
antibodies or functional equivalents are capable of preventing
and/or counteracting RSV-infection and/or the adverse effects of an
RSV-infection. A nucleic acid sequence, functional part, derivative
and/or analogue according to the invention for use as a medicament
and/or prophylactic agent is therefore also herewith provided. Said
nucleic acid is preferably used for counteracting RSV. Further
provided is therefore a use of a nucleic acid sequence, functional
part, derivative and/or analogue according to the invention for the
preparation of a medicament and/or prophylactic agent for at least
in part treating and/or preventing a RSV-related disorder.
[0095] By at least a functional part of a nucleic acid of the
invention is meant a part 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, comprising at least one expression
characteristic (in kind not necessarily in amount) as a nucleic
acid of the invention. Said functional part at least encodes an
amino acid sequence comprising a sequence which is at least 70%
identical to a CDR sequence as depicted in FIGS. 11A-C, FIG. 14A,
FIG. 14B and/or FIG. 14C.
[0096] The invention furthermore provides an isolated antibody
producing cell capable of producing an antibody, functional part,
derivative or analogue according to the invention. Possible (but
not limiting) ways of obtaining such antibody producing cells are
outlined in detail in the examples. The inventors have developed
and used a new method in order to improve the stability of
RSV-specific antibody producing cells. Using this method,
RSV-specific antibody producing cells are generated which are
stable for at least six months. An RSV-specific antibody producing
cell according to the invention, which is stable for at least nine
weeks, preferably for at least three months, more preferably for at
least six months is therefore also herewith provided.
[0097] The present inventors have used their insight that the
stability of an RSV-specific antibody producing cell is influenced
by influencing the amount of BCL6 and/or Blimp-1 expression product
within said antibody producing cell. The amount of BCL6 and/or
Blimp-1 expression product is either directly or indirectly
influenced. Preferably the amounts of both BCL6 and Blimp-1
expression products within said antibody producing cell are
regulated, since both expression products are involved in the
stability of an antibody producing cell. The stability of an
antibody producing cell is defined as the capability of said
antibody producing cell to remain in a certain developmental stage
(preferably after said cell has been brought into said stage).
Different developmental stages of a cell involve at least one
different characteristic of said cell. For instance, a memory B
cell is known to differentiate upon stimulation into an
antibody-secreting plasma cell via a stage which some researchers
call a plasmablast. A memory B cell, a plasmablast and a plasma
cell are different developmental stages of a B cell, wherein the B
cell has different characteristics. A memory B cell exhibits low
proliferation and antibody secretion. A plasmablast exhibits both
higher proliferation and higher antibody secretion levels as
compared to a memory B cell, whereas a plasma cell secretes high
antibody levels but is not capable of proliferating. With a method
of the present inventors it has become possible to regulate the
replicative life span of an antibody producing cell. A replicative
life span of an antibody producing cell is defined herein as the
time span wherein a B cell and its progeny cells are capable of
replicating while maintaining their capability of producing
antibody and/or developing into a cell that produces antibody.
Preferably the replicative life span of an antibody producing cell
is prolonged, meaning that said antibody producing cell will not
terminally differentiate--or only after a longer period as compared
to the same kind of antibody producing cells that are currently
used--and continue to proliferate in vitro. According to the
inventors it is possible to regulate the amount of BCL6 and/or
Blimp-1 expression product in an antibody producing cell to such
extent that the antibody producing cell is brought into, and/or
kept in, a predetermined developmental state in which the cells
continue to proliferate. With a method of the inventors it has
therefore become possible to increase the replicative life span of
an antibody producing cell since it is possible to maintain a B
cell in a certain developmental stage wherein replication occurs.
Reference is made to PCT/NL2006/000625, filed by the same
applicant. The present invention provides means and methods for
producing stable RSV-specific antibody producing cells.
[0098] An antibody producing cell is defined as a cell which cell
is capable of producing and/or secreting antibody or a functional
equivalent thereof, and/or which cell is capable of developing into
a cell which is capable of producing and/or secreting antibody or a
functional equivalent thereof. An RSV-specific antibody producing
cell is defined herein as a cell capable of producing and/or
secreting antibodies or functional equivalents thereof which are
capable of specifically binding RSV and/or a component of RSV, such
as for instance an epitope of the RSV F (fusion) protein, the RSV G
(attachment) protein or RSV SH (small hydrophobic) protein.
Preferably, said RSV-specific antibody producing cell comprises a B
cell and/or a B cell-derived plasma cell. A B cell is called herein
an antibody producing cell, even when the B cell is in a stage
wherein antibody production is low or not present at all, such as a
naive B cell or a memory B cell, being activated or not, because
such cells are capable of developing into cells that produce
antibody, such as a plasmablast and/or plasma cell.
[0099] An RSV-specific antibody producing cell according to the
invention preferably comprises a mammalian cell. Non-limiting
examples include antibody producing cells derived from a human
individual, rodent, rabbit, llama, pig, cow, goat, horse, ape,
gorilla. Preferably, said antibody producing cell comprises a human
cell, a murine cell, a rabbit cell and/or a llama cell.
[0100] BCL6 encodes a transcriptional repressor which is required
for normal B cell and T cell development and maturation and which
is required for the formation of germinal centers. (Ye, 1997). BCL6
is highly expressed in germinal center B cells whereas it is hardly
expressed in plasma cells. BCL6 inhibits differentiation of
activated B cells into plasma cells. The transcriptional repressor
B lymphocyte induced maturation protein-1 (Blimp-1) is required for
development of a B cell into a plasma cell. The human variant of
Blimp-1 is named Prdm1. As used herein, any reference to Blimp-1
includes a reference to Prdm1. Blimp-1 drives plasma cell
differentiation. BCL6 and Blimp-1 repress expression of the other;
thus in a natural situation when one reaches an higher expression
level than the other, the stage of differentiation is enforced. In
the human body, differentiation of plasma cells from activated
naive or memory B cells involves downregulation of BCL6 and
upregulation of Blimp-1. In germinal center cells BCL6 expression
is high and Blimp-1 expression is low. In resting memory cells
expression of BCL6 and Blimp-1 are low. Signals that trigger
differentiation cause an upregulation of Blimp-1, and this Blimp-1
counteracts the expression of BCL6. The stage where both BCL6 and
Blimp-1 are expressed is short-lived and is called a plasmablast.
With progressively increasing Blimp-1 levels, BCL6 expression is
extinguished, resulting in a plasma cell.
[0101] In one embodiment of the present invention, an RSV-specific
antibody producing cell is provided wherein BCL6 and Blimp-1 are
co-expressed (meaning that both BCL6 and Blimp-1 are expressed in
said antibody producing cell for at least 1 day, preferably at
least one week, more preferably at least six weeks, most preferably
at least three months. Said RSV-specific antibody producing cell is
capable of proliferating when an appropriate signal is provided. It
has been found that co-expression of BCL6 and Blimp-1 results in an
antibody producing cell which is capable of both proliferating and
producing antibody. BCL6 and Blimp-1 are preferably co-expressed in
a B cell, preferably a human B cell. Co-expression of BCL6 and
Blimp-1 in a B cell results in stabilization of said B cell in a
plasmablast-like stage, Plasmablasts, like plasma cells, are
capable of secreting antibody. However, plasmablasts are still
capable of proliferating, whereas plasma cells have lost their
capability of proliferating. Plasma cells are therefore unsuitable
for culturing antibody-producing cell lines.
[0102] One preferred embodiment provides an RSV-specific antibody
producing cell comprising an exogenous nucleic acid sequence
encoding BCL6 or a functional part, derivative and/or analogue
thereof. An exogenous nucleic acid is defined herein as a nucleic
acid sequence which does not naturally belong to the genome of a
cell. With such exogenous nucleic acid molecule it is possible to
regulate a BCL6 concentration in an antibody producing cell
independently from expression of endogenous BCL6. Hence, even if
expression of endogenous BCL6 is low or absent, for instance caused
by Blimp-1, an exogenous nucleic acid sequence encoding BCL6 or a
functional part, derivative and/or analogue thereof is still
capable of producing a concentration of BCL6 which is sufficient
for influencing the stability of an antibody producing cell.
Preferably, said nucleic acid sequence encoding BCL6 or a
functional part, derivative and/or analogue thereof is
constitutively active, so that BCL6 expression is maintained even
when endogenous BCL6 expression of said cell is inhibited by an
endogenous repressor such as Blimp-1. Most preferably, expression
of said nucleic acid sequence encoding BCL6 or a functional part,
derivative and/or analogue thereof is regulated by an exogenous
inducer of repressor, so that the extent of BCL6 expression is
regulated at will.
[0103] Preferably, as outlined below in more detail, an
RSV-specific antibody producing cell according to the invention
comprises an exogenous nucleic acid sequence encoding Bcl-xL or a
functional part, derivative and/or analogue thereof. If Bcl-xL or a
functional part, derivative and/or analogue thereof is present, it
is possible to grow plasmablasts under conditions of low cell
density. Expression of said nucleic acid sequence encoding Bcl-xL
or a functional part, derivative and/or analogue thereof is
preferably regulated by an exogenous inducer of repressor, so that
the extent of Bcl-xL expression is regulated at will. A preferred
embodiment therefore provides an RSV-specific antibody producing
cell comprising:
[0104] an exogenous nucleic acid sequence encoding BCL6 or a
functional part, derivative and/or analogue thereof, and/or
[0105] an exogenous nucleic acid sequence encoding Bcl-xL or a
functional part, derivative and/or analogue thereof. Said
RSV-specific antibody producing cell preferably comprises both an
exogenous nucleic acid sequence encoding BCL6--or a functional
part, derivative and/or analogue thereof--and an exogenous nucleic
acid sequence encoding Bcl-xL--or a functional part, derivative
and/or analogue thereof. Preferably, expression of said nucleic
acid sequence encoding BCL6, Bcl-xL or a functional part,
derivative and/or analogue of BCL6 or Bcl-xL is regulated by an
activator and/or repressor that is inducible by an exogenous
compound. For instance, an inducible promoter system is used such
as a Tet-on or Tet-off system.
[0106] A stable RSV-specific antibody producing cell according to
the invention is preferably generated by co-expressing BCL6 and
Blimp-1 in an RSV-specific antibody producing cell. An RSV-specific
antibody producing cell is preferably obtained from an individual
who has been exposed to RSV. Methods for isolating antibody
producing cells are well known in the art. For instance,
RSV-derived compounds that are marked with a label and/or tag are
incubated with a sample of an individual who has been exposed to
RSV, which sample comprises antibody producing cells. RSV-specific
antibody producing cells that recognize the tagged RSV-derived
compounds are isolated while unbound cells are washed away. The
resulting RSV-specific antibody producing cells are subsequently
stabilized by co-expressing BCL6 as well as Blimp-1.
[0107] One embodiment involves first stabilizing total
antibody-producing cells from an RSV exposed donor and then
isolating cells that recognize the tagged RSV-derived compound. In
another embodiment antibody producing cells are equipped with a
(fluorescent) marker downstream their B cell receptor (BCR,
membrane expressed form of the antibody) that signals when the
antibody producing cell binds an un-tagged/unlabeled antigen via
the BCR. Antibody producing cells in which the marker is turn are
selected and are subsequently stabilized by co-expressing BCL6 as
well as Blimp-1. In another embodiment, when there are no
antigen-derived compounds available but when there are assays
available to screen for unique antibodies, total/bulk antibody
producing cells are stabilized by co-expressing BCL6 as well as
Blimp-1 and, optionally, also Bcl-XL. According to this embodiment,
cells are cultured at low densities, preferably between 10 and 100
cells per 96-well, in the presence of L-cells (mini bulk cultures,
MBC). Culture supernatants can be used directly in screenings
assays, like ELISA, Western blot or functional assays like ELISPOT,
neutralization assays or cell migration assays.
[0108] In one embodiment MBC are selected and, to obtain monoclonal
cell lines of the antibody producing cell of interest, limiting
dilution cultures are preformed and, preferably 2-3 weeks later,
supernatants of those cultures are screened again in the preferred
assay.
[0109] As is well known by the skilled person, many alternative
methods are available in the art. The above mentioned embodiments
are non-limiting.
[0110] Further provided is therefore a method for producing an
antibody producing cell, which is stable for at least three months
and which is capable of producing RSV-specific antibodies or
functional equivalents thereof, the method comprising:
[0111] increasing an expression level of Blimp-1 in a cell which is
capable of producing RSV-specific antibodies or functional
equivalents thereof; and
[0112] increasing and/or maintaining a BCL6 expression level in
said cell.
[0113] With a method according to the invention it has become
possible to convert an RSV-specific memory B cell into a
plasmablast-like cell and to stabilize said cell, so that rapid
differentiation into a plasma cell does not occur. This is contrary
to natural development of plasma cells, wherein expression of
Blimp-1 in a memory B cell results in rapid development into a
plasma cell, thereby inhibiting BCL6 expression so that the
resulting plasma cell hardly expresses BCL6. One embodiment of the
present invention thus involves co-expression of both BCL6 and
Blimp-1 in an RSV-specific B cell, resulting in a cell that is
capable of both proliferating and producing antibody. The BCL6
expression level in said RSV-specific B-cell is preferably brought
to, and maintained at, essentially the same level or at a higher
level as compared to a plasmablast. This way a stable culture of
RSV-specific B cells is generated, which cells remain capable of
producing RSV-specific antibodies. These RSV-specific B cells that
co-express BCL6 and Blimp-1 are preferably further stabilized
through the addition of the anti-apoptotic gene Bcl-xL. With the
introduction of Bcl-xL it is now possible to grow plasmablasts
under conditions of low cell density. Hence, the invention also
provides a method to culture plasmablasts under conditions of low
cell density comprising generating an RSV-specific antibody
producing cell with expression levels of BCL6, Blimp-1 and Bcl-xL
with any of the herein described methods.
[0114] The amount of BCL6 expression product (preferably a BCL6
protein) in an RSV-specific antibody producing cell is regulated in
a variety of ways.
[0115] In one embodiment an antibody producing cell is provided
with a compound capable of directly or indirectly influencing BCL6
expression. An antibody producing cell is preferably provided with
a compound capable of enhancing BCL6 expression, in order to
counteract downregulation of BCL6 during expression of Blimp-1.
Such compound preferably comprises a Signal Transducer of
Activation and Transcription 5 (STAT5) protein or a functional
part, derivative and/or analogue thereof, and/or a nucleic acid
sequence coding therefore. STAT5 is a signal transducer capable of
enhancing BCL6 expression. There are two known forms of STAT5,
STAT5a and STAT5b, which are encoded by two different, tandemly
linked genes. Administration and/or activation of STAT5 results in
enhanced BCL6 levels. Hence, downregulation of BCL6 by Blimp-1 is
at least in part compensated by upregulation expression of BCL6 by
STAT5 or a functional part, derivative and/or analogue thereof.
Hence, STAT5 or a functional part, derivative and/or analogue
thereof is capable of directly influencing BCL6 expression. It is
also possible to indirectly influence BCL6 expression. This is for
instance done by regulating the amount of a compound which in turn
is capable of directly or indirectly activating STAT5 and/or
regulating STAT5 expression. Hence, in one embodiment the
expression and/or activity of endogenous and/or exogenous STAT5 is
increased. It is for instance possible to indirectly enhance BCL6
expression by culturing an antibody producing cell in the presence
of interleukin (IL) 2 and/or IL 4 which are capable of activating
STAT5.
[0116] In one embodiment, an RSV-specific antibody producing cell
is provided with a nucleic acid sequence encoding STAT5 or a
functional part, derivative and/or analogue thereof, wherein said
nucleic acid sequence is constitutively active, meaning that STAT5
is continuously expressed, independent of the presence of
(endogenous) regulators. In case that endogenous STAT5 expression
is low, or absent, an exogenous constitutively active nucleic acid
sequence encoding STAT5 or a functional part, derivative and/or
analogue thereof is preferably applied resulting in a concentration
of STAT5 or a functional part, derivative and/or analogue thereof
which is sufficient to enhance BCL6 expression. Most preferably, an
RSV-specific antibody producing cell is provided with a nucleic
acid sequence encoding a compound comprising STAT5 or a functional
part, derivative and/or analogue thereof, preferably a fusion
protein, whose activity is regulated by an exogenous inducer of
repressor, so that the extent of activation of BCL6 expression is
regulated at will. Another system that allows for induction of
BCL-6 is provided by a Tet-on system in which addition of
tetracycline and/or derivatives of tetracycline induce activity of
a transactivator that induced BCL6 gene transcription followed by
BCL protein synthesis. In one preferred embodiment, an antibody
producing cell is provided with a nucleic acid sequence encoding an
estrogen receptor (ER) and STAT5 as a fusion protein ER-STAT5. This
fusion protein is inactive because it forms a complex with heat
shock proteins in the cytosol. This way, STAT5 is unable to reach
the nucleus and BCL6 expression is not enhanced. Upon
administration of the exogenous inducer 4 hydroxy-tamoxifen (4HT),
the fusion protein ER-STAT5 dissociates from the heat shock
proteins, so that STAT5 is capable of entering the nucleus and
activating BCL6 expression.
[0117] Additionally, or alternatively, BCL6 expression in an
RSV-specific antibody producing cell is enhanced by culturing said
antibody producing cell in the presence of a compound capable of
directly or indirectly enhancing BCL6 expression.
[0118] One embodiment therefore provides a method for producing an
RSV-specific antibody producing cell comprising:
[0119] providing an RSV-specific antibody producing cell with a
compound capable of directly or indirectly enhancing BCL6
expression; and/or
[0120] culturing an RSV-specific antibody producing cell in the
presence of a compound capable of directly or indirectly enhancing
BCL6 expression. Said compound capable of directly or indirectly
enhancing BCL6 expression preferably comprises STAT5 or a
functional part, derivative and/or analogue thereof. Provided is
therefore a method according to the invention comprising providing
said RSV-specific antibody producing cell with STAT5 or a
functional part, derivative and/or analogue thereof, or with a
nucleic acid sequence encoding STAT5 or a functional part,
derivative and/or analogue thereof. In one embodiment said antibody
producing cell is cultured after introduction of a nucleic acid
sequence encoding STAT5 or a functional part, derivative and/or
analogue thereof into said cell. Said nucleic acid sequence is for
instance introduced into said cell by transfection and/or
virus-mediated gene transfer. Many alternative methods for
introducing a nucleic acid sequence into a cell are available in
the art which need no further explanation here.
[0121] With a compound capable of directly or indirectly enhancing
BCL6 expression it is possible to enhance expression of endogenous
BCL6. In one preferred embodiment however an antibody producing
cell is provided with a nucleic acid sequence encoding BCL6 or a
functional part, derivative and/or analogue thereof. As explained
herein before, an exogenous nucleic acid encoding BCL6 is preferred
because this allows regulation of a BCL6 concentration within a
cell independently from expression of endogenous BCL6. Hence, even
if expression of endogenous BCL6 is low or absent, for instance
caused by Blimp-1, an exogenous nucleic acid sequence encoding BCL6
or a functional part, derivative and/or analogue thereof is still
capable of producing a concentration of BCL6 which is sufficient
for influencing the stability of an antibody producing cell. Also
provided is therefore a method according to the invention
comprising providing an RSV-specific antibody producing cell with a
nucleic acid sequence encoding BCL6 or a functional part,
derivative and/or analogue thereof. Preferably, said antibody
producing cell is provided with a constitutively active nucleic
acid sequence encoding BCL6 or a functional part, derivative and/or
analogue thereof, so that BCL6 expression is maintained even when
endogenous BCL6 expression of said cell is inhibited by an
endogenous repressor such as Blimp-1. Most preferably, expression
of said nucleic acid sequence encoding BCL6 or a functional part,
derivative and/or analogue thereof is regulated by an exogenous
inducer of repressor, so that the extent of BCL6 expression is
regulated at will. For instance, an inducible promoter system is
used such as a Tet-on or Tet-off system, as already described.
[0122] In another preferred embodiment, the invention provides a
method wherein the amount of BCL6 is indirectly regulated by
providing an RSV-specific antibody producing cell with a nucleic
acid sequence encoding E47 or a functional part, derivative and/or
analogue thereof E47 encodes a transcription factor that belongs to
a family of helix-loop-helix proteins, named E-proteins. There are
four E-proteins, E12, E47, E2-2 and HEB, which are involved in
lymphocyte development. E12 and E47 are encoded by one gene, named
E2A, which is spliced differently. E-proteins can be inhibited by
the E protein inhibitor Id2, and Id3, and by ABF-1 (Mathas S.,
2006). E proteins have been described as tumor suppressors and
overexpression has been shown to induce apoptosis. One of the
specific targets of E47 are the Socs1 and Socs3 genes. Those Socs
genes are known as negative regulators of STAT5b and thus
indirectly of BCL6. In other words, expression of E47 within a B
cell enhances Blimp-1 expression which results in B-cell
differentiation towards an antibody producing phenotype
(plasmacell).
[0123] The amount of Blimp-1 expression in an RSV-specific antibody
producing cell is also regulated in a variety of ways. In one
embodiment an RSV-specific antibody producing cell is provided with
a compound capable of directly or indirectly influencing Blimp-1
expression. Additionally, or alternatively, an antibody producing
cell is cultured in the presence of a compound capable of directly
or indirectly influencing Blimp-1 expression. Further provided is
therefore a method according to the invention comprising providing
an RSV-specific antibody producing cell with a compound capable of
directly or indirectly influencing Blimp-1 expression. Further
provided is a method according to the invention comprising
culturing said antibody producing cell in the presence of a
compound capable of directly or indirectly influencing Blimp-1
expression. Preferably, a compound is used that is capable of
enhancing Blimp-1 expression in order to counteract downregulation
of Blimp-1 during expression of BCL6. Said compound most preferably
comprises IL-21.
[0124] In one preferred embodiment said compound capable of
directly or indirectly influencing Blimp-1 expression comprises a
Signal Transducer of Activation and Transcription 3 (STAT3) protein
or a functional part, derivative and/or analogue thereof, and/or a
nucleic acid sequence coding therefore. STAT3 is a signal
transducer which is involved in B cell development and
differentiation. STAT3 is capable of upregulating Blimp-1
expression, Further provided is therefore a method according to the
invention wherein said compound capable of directly or indirectly
influencing Blimp-1 expression comprises STAT3 or a functional
part, derivative and/or analogue thereof, or a nucleic acid
sequence encoding STAT3 or a functional part, derivative and/or
analogue thereof. Most preferably, expression of said nucleic acid
sequence encoding STAT3 or a functional part, derivative and/or
analogue thereof is regulated by an exogenous inducer of repressor,
so that the extent of STAT3 expression is regulated at will. For
instance, an inducible promoter system is used such as for instance
a Tet-on or Tet-off system. In one embodiment a fusion product
comprising of STAT3, a derivative or analogue, and ER is introduced
in said cell allowing regulation of STAT3 expression by
hydroxytamoxifen.
[0125] Since STAT3 is capable of influencing Blimp-1 expression, it
is also possible to indirectly regulate Blimp-1 expression by
administering a compound capable of directly or indirectly
regulating the activity and/or expression of STAT3. In one
embodiment an antibody producing cell is provided with a compound
that is capable of enhancing the activity of STAT3, so that Blimp-1
expression is indirectly enhanced as well. Further provided is
therefore a method according to the invention, wherein an antibody
producing cell is provided with a compound capable of directly or
indirectly enhancing activity of STAT3.
[0126] Hence, in one embodiment an antibody producing cell is
provided with a compound capable of directly or indirectly
activating STAT3, in order to enhance Blimp-1 expression.
[0127] STAT3 is activated in a variety of ways. Preferably, STAT3
is activated by providing an antibody producing cell with a
cytokine. Cytokines, being naturally involved in B cell
differentiation, are very effective in regulating STAT proteins.
Very effective activators of STAT3 are IL-21 and IL-6, but also
IL-2, IL-7, IL-10, IL-15 and IL-27 are known to activate STAT3.
Moreover, Toll-like receptors (TLRs) which are involved in innate
immunity are also capable of activating STAT3. One embodiment
therefore provides a method of the invention, wherein said compound
capable of directly or indirectly influencing Blimp-1 expression
comprises IL-21, IL-2, IL-6, IL-7, IL-10, IL-15 and/or IL-27. Most
preferably IL-21 is used, since IL-21 is particularly suitable for
influencing the stability of an antibody producing cell. IL-21 is
capable of upregulating Blimp-1 expression even when Blimp-1
expression is counteracted by BCL6.
[0128] Additionally, or alternatively a mutated Janus kinase (JAK)
is used in order to activate STAT3. Naturally, a JAK is capable of
phosphorylating STAT3 after it has itself been activated by at
least one cytokine. A mutated Janus kinase capable of activating
STAT3, independent of the presence of cytokines, is particularly
suitable in a method according to the present invention.
[0129] As already explained before, a compound capable of enhancing
Blimp-1 expression in one embodiment comprises a nucleic acid
sequence encoding STAT3 or a functional part, derivative and/or
analogue thereof. The presence of an exogenous nucleic acid
sequence encoding STAT3 or a functional part, derivative and/or
analogue thereof allows for a continuous presence of STAT3 or a
functional part, derivative and/or analogue thereof even when
expression of endogenous STAT3 is very low or absent.
[0130] It is also possible to decrease expression and/or activity
of STAT5 in order to upregulate Blimp-1. If the amount and/or
activity of STAT5 is decreased, activation of BCL6 expression is
decreased as well, which results in a decreased amount of BCL6
expression product. Since BCL6 and Blimp-1 counteract each other's
expression, a decreased amount of BCL6 expression product results
in an increased amount of Blimp-1 expression product. Compounds
capable of downregulating the activity of STAT5 are thus capable of
indirectly upregulating Blimp-1. Such compounds for instance
comprise members of the suppressor of cytokine signalling (SOCS)
proteins. In one embodiment the amount of Blimp-1 expression
product in an RSV-specific antibody producing cell is therefore
upregulated by providing said cell with a SOCS protein, and/or by
activating a SOCS protein within said cell.
[0131] In one preferred embodiment the expression and/or activity
of STAT5 is decreased when an RSV-specific antibody-producing cell
is provided with a nucleic acid sequence encoding E47 or a
functional part, derivative and/or analogue thereof. Expression of
E47 within B cells expressing high levels of STAT5b intervenes with
differentiation and proliferation, i.e. blocking of STAT5 via E47
and SOCS results in decreased BCL6 levels and subsequently in
increased Blimp-1 levels. Upregulated levels of Blimp-1 result in a
decreased proliferation and in a differentiation of the involved
cell towards an antibody-producing cell. In other words, expression
of E47 within a B cell enhances Blimp-1 expression which results in
B-cell differentiation towards an antibody producing phenotype
(plasma cell).
[0132] By at least a functional part of a STAT5 protein, a STAT3
protein, Bcl-xL and/or BCL6 is meant a proteinaceous molecule that
has the same capability--in kind, not necessarily in amount--of
influencing the stability of an antibody producing cell as compared
to a STAT5 protein, a STAT3 protein, Bcl-xL and/or BCL6,
respectively. A functional part of a STAT5 protein or a STAT3
protein is for instance devoid of amino acids that are not, or only
very little, involved in said capability. A derivative of a STAT5
protein, a STAT3 protein, Bcl-xL and/or BCL6 is defined as a
protein which has been altered such that the capability of said
protein of influencing the stability of an antibody producing cell
is essentially the same in kind, not necessarily in amount. A
derivative is provided in many ways, for instance through
conservative amino acid substitution wherein one amino acid is
substituted by another amino acid with generally similar properties
(size, hydrophobicity, etc), such that the overall functioning is
likely not to be seriously affected. A derivative for instance
comprises a fusion protein, such as a STAT5-ER or STAT3-ER fusion
protein whose activity depends on the presence of 4
hydroxy-tamoxifen (4HT). An analogue of a STAT5 protein, a STAT3
protein, Bcl-xL and/or BCL6 is defined as a molecule having the
same capability of influencing the stability of an antibody
producing cell in kind, not necessarily in amount. Said analogue is
not necessarily derived from said STAT5 protein, STAT3 protein,
Bcl-xL and/or BCL6.
[0133] In one preferred embodiment said RSV-specific antibody
producing cell is cultured in the presence of IL-21 before said
antibody producing cell is provided with a nucleic acid sequence
encoding BCL6 or a functional part, derivative and/or analogue
thereof. Culturing RSV-specific antibody producing cells,
preferably B cells, in the presence of IL-21 before said cell is
provided with a nucleic acid sequence encoding BCL6 or a functional
part, derivative and/or analogue thereof is preferred, because in
these embodiments stability, proliferation and/or antibody
production is particularly well improved.
[0134] In a preferred embodiment, the invention provides a method
for influencing the stability of an RSV-specific antibody producing
cell as described herein, further comprising directly or indirectly
increasing the amount of Bcl-xL expression product within said
antibody producing cell. This is for example accomplished by
providing said antibody producing cell with a nucleic acid sequence
encoding Bcl-xL or a functional part, derivative and/or analogue
thereof or with nucleic acid sequences encoding other
anti-apoptotic genes including but not limited to Bcl-2. In yet
another embodiment this is accomplished by providing said antibody
producing cell with a compound capable of directly or indirectly
enhancing Bcl-xL expression, preferably said compound comprises
APRIL, BAFF, CD40, BCR stimulation, cytokines, growth factors or
downstream effectors like JNK and AKT (PKB).
[0135] Bcl-xL is a member of the anti-apoptotic Bcl-2 family,
Bcl2-proteins interact with and counteract so-called Bcl-2 homology
domain 3 (BH3)-only family members such as Bax, Bak, Bim, and Bad,
which induce cytochome c release following intrinsic death stimuli
(Boise, L. H., 1993). Thus, protection of mitochondrial membrane
integrity through proteins like Bcl-xL is critical for cell
survival.
[0136] STAT5 activation has been shown to protect cells from cell
death. STAT5 has been shown to regulate the expression of Bcl-xL,
supporting an anti-apoptotic role for STAT5. STAT5 positively
regulates the Bcl-xL expression through STAT binding elements
within the Bcl-xL promoter. In vivo, Bcl-xL expression is absent in
bone marrow of STAT5A/B-doubly deficient mice. Furthermore,
STAT5-mediated erythroblast survival is dependent upon upregulation
of Bcl-xL. Recently, it has been shown that transgenic
overexpression of Bcl-xL in mouse B cells promotes B cell survival
and nonmalignant plasma cell foci.
[0137] A method according to the invention is particularly suitable
for producing a cell culture comprising RSV-specific antibody
producing cells that are capable of proliferating and secreting
antibody. In one embodiment, an RSV-specific memory B cell is used
in order to produce an ex vivo B cell culture. Said memory B cell
is preferably human so that human antibodies are produced. Said B
cell preferably originates from an individual, which individual had
been previously exposed to Respiratory Syncytial Virus. In one
embodiment RSV-specific B cells are isolated from a peripheral
blood sample and/or a tonsil sample, using methods known in the
art. Memory B cells are for instance isolated by selection
(magnetic beads sorting) for the B cell marker CD 19 and/or CD22
and (subsequent) selection for cell surface IgG and/or CD27 and/or
by negative selection for IgM, IgD and/or IgA. In a germinal center
B cell, BCL6 expression is high whereas Blimp-1 expression is low.
Natural development into an antibody secreting cell involves
upregulation of Blimp-1 expression. Since Blimp-1 represses BCL6
expression, upregulation of Blimp-1 results in downregulation of
BCL6 in a natural situation. In a preferred embodiment of the
present invention however, Blimp-1 expression is upregulated while
BCL6 expression is at least in part maintained. This results in an
RSV-specific antibody producing cell wherein BCL6 and Blimp-1 are
co-expressed. Said RSV-specific antibody producing cell is capable
of proliferating and secreting anti-RSV antibodies and is therefore
suitable for use in an ex vivo B cell culture. In a further
preferred embodiment, said antibody producing cell is protected by
apoptosis by Bcl-xL. An RSV-specific antibody producing cell
according to the present invention provides the advantage that it
is stable and does not undergo terminal differentiation during a
prolonged period. Said antibody producing cell according to the
invention 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. A B cell according to the
invention is preferably cultured in the presence of CD40L since
replication of most B cells is favoured by CD40L.
[0138] In one embodiment BCL6 expression is maintained at
essentially the same level, or at a higher level, as compared to a
germinal center B cell since a significant BCL6 expression,
together with Blimp-1 expression, results in an antibody producing
cell with preferred proliferation and antibody production
properties and/or stability. In a preferred embodiment, said BCL6
expression and/or Blimp-1 expression are accompanied by Bcl-xL
expression, resulting in even more preferred proliferation and
antibody production properties and/or stability.
[0139] One embodiment therefore provides a method for producing an
RSV-specific antibody producing cell which 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, the
method comprising:
[0140] providing an RSV-specific memory B cell;
[0141] increasing an expression level of Blimp-1 in said cell;
and
[0142] increasing and/or maintaining a BCL6 expression level in
said cell. An ex vivo method for producing an RSV-specific antibody
producing cell comprising increasing an expression level of Blimp-1
in an RSV-specific memory B cell and increasing and/or maintaining
a BCL6 expression level in said cell is also provided. Said BCL6
and Blimp-1 expression levels are preferably brought to, and/or
maintained at, essentially the same level, or at a higher level, as
compared to a plasmablast. In a preferred embodiment said B cell is
transduced with BCL6 and Bcl-xL. Further provided is therefore a
method for producing an RSV-specific antibody producing cell which
is stable for at least three months, comprising:
[0143] providing a B cell capable of producing RSV-specific
antibodies with BCL6, or a functional part, derivative and/or
analogue thereof; and
[0144] providing said B cell with Bcl-xL or a functional part,
derivative and/or analogue thereof; and
[0145] culturing said B cell.
[0146] Said B cell is preferably provided with a nucleic acid
sequence encoding BCL6, or a functional part, derivative and/or
analogue thereof, and with a nucleic acid sequence Bcl-xL or a
functional part, derivative and/or analogue thereof.
[0147] Said B cell is preferably cultured in the presence of a
compound capable of enhancing Blimp-1 expression, such as for
instance IL-21, IL-2, IL-6, IL-7, IL-10, IL-15, IL-27, or a mutated
Janus kinase. Preferably, IL-21 is used because this cytokine is
particularly suitable for enhancing Blimp-1 expression and
stabilizing an antibody producing cell with a method according to
the present invention. Moreover, in order to enhance transduction
efficacy, said B cell is preferably cultured in the presence of
IL-21 before said B cell is transduced with a nucleic acid sequence
encoding BCL6 and/or Bcl-xL, or a functional part, derivative
and/or analogue thereof.
[0148] In one embodiment said B cell is provided with a SOCS
protein or a functional part, derivative and/or analogue thereof,
or a nucleic acid coding therefore, since a SOCS protein or a
functional part, derivative and/or analogue thereof is capable of
indirectly enhancing Blimp-1 expression. In another alternative or
additional embodiment, said B-cell is provided with E47 or a
functional part, derivative and/or analogue thereof, or a nucleic
acid coding therefore. As already outlined earlier, as a result of
an increased level of E47 or a functional part, derivative and/or
analogue thereof, SOCS protein function is enhanced and Blimp-1
expression is indirectly increased.
[0149] In the Examples particularly preferred embodiments are
shown. According to one particularly preferred embodiment,
RSV-specific B cells are firstly cultured in the presence of IL-21.
Subsequently the B cells are subjected to a transduction reaction
using a nucleic acid encoding BCL6 and a nucleic acid encoding
Bcl-xL. Preferably spin transduction is used. Most preferably, B
cells and virus comprising at least one nucleic acid of interest
are mixed, where after the mixture is spinned in order to achieve a
high transduction efficacy. After transduction, the B cells are
cultured in the absence of IL-21 and in the presence of IL-4 and
L-cells during 3-5 days in order to allow BCL6 expression.
Subsequently, according to this preferred embodiment, the B cells
are subjected again to a transduction reaction using a nucleic acid
encoding BCL6 and a nucleic acid encoding Bcl-xL. Afterwards, the B
cells are again cultured in the absence of IL-21 and in the
presence of IL-4 and L-cells during 3-5 days in order to allow BCL6
expression. Subsequently, cells expressing BCL6 and Bcl-xL are
isolated and IL-21 is administered again to the culture in order to
enhance replication and antibody production. Antibodies that are
secreted by Bcl-6, Blimp 1 and Bcl-XL expressing cells in the
culture supernatant are preferably screened for in vitro
neutralizing capacity/activity/reactivity to RSV. Antibody
producing cells that produce those antibodies are preferably
further selected, for instance by limiting dilution culture. Stable
RSV-specific B cells are thus obtained wherein BCL6 and Blimp-1 are
co-expressed. Said B cells are capable of replicating and producing
antibody in an in vitro culture during at least six months,
[0150] One embodiment provides a method according to the invention
further comprising selecting and/or isolating an RSV-specific
antibody or a functional equivalent thereof. In one embodiment IgM
producing cells and IgG producing cells are selected and/or
isolated. Preferably an IgG producing cell is selected and/or
isolated,
[0151] RSV-specific antibody producing cells generated with a
method according to the invention are suitable for producing
antibodies against RSV. In one preferred embodiment however, the
genes encoding the Ig heavy and/or light chains are isolated from
said cell and expressed in a second cell, such as for instance
cells of a Chinese hamster ovary (CHO) cell line or 293(T) cells.
Said second cell, also called herein a producer cell, is preferably
adapted to commercial antibody production. Proliferation of said
producer cell results in a producer cell line capable of producing
RSV-specific antibodies. Preferably, said producer cell line is
suitable for producing compounds for use in humans. Hence, said
producer cell line is preferably free of pathogenic agents such as
pathogenic microorganisms.
[0152] A method according to the invention is preferably used for
generating 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
commercial antibody production has become possible. Most preferably
a stable cell line capable of producing monoclonal antibodies is
produced. This is preferably performed by using memory B cells that
have for instance been isolated from a sample by selection for CD
19 and/or CD22 (B cell marker) and cell surface IgG and/or CD27 (to
mark memory cells) and/or by negative selection for IgM, IgD and/or
IgA. Furthermore, an RSV-specific antibody producing cell is for
instance selected in a binding assay using RSV or a component
derived from RSV, such as for instance the RSV F protein, G protein
and/or SH protein. Subsequently, according to this preferred
embodiment Blimp-1 and BCL6 are co-expressed in said RSV-specific
antibody producing cell, resulting in a culture of cells capable of
specifically binding (a component of) RSV. In yet another preferred
embodiment, said B cell is further provided with Bcl-xL or a
functional part, derivative and/or analogue thereof.
[0153] If only one memory cell is used, a cell line according to
the invention which produces monoclonal antibodies is obtained. It
is also possible to generate a monoclonal antibody producing cell
line starting with B cells capable of producing antibodies against
RSV. After a stable B cell culture has been produced with a method
according to the invention, a B cell capable of producing
antibodies against a specific antigen of RSV is isolated and at
least a functional part of a gene encoding the Ig heavy chain
and/or light chain from said B cell is preferably expressed in a
second cell line. Preferably at least a functional part of the gene
encoding the Ig heavy chain and at least a functional part of the
gene encoding the Ig light chain from said B cell are expressed in
a second cell line.
[0154] In one embodiment an antibody producing cell, preferably but
not necessarily a memory B cell, that has been obtained from an
individual which had been previously exposed to RSV, is used in a
method according to the invention. This way, it has become possible
to produce human antibodies of interest ex vivo.
[0155] Further provided is therefore a method for producing
antibodies which are capable of specifically binding and/or
neutralizing Respiratory Syncytial Virus, the method
comprising:
[0156] producing an antibody producing cell capable of producing
RSV-specific antibodies with a method according to the invention;
and
[0157] obtaining antibodies produced by said antibody producing
cell.
[0158] An isolated or recombinant antibody, as well as an isolated
or recombinant antibody producing cell, obtainable by a method
according to the invention, or a functional equivalent thereof, is
also provided. Said antibody preferably comprises antibody D25,
AM14, AM16 and/or AM23, or a functional part, derivative or
analogue thereof.
[0159] Once an RSV-specific antibody producing cell according to
the invention is obtained, at least a functional part of a gene
encoding the Ig heavy chain and/or light chain of said cell is
preferably isolated and/or generated artificially. In one
embodiment a nucleic acid sequence comprising at least a functional
part of a nucleic acid sequence as depicted in FIG. 11A, FIG. 12,
FIG. 14A, FIG. 14B and/or FIG. 14C is provided. Said functional
part preferably comprises at least one nucleic acid sequence as
depicted in FIG. 11A, FIG. 12, FIG. 14A, FIG. 14B and/or FIG. 14C.
Said functional part preferably encodes at least one CDR as
depicted in FIGS. 11B and 11C, FIG. 12, FIG. 14A, FIG. 14B and/or
FIG. 14C.
[0160] Further provided is an isolated, synthetic or recombinant
nucleic acid sequence comprising a heavy chain sequence which is at
least 70%, preferably at least 80%, more preferably at least 90%
homologous to at least part of the sequence
CAGGTGCAGCTGGTACAGTCTGGGGCTGAAGTGAAGAAGCCTGGGTCCTCGGTGATGGTCTC
CTGCCAGGCCTCTGGAGGCCCCCTCAGAA (SEQ ID NO: 59), ACTATATTATCAAC (SEQ
ID NO: 60), TGGCTACGACAGGCCCCTGGACAAGGCCCTGAGTGGATGGGA (SEQ ID NO:
61), GGGATCATTCCTGTCTTGGGTACAGTACACTACGCACCGAAGTTCCAGGGC (SEQ ID
NO: 62),
AGAGTCACGATTACCGCGGACGAATCCACAGACACAGCCTACATCCATCTGATCAGCCTGAG
ATCTGAGGACACGGCCATGTATTACTGTGCGACG (SEQ ID NO: 63),
GAAACAGCTCTGGTTGTATCTACTACCTACCTACCACACTACTTTGACAAC (SEQ ID NO:
64), TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG (SEQ ID NO: 65), and/or
CAGGTGCAGCTGGTACAGTCTGGGGCTGAAGTGAAGAAGCCTGGGTCCTCGGTGATGGTCTC
CTGCCAGGCCTCTGGAGGCCCCCTCAGAAACTATATTATCAACTGGCTACGACAGGCCCCTG
GACAAGGCCCTGAGTGGATGGGAGGGATCATTCCTGTCTTGGGTACAGTACACTACGCACCG
AAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACAGACACAGCCTACATCCATCT
GATCAGCCTGAGATCTGAGGACACGGCCATGTATTACTGTGCGACGGAAACAGCTCTGGTTG
TATCTACTACCTACCTACCACACTACTTTGACAACTGGGGCCAGGGAACCCTGGTCACCGTC
TCCTCAG (SEQ ID NO: 9), said part having at least 15 nucleotides.
Said heavy chain sequence is preferably derived from antibody D25.
Said heavy chain sequence preferably comprises a sequence which is
at least 70%, preferably at least 80%, more preferably at least 90%
homologous to a sequence as depicted in FIG. 11A. An isolated,
synthetic or recombinant nucleic acid sequence comprising a heavy
chain sequence consisting of any of the above mentioned heavy chain
sequences is also herewith provided.
[0161] An isolated, synthetic or recombinant nucleic acid sequence
comprising a light chain sequence which is at least 70%, preferably
at least 80%, more preferably at least 90% homologous to a least
part of the sequence
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCAGCTGTAGGAGACAGAGTCACCAT
CACTTGC (SEQ ID NO: 66), CAGGCGAGTCAGGACATTGTCAACTATTTAAAT (SEQ ID
NO: 67), TGGTATCAACAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTAC (SEQ ID NO:
68), GTTGCATCCAATTTGGAGACA (SEQ ID NO: 69),
GGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTAGTCTCACCATCAGCAG
CCTGCAGCCTGAAGATGTTGCAACATATTATTGT (SEQ ID NO: 70),
CAACAATATGATAATCTCCCA (SEQ ID NO: 71),
CTCACATTCGGCGGAGGGACCAAGGTTGAGATCAAAAGA (SEQ ID NO: 72) and/or
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCAGCTGTAGGAGACAGAGTCACCAT
CACTTGCCAGGCGAGTCAGGACATTGTCAACTATTTAAATTGGTATCAACAGAAACCAGGGA
AAGCCCCTAAGCTCCTGATCTACGTTGCATCCAATTTGGAGACAGGGGTCCCATCAAGGTTC
AGTGGAAGTGGATCTGGGACAGATTTTAGTCTCACCATCAGCAGCCTGCAGCCTGAAGATGT
TGCAACATATTATTGTCAACAATATGATAATCTCCCACTCACATTCGGCGGAGGGACCAAGG
TTGAGATCAAAAGA (SEQ ID NO: 10), said part having at least 15
nucleotides, is also provided. Said light chain sequence is
preferably derived from antibody D25.
[0162] Said light chain sequence preferably comprises a sequence
which is at least 70%, preferably at least 80%, more preferably at
least 90% homologous to a sequence as depicted in FIG. 11A. An
isolated, synthetic or recombinant nucleic acid sequence comprising
a heavy chain sequence consisting of any of the above mentioned
light chain sequences is also herewith provided.
[0163] Further provided is an isolated, synthetic or recombinant
nucleic acid sequence comprising a heavy chain sequence which is at
least 70%, preferably at least 80%, more preferably at least 90%
homologous to at least part of the sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTC
CTGTGCGGCCTCT (SEQ ID NO: 94), GGATTCAGCTTCAGTCACTATGCC (SEQ ID NO:
95), ATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCAGTT (SEQ ID
NO: 96), ATATCTTATGATGGAGAAAATACA (SEQ ID NO: 97),
TATTACGCAGACTCCGTGAAGGGCCGATTCTCCATCTCCAGAGACAATTCCAAGAACACAGT
GTCTCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTCTATATTACTGT (SEQ ID NO:
98), GCGAGAGACCGCATAGTGGACGACTACTACTACTACGGTATGGACGTC (SEQ ID NO:
99), TGGGGCCAAGGGGCCACGGTCACCGTCTCCTCAG (SEQ ID NO: 100) and/or
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTC
CTGTGCGGCCTCTGGATTCAGCTTCAGTCACTATGCCATGCACTGGGTCCGCCAGGCTCCAG
GCAAGGGACTGGAGTGGGTGGCAGTTATATCTTATGATGGAGAAAATACATATTACGCAGAC
TCCGTGAAGGGCCGATTCTCCATCTCCAGAGACAATTCCAAGAACACAGTGTCTCTGCAAAT
GAACAGCCTGAGACCTGAGGACACGGCTCTATATTACTGTGCGAGAGACCGCATAGTGGACG
ACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGGCCACGGTCACCGTCTCCTCA (SEQ ID
NO: 101), said part having at least 15 nucleotides. Said heavy
chain sequence is preferably derived from antibody AM14. An
isolated, synthetic or recombinant nucleic acid sequence comprising
a heavy chain sequence consisting of any of the above mentioned
heavy chain sequences is also herewith provided.
[0164] An isolated, synthetic or recombinant nucleic acid sequence
comprising a light chain sequence which is at least 70%, preferably
at least 80%, more preferably at least 90% homologous to a least
part of the sequence
GACATCCAGATGACCCAGTCTCCATCTTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCAT
CACTTGCCAGGCGAGT (SEQ ID NO: 102), CAGGACATTAAGAAGTAT (SEQ ID NO:
103), TTAAATTGGTATCATCAGAAACCAGGGAAAGTCCCTGAGCTCCTGATGCAC (SEQ ID
NO: 104), GATGCATCC,
AATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGCAGGGGATCTGGGACAGATTTTACTCT
CACCATTAGCAGCCTGCAGCCTGAAGATATTGGAACATATTACTGT (SEQ ID NO: 105),
CAACAGTATGATAATCTGCCTCCGCTCACT (SEQ ID NO: 106),
TTCGGCGGAGGGACCAAGGTGGAGATCAAAC (SEQ ID NO: 107) and/or
GACATCCAGATGACCCAGTCTCCATCTTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCAT
CACTTGCCAGGCGAGTCAGGACATTAAGAAGTATTTAAATTGGTATCATCAGAAACCAGGGA
AAGTCCCTGAGCTCCTGATGCACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTC
AGTGGCAGGGGATCTGGGACAGATTTTACTCTCACCATTAGCAGCCTGCAGCCTGAAGATAT
TGGAACATATTACTGTCAACAGTATGATAATCTGCCTCCGCTCACTTTCGGCGGAGGGACCA
AGGTGGAGATCAAACGAACTGTG (SEQ ID NO: 108), said part having at least
15 nucleotides, is also provided. Said light chain sequence is
preferably derived from antibody AM14. An isolated, synthetic or
recombinant nucleic acid sequence comprising a heavy chain sequence
consisting of any of the above mentioned light chain sequences is
also herewith provided.
[0165] Further provided is an isolated, synthetic or recombinant
nucleic acid sequence comprising a heavy chain sequence which is at
least 70%, preferably at least 80%, more preferably at least 90%
homologous to at least part of the sequence
GAGGTGCAGCTGGTGGAGACCGGGGGAGGCCTGGCCCAGCCTGGGGGGTCCCTGAGACTCTC
CTGTGCAGCCTCT (SEQ ID NO: 109), GGATTCACATTCAGTAGTTATAAC (SEQ ID
NO: 110), ATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCACAC (SEQ
ID NO: 111), ATTAGTGCGGGTAGTAGTTACATA (SEQ ID NO: 112),
TACTACTCAGACTCAGTGAAGGGCCGATTCACCGTCTCCAGAGACAACGTCAGGAACTCAGT
ATATCTGCAAATGAACAGCCTGAGAGCCGCTGACACGGCTGTGTATTACTGT (SEQ ID NO:
113), GCGAGAGAGGATTATGGTCCGGGAAATTATTATAGTCCTAACTGGTTCGACCCC (SEQ
ID NO: 114), TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG (SEQ ID NO: 115)
and/or
GAGGTGCAGCTGGTGGAGACCGGGGGAGGCCTGGCCCAGCCTGGGGGGTCCCTGAGACTCTC
CTGTGCAGCCTCTGGATTCACATTCAGTAGTTATAACATGAACTGGGTCCGCCAGGCTCCAG
GGAAGGGGCTGGAGTGGGTCTCACACATTAGTGCGGGTAGTAGTTACATATACTACTCAGAC
TCAGTGAAGGGCCGATTCACCGTCTCCAGAGACAACGTCAGGAACTCAGTATATCTGCAAAT
GAACAGCCTGAGAGCCGCTGACACGGCTGTGTATTACTGTGCGAGAGAGGATTATGGTCCGG
GAAATTATTATAGTCCTAACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCC TCA
(SEQ ID NO: 116), said part having at least 15 nucleotides. Said
heavy chain sequence is preferably derived from antibody AM16. An
isolated, synthetic or recombinant nucleic acid sequence comprising
a heavy chain sequence consisting of any of the above mentioned
heavy chain sequences is also herewith provided.
[0166] An isolated, synthetic or recombinant nucleic acid sequence
comprising a light chain sequence which is at least 70%, preferably
at least 80%, more preferably at least 90% homologous to a least
part of the sequence
CAGTCTGTCGTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGAGTCACCATCTC
CTGCACTGGGAGC (SEQ ID NO: 117), AGCTCCAACATCGGGGCAGGTTATGAT (SEQ ID
NO: 118), GTACACTGGTACCAGCAGCTTCCAGGAACAGCCCCCAAACTCCTCATCTAT (SEQ
ID NO: 119), GGCAACACT,
AATCGGCCCTCAGGGGTCTCCGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCT
GGCCATCACTGGACTCCAGGCTGAGGATGAGGCTGATTATTACTGC (SEQ ID NO: 120),
CACTCCTATGACAGAAGCCTGAGTGGT (SEQ ID NO: 121),
TCAGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAG (SEQ ID NO: 122) and/or
CAGTCTGTCGTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGAGTCACCATCTC
CTGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGATGTACACTGGTACCAGCAGCTTC
CAGGAACAGCCCCCAAACTCCTCATCTATGGCAACACTAATCGGCCCTCAGGGGTCTCCGAC
CGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGACTCCAGGCTGA
GGATGAGGCTGATTATTACTGCCACTCCTATGACAGAAGCCTGAGTGGTTCAGTATTCGGCG
GAGGGACCAAGCTGACCGTC (SEQ ID NO: 123), said part having at least 15
nucleotides, is also provided. Said light chain sequence is
preferably derived from antibody AM16. An isolated, synthetic or
recombinant nucleic acid sequence comprising a heavy chain sequence
consisting of any of the above mentioned light chain sequences is
also herewith provided.
[0167] Further provided is an isolated, synthetic or recombinant
nucleic acid sequence comprising a heavy chain sequence which is at
least 70%, preferably at least 80%, more preferably at least 90%
homologous to at least part of the sequence
CAGGTGCAACTGGTGGAGTCTGGGGGAAATGTGGTCAAGCCTGGGACGTCCCTGAGACTGTC
CTGTGCAGCGACT (SEQ ID NO: 124), GGATTCAACTTCCATAACTACGGC (SEQ ID
NO: 125), ATGAACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCGGTT (SEQ
ID NO: 126), GTTTGGTATGATGGAAGTAAGAAA (SEQ ID NO: 127),
TACTATGCAGACTCCGTGACGGGCCGATTCGCCATCTCCAGAGACAATTCCAAGAACACTCT
GTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCTGTTTATTATTGT (SEQ ID NO:
128), GTGAGAGATAAAGTGGGACCGACTCCCTACTTTGACTCC (SEQ ID NO: 129),
TGGGGCCAGGGAACCCTGGTCACCGTATCCTCAG (SEQ ID NO: 130) and/or
GAGGTGCAGCTGGTGGAGTCTGGGGGAAATGTGGTCAAGCCTGGGACGTCCCTGAGACTGTC
CTGTGCAGCGACTGGATTCAACTTCCATAACTACGGCATGAACTGGGTCCGCCAGGCTCCAG
GCAAGGGGCTGGAGTGGGTGGCGGTTGTTTGGTATGATGGAAGTAAGAAATACTATGCAGAC
TCCGTGACGGGCCGATTCGCCATCTCCAGAGACAATTCCAAGAACACTCTGTATCTGCAAAT
GAACAGCCTGAGAGTCGAGGACACGGCTGTTTATTATTGTGTGAGAGATAAAGTGGGACCGA
CTCCCTACTTTGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO:
131), said part having at least 15 nucleotides. Said heavy chain
sequence is preferably derived from antibody AM23. An isolated,
synthetic or recombinant nucleic acid sequence comprising a heavy
chain sequence consisting of any of the above mentioned heavy chain
sequences is also herewith provided.
[0168] An isolated, synthetic or recombinant nucleic acid sequence
comprising a light chain sequence which is at least 70%, preferably
at least 80%, more preferably at least 90% homologous to a least
part of the sequence
TCCTATGTGCTGACTCAGCCACCCTCGGTGTCACTGGCCCCAGGAGGGACGGCCGCGATCAC
CTGTGGAAGAAAC (SEQ ID NO: 132), AACATTGGAAGTGAAACT (SEQ ID NO:
133), GTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCGTCTAT (SEQ ID
NO: 134), GATGATGAC,
GACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCT
GACCATCAGCAGGGTCGAGGCCGGGGATGAGGCCGACTATTACTGT (SEQ ID NO: 135),
CAGGTGTGGGATAGGAGTAATTATCATCAGGTA (SEQ ID NO: 136),
TTCGGCGGAGGGACCAAGTTGACCGTCCT AG (SEQ ID NO: 137) and/or
TCCTATGTGCTGACTCAGCCCCCCTCGGTGTCACTGGCCCCAGGAGGGACGGCCGCGATCAC
CTGTGGAAGAAACAACATTGGAAGTGAAACTGTGCACTGGTACCAGCAGAAGCCAGGCCAGG
CCCCTGTGCTGGTCGTCTATGATGATGACGACCGGCCCTCAGGGATCCCTGAGCGATTCTCT
GGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAGGCCGGGGATGAGGC
CGACTATTACTGTCAGGTGTGGGATAGGAGTAATTATCATCAGGTATTCGGCGGAGGGACCA
AGCTGACCGTC (SEQ ID NO: 138), said part having at least 15
nucleotides, is also provided. Said light chain sequence is
preferably derived from antibody AM23. An isolated, synthetic or
recombinant nucleic acid sequence comprising a heavy chain sequence
consisting of any of the above mentioned heavy chain sequences is
also herewith provided.
[0169] A nucleic acid sequence encoding an amino acid sequence
which is at least 70%, preferably at least 80%, more preferably at
least 90% identical to at least a functional part of an amino acid
sequence as depicted in FIG. 11, FIG. 14A, FIG. 14B and/or FIG.
14C, said part having at least 5 amino acid residues is also
provided. Said nucleic acid sequence preferably encodes an amino
acid sequence which is at least 80% identical to heavy chain CDR
sequence 1, 2 and/or 3 and/or light chain CDR sequence 1 or 2
depicted in FIGS. 11B and 11C. In another preferred embodiment said
nucleic acid sequence encodes an amino acid sequence which is at
least 80% identical to at least one of the CDR sequences depicted
in FIG. 14A, in FIG. 14B and/or in FIG. 14C. In one preferred
embodiment said nucleic acid sequence encodes an amino acid
sequence which is at least 70% identical to a heavy chain sequence
depicted in FIG. 11A, to a heavy chain sequence depicted in FIG.
14A, to a heavy chain sequence depicted in FIG. 11B, to a heavy
chain sequence depicted in FIG. 14C, to a light chain sequence
depicted in FIG. 11A, to a light chain sequence depicted in FIG.
14A, to a light chain sequence depicted in FIG. 14B, and/or to a
light chain sequence depicted in FIG. 14C.
[0170] Further provided is therefore an isolated, synthetic or
recombinant nucleic acid sequence comprising a sequence encoding an
amino acid sequence which is at least 70%, preferably at least 80%,
more preferably at least 85% identical to an amino acid sequence as
depicted in FIG. 11. Said nucleic acid sequence preferably encodes
an amino acid sequence which is at least 80% identical to heavy
chain CDR sequence 1, 2 and/or 3 and/or light chain CDR sequence 1
or 2 as depicted in FIGS. 11B and 11C. One embodiment provides an
isolated, synthetic or recombinant nucleic acid sequence comprising
a sequence encoding an amino acid sequence which is at least 70%
identical to the amino acid sequence NYIIN (SEQ ID NO: 1), and/or
at least 75% identical to the sequence GIIPVLGTVHYAPKFQG (SEQ ID
NO: 2), and/or at least 70% identical to the sequence
ETALVVSTTYLPHYFDN (SEQ ID NO: 3), and/or at least 85% identical to
the sequence QASQDIVNYLN (SEQ ID NO: 4), and/or at least 70%
identical to the sequence VASNLET (SEQ ID NO: 5), and/or at least
70% identical to the sequence
QVQLVQSGAEVKKPGSSVMVSCQASGGPLRNYIINWLRQAPGQGPEWMGGIIPVLGT
VHYAPKFQGRVTITADESTDTAYIHLISLRSEDTAMYYCATETALVVSTTYLPHYFDN
WGQGTLVTVSS (SEQ ID NO: 7), and/or at least 70% identical to the
sequence DIQMTQSPSSLSAAVGDRVTITCQASQDIVNYLNWYQQKPGKAPKLLIYVASNLETG
VPSRFSGSGSGTDFSLTISSLQPEDVATYYCQQYDNLPLTFGGGTKVEIKRTV (SEQ ID NO:
8).
[0171] A nucleic acid sequence according to the invention is
preferably at least 80%, more preferably at least 85%, more
preferably at least 90%, most preferably at least 95% homologous to
any of the above recited sequences.
[0172] Further provided is an isolated, synthetic or recombinant
nucleic acid sequence comprising a sequence encoding an amino acid
sequence which is at least 70%, preferably at least 80%, more
preferably at least 85% identical to an amino acid sequence as
depicted in FIG. 14A-C. Said nucleic acid sequence preferably
encodes an amino acid sequence which is at least 70% identical to a
CDR sequence as depicted in FIG. 14A, 14B and/or 14C. One
embodiment provides an isolated, synthetic or recombinant nucleic
acid sequence comprising a sequence encoding an amino acid sequence
which is at least 70% identical to an amino acid sequence selected
from the group consisting of: GFSFSHYA (SEQ ID NO: 73), ISYDGENT
(SEQ ID NO: 74), ARDRIVDDYYYYGMDV (SEQ ID NO: 75), QDIKKY (SEQ ID
NO: 76), DAS, QQYDNLPPLT (SEQ ID NO: 77),
EVQLVESGGGVVQPGRSLRLSCAASGFSFSHYAMHWVRQAPGKGLEWVAVISYDGE
NTYYADSVKGRFSISRDNSKNTVSLQMNSLRPEDTALYYCARDRIVDDYYYYGMDV
WGQGATVTVSS (SEQ ID NO: 78),
DIQMTQSPSSLSASVGDRVTITCQASQDIKKYLNWYHQKPGKVPELLMHDASNLETG
VPSRFSGRGSGTDFTLTISSLQPEDIGTYYCQQYDNLPPLTFGGGTKVEIKRTV (SEQ ID NO:
79), GFTFSSYN (SEQ ID NO: 80), ISAGSSYI (SEQ ID NO: 81),
AREDYGPGNYYSPNWFDP (SEQ ID NO: 82), SSNIGAGYD (SEQ ID NO: 83), GNT,
HSYDRSLSG (SEQ ID NO: 84),
EVQLVETGGGLAQPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSHISAGSS
YIYYSDSVKGRFTVSRDNVRNSVYLQMNSLRAADTAVYYCAREDYGPGNYYSPNW
FDPWGQGTLVTVSS (SEQ ID NO: 85),
QSVVTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKWYGNTNRPS
GVSDRFSGSKSGTSASLAITGLQAEDEADYYCHSYDRSLSGSVFGGGTKLTV (SEQ ID NO:
86), GFNFHNYG (SEQ ID NO: 87), VWYDGSKK (SEQ ID NO: 88),
VRDKVGPTPYFDS (SEQ ID NO: 89), NIGSET (SEQ ID NO: 90), DDD,
QVWDRSNYHQV (SEQ ID NO: 91),
EVQLVESGGNVVKPGTSLRLSCAATGFNFHNYGMNWVRQAPGKGLEWVAVVWYD
GSKKYYADSVTGRFAISRDNSKNTLYLQMNSLRVEDTAVYYCVRDKVGPTPYFDSW GQGTLVTVSS
(SEQ ID NO: 92), and
SYVLTQPPSVSLAPGGTAAITCGRNNIGSETVHWYQQKPGQAPVLVVYDDDDRPSGI
PERFSGSNSGNTATLTISRVEAGDEADYYCQVWDRSNYHQVFGGGTKLTV (SEQ ID NO:
93).
[0173] A nucleic acid sequence according to the invention is
preferably at least 80%, more preferably at least 85%, more
preferably at least 90%, most preferably at least 95% homologous to
any of the above recited sequences.
[0174] As already explained herein before, nucleic acid sequences
according to the present invention are particularly suitable for
expressing an antibody or a functional part, derivative or analogue
thereof according to the invention, preferably D25, AM14, AM16,
AM23 or a functional part, derivative and/or analogue thereof, in a
nucleic acid expression system. A nucleic acid sequence according
to the present invention is preferably expressed in a cell, more
preferably in a producer cell adapted for antibody production.
[0175] The invention is further explained in the following
examples. These examples do not limit the scope of the invention,
but merely serve to clarify the invention.
EXAMPLES
Materials and Methods
Maintenance and Isolation of Human B Cells
[0176] Using standard procedures, CD19 positive human B cells were
isolated from bloodbank derived buffy coat (other sources can be
fresh blood with an anticoagulation factor, or a lymphoid organ for
example tonsil or spleen). In brief, total peripheral blood
mononuclear cells (PBMC) were isolated using ficoll density
separation (Amersham, Buckinghamshire, UK). CD22 labeled beads were
used to positively selected B cells by MACS cell sorting technique
as described by the manufacturer (Miltenyi, Utrecht, Netherlands).
Cells were subsequently stained with appropriate combinations of
monoclonal antibodies (mAbs) to CD19, CD27, IgD, IgM and IgA
(Becton Dickinson (BD), Franklin Lakes, N.J., USA). Memory B cells
that are positive for CD 19 and CD27 and negative for IgM, IgA and
IgD were then sorted using the FACSAria (BD) (FIG. 1). Besides
memory B cells, other B cells subsets, like naive, naive,
follicular, memory, antibody producing, centroblast, centrocyte,
germinal center, plasma blast, plasma cell, marginal zone,
perisinusoidal or transitional B cells (many of those subsets have
only been determined in mice) can be isolated using appropriate
markers.
Cell Culture
[0177] Sorted cells were washed and cultured in 24 well plates (1.5
to 2.times.10.sup.5 cells/ml) on 80 Gray, irradiated
CD40L-expressing L-cells (5.times.10.sup.4 cells/ml; provided by
DR. J. Banchereau, Schering Plough France, Dardilly France), in
complete medium (Iscove's Modified D Minimal Essential Medium
containing 8% fetal calf serum (FCS) and Penicillin/Streptomycin).
Unless mentioned otherwise, these CD40L-expressing L-cells are
always present in the cultures in combination with 8% FCS. To
prepare the B cell for retroviral transduction cells were cultured
for 36 hours in the presence of mouse IL-21 (50 ng/ml, R&D,
Minneapolis, Minn., USA). After transduction cells are
preferentially cultured in the presence of IL-21, however cells do
respond to IL-4, 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 can be required in some
experiments.
Retroviral Constructs and Production of Recombinant Retrovirus
[0178] Constitutive active mutants of STAT5a and b have been
described previously. DNAs encoding these mutants and wildtype
STAT5b were obtained from T. Kitamura (IMSUT, Tokyo, Japan). Bcl-6
was identified in a senescence rescue screen in murine fibroblasts
as an inhibitor of anti-proliferative p19ARF-p53 signaling. Bcl-XL
was identified as an anti-apoptose factor, which was kindly
provided by Dr Korsmeyer (Howard Hughes Medical Institute, Boston,
US). These DNAs were ligated into LZRS-linker-IRES-GFP (or IRES-YFP
or IRES-NGFR) vector that was described previously (Heemskerk et
al., 1997; Heemskerk et al., 1999). Instead of the IRES-GFP (Green
Fluorescent Protein) marker also an IRES-YFP (Yellow Fluorescent
Protein) or an IRES-NGFR (Nerve Growth Factor Receptor) was used.
NGFR is a signaling-incompetent mutant of the NGFR, kindly provided
by Dr. C. Bonini A monoclonal antibody against NGFR (Chromaprobe,
Mountain View, Calif., US or Miltenyi) was used to visualize
NGFR-expressing cells.
[0179] For production of recombinant retrovirus, the retroviral
plasmids were transfected into a helper-virus free amphotropic
producer cell line Phoenix-A, a derivative of the human embryonic
kidney cell line 293 (Kinsella and Nolan, 1996) (a kind gift of Dr.
G. Nolan, Stanford University, Palo Alto, Calif.), using Fugene-6
(Roche Diagnostics Netherlands, Almere, Netherlands) according to
manufacturers protocols. Two days later selection of transfected
cells started by the addition of 2 .mu.g/ml puromycin (Becton
Dickinson Clontech Laboratories, Palo Alto, Calif.). Ten to 14 days
after transfection 6.times.10.sup.6 cells were plated per 10 cm
petridish (Becton Dickinson Discovery Labware, Bedford, Mass.) in
10 ml complete medium without puromycin. The next day the medium
was refreshed and on the following day retroviral supernatant was
harvested, centrifuged and frozen in cell free aliquots at
-70.degree. C. This approach affords a reproducible rapid, large
scale and high titer retroviral production of over 3.times.10.sup.6
infectious virus particles/ml.
Retroviral Transduction
[0180] The recombinant human fibronectin fragments CH-296
transduction procedure (RetroNectin.TM.; Takara, Otsu, Japan) was
performed as described previously (Heemskerk et al., 1997;
Heemskerk et al., 1999). Non-tissue culture-treated 24 wells plates
(Costar, Badhoevedorp, Netherlands) were coated with 0.3 ml of 30
.mu.g/ml recombinant human fibronectin fragment CH-296 at room
temperature for 2 hours or overnight at 4.degree. C. When different
sized non-tissue culture plates were used, reagents were used
proportionately. The CH-296 solution was removed, followed by
incubation with 2% human serum albumin (HSA) in phosphate buffered
saline (PBS) for 30 min at room temperature, followed by washing
once with PBS. 5.times.10.sup.5 B cells, which were prepared for
retroviral transduction were plated in 0.25 ml RPMI without FCS and
L-cells and mixed with 0.25 ml of thawed retroviral supernatant.
For the Bcl-6 Bcl-XL double transduction 125 .mu.l of
Bcl-6-IRES-NGFR (or IRES-YFP) (Shvarts A. et al. Genes Dev., 2002)
and 125 .mu.l of Bcl-XL-IRES-GFP (provided by S. Korsmeyer, Howard
Hughes Medical Institute, Childrens Hospital, Boston, USA) were
mixed and added to the cells. The culture was subsequently
centrifuged at 1800 rpm at 25.degree. C. for 60 minutes and
incubated for 6 hours at 37.degree. C. Next 0.25 ml of supernatant
was removed and 0.25 ml of fresh retroviral supernatant was added.
The culture was again centrifuged at 1800 rpm at 25.degree. C. for
60 minutes and incubated at 37.degree. C. overnight. The next
morning cells were transferred to 24 wells tissue culture treated
plate (Costar) 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, Minn., USA). Transduction efficiency was
determined by antibody staining of a truncated, signaling
incompetent mutant of Nerve Growth Factor Receptor (ANGFR, provided
by C. Bonini, St. Raphael Hospital, Milan, Italy) or (co)
expression of GFP and or YFP. The cells containing the transgene(s)
of interest are then selected for further experiments.
Flowcytometry
[0181] Antibodies against the human molecules IgD, IgG, CD3, CD 19,
CD20, CD27, CD38, CD40, CD45, CD56, CD70, CD80, CD86, HLA-DR (BD)
directly labeled with FITC, PE, PERCP, PE-Cy5, APC or APC-Cy7 and
IgM, kappa light chain, lambda light chain, CD 138, directly
labeled with PE (DAKO) were used for flowcytometry analysis.
Stained cells were analyzed using a LSRII (BD) and FACS data was
processed with Flow Jo computer software (Tree Star, Inc).
Proliferation Experiment
[0182] Naive and memory B cells were isolated from fresh PBMC on
the FACSAria: Naive B cells: CD19-Pe-Cy7 pos, CD27-APC neg, IgD-PE
pos
[0183] Memory B cells: CD19-Pe-Cy7 pos, CD27-APC pos, IgD-PE neg,
IgA-FITC neg Cells were washed in PBS and resuspended in 0.5 ml
RPMI (37.degree. C.) without FCS. An equal amount of IMDM
containing 2 .mu.M Carboxyfluorescein succinimidyl ester (CFSE) was
added to the cell mixture and incubated for 7 min at 37.degree. C.
Up labeling of the cells was stopped by washing the cell with cold
FCS. Cells were resuspended in 500 .mu.l IMDM-8% FCS and cultured
with L-cells and in the absence or presence of IL-21. Non-labeled
cells were used as control. After 36 hrs (immediately before
transduction) a proportion of cells was analyzed for their CFSE
content. Remaining cells were spin transduced with Bcl-6-IRES-NGFR,
cultured for 3 days, and analyzed for their CFSE content using the
LSRII. Data was analyzed using FlowJo software (Treestar)
[0184] Isolation of antigen specific human B cells using high speed
single cell sorting In addition to the memory B cell isolation
method described above starting with MBC (i.e. 100 cell/well
cultures), 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
phycoerythrin (PE) labeled Tetanus Toxoid (provided by A. Radbruch,
Berlin, Germany) (FIG. 4). Cells were cultured at 1 cell/well and
checked for TT binding. Notwithstanding that any other labeled
antigen can be used.
[0185] Determining the B Cell Receptor (BCR) Expression Alter Long
Term Culture of Bcl-6 and Bcl-XL Transduced Cells
[0186] It is known that B cells that differentiate during in vitro
culture lose their BCR membrane expression, 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, CD 19, Kappa and/or Lambda or IgG or with labeled
Tetanus Toxoid. To show the usefulness of the BCR expression we
sorted TT-PE (Radbruch) binding cells using the FACSAria (BD) at 1
cell/well in 96-well plates, which were seeded with L-cells and
IL-21 containing culture medium. After three weeks Tetanus Toxoid
binding of outgrowing clones was checked using the FACS Canto (BD).
Therefore cells were harvested and stained in 96-well plates with
GFP, NGFR, CD19 and TT-PE.
[0187] Development of Bcl-6 and Bcl-XL Double Positive B Cell Lines
that Secrete Antibodies
[0188] B cell lines were created that produces monoclonal
antibodies and are 100% Bcl-6 and Bcl-XL double positive. First
this was achieved by inducing proliferation and differentiation
using IL-21. Meanwhile these cells are transduced with the
Bcl-6-IRES-NGFR and Bcl-XL-IRES-GFP retroviruses. The cells are
maintained on IL-4 for 3-4 days. The cells that are transduced with
either one or both retroviruses then express the transgene and will
therefore express the NGFR or GFP protein. The expression of NGFR
and/or GFP can be visualized by using the LSRII (BD). If necessary,
cells can be transduced again to obtain higher numbers of cells
expressing both transgenes. Irrespective of a second transduction
the cells that express both transgenes are sorted using the FACS
Aria (BD) and cultured at a cell density ranging from 10-500
cells/well in 96-well plates in the presence of IL-21 and 2500 to
5000 L-cells/well. These mini-bulk-cultures (MBC) secrete
relatively large amounts of antibody in the culture supernatant
already at day 5 which then can be used for screening purposes.
Screening can be based on techniques available for the antigen of
interest e.g. ELISA/EIA/RIA, Western blot or direct functional
assays like neutralization of cytokine blocking experiments. After
screening and selection of MBC that recognize the antigen of
interest (TT and RSV in our experiments), cells are subcloned at
0.5-1 cell/well in 96 well in the presence of IL-21. Subcloning
normally takes 2-3 weeks and can be performed by limiting dilution
(LD) cultures or single cell sorting using flow cytometry
(FACSAria).
RSV A-2 Virus Stock and HEp2 cNaiveell Line
[0189] The RSV A-2 virus (kindly provided by G. van Bleek, WKZ,
Utrecht) and HEp2 cell line (Clinical Laboratory, AMC, Amsterdam),
were cultured in large quantities and frozen in liquid
nitrogen.
[0190] The adherent HEp2 cell line was cultured in normal medium in
T175 Falcon bottles before aliquots were frozen.
[0191] To obtain a high titer. RSV stock, HEp2 cells were seeded
and cultured to reach 50-60% confluence. The original RSV stock was
added (1/20 dilution total volume 5 ml) for 45' at RT on the HEp2
cells. 15 ml fresh medium was added and cells were left o/n at
37.degree. C., 5% CO.sub.2 with the coverlid open. The next morning
culture supernatant was carefully removed and 15 ml medium
containing 1% FCS was added. Cells were left for 24 to 36 hours at
37.degree. C., 5% CO.sub.2 with the coverlid closed. When RSV
induced syncytia were clearly visible and the majority of the
syncytia were still intact, the medium was harvested, filtered
(0.22 .mu.m) and spin at 1450 rpm at RT before samples were snap
frozen and stored in liquid nitrogen. A 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
[0192] HEp2 cells that were infected with RSV A-2 to obtain virus
stocks were used to isolate RSV proteins. First cell were carefully
washed with PBS and trypsinized Trypsin (Gibco) was washed away and
the cell pellet was lysed with 1% octylglucoside (cell pellet of
one Tl 75 flask was treated with 2 ml octylglucoside). Suspension
was homogenized with syringe and needle (10 times up and down),
incubated for 1 hour on ice and then dialyzed against 2 L TBS
buffer pH 7.4, o/n at 4.degree. C. Supernatant was obtained after
spin down of cell debris. The protein content was determined at 3.6
mg/ml and was used at 20 .mu.g/ml (50 .mu.l) in ELISAs.
Determining TCID50 and PFU of RSV Stocks
[0193] To determine the TCID50, 10.sup.4 HEp2 were seeded in 96
well plates and infected with a 2 or 10 step serial dilution of RSV
virus in 4-plo. 2-3 days later culture supernatant were removed and
cells were fixed with 80% acetone for 10' at RT. After removal of
the acetone, the fixed cell layer was dried and kept at 4.degree.
C. or frozen at -20.degree. C. To stain RSV HEp2 cells the plates
were first blocked with 5% milkpower in PBS 0.1% Tween 20. Then
plates were washed 3 times before being incubated for 3-5 hours at
37.degree. C. with polyclonal goat anti-RSV-HRP (1:500, Biodesign,
Saco, Me., US) and washed extensively. Next the wells were
incubated with AEC substrate for 30' at RT. Infected foci stain red
and can be observed by eye using a light microscope and can be
counted. Standard Excel software was used to determine the
TCID.sub.50.
[0194] To determine the amount of plaque forming units (PFU) of the
virus, 1.times.10.sup.5/ml of HEp2 cells in 24 well plates were
incubated with 10-fold serial dilutions (10.sup.-3-10.sup.-7) of
RSV virus stock in medium with 1% FCS at 37.degree. C. for 45' (200
.mu.l) before cells and virus were covered with 0.5 ml hand warm
0.25% seaplaque agar (Biozyme). The agarose layer prevents the
spreading of the virus to uninfected cells through the culture
medium. Thereby the virus can infect only neighboring cells, which
eventually are killed by the virus creating plaques in the
monolayer of HEp2 cells. Those 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 individuals) and the PFU value can be
determined.
Selection of Respiratory Syncytial Virus (RSV) Neutralizing
Antibodies
[0195] To obtain anti-respiratory syncytial virus (RSV) B cell
clones, peripheral blood cells (PBMC) from two donors were isolated
from bloodbank derived buffy coats (donor B62 and B63). Before
sorting CD19.sup.posIgM.sup.negIgD.sup.negIgA.sup.negCD27.sup.pos
cells using the FACSAria (BD) (FIG. 1), CD22+ cells were isolated
using MACS beads and columns (Miltenyi). Only if mentioned
differently, cells were cultured with L-cells. Cells were cultured
for 36 hours in the presence IL-21 before being transduced with
Bcl-6-IRES-NGFR only. After 12 h cells were harvested and cultured
for 3 days in the presence of IL-4 before NGFR expressing cells
were sorted using MACS beads (Miltenyi) and immediately transduced
with Bcl-XL-IRES-GFP. The B cells that did not bind to the MACS
beads were washed and transduced with Bcl-6 and Bcl-XL at the same
time. After 12 h cells were harvested, pooled and cultured for 3
days in the presence of IL-4 before being sorted on GFP and NGFR
expression on the FACSAria. Cells were washed and cultured at 100
cell/well density in 96 well plates (Costar) in the presence of
IL-21.
[0196] The double transduced Bcl-6 and Bcl-XL B cell cultures were
screened for RSV binding using a RSV-infected HEp2 cell lysate
ELISA and were tested in parallel using a RSV microneutralization
experiment. In brief, 10.sup.4 HEp2 cells arc seeded in flat bottom
96 well plates (Costar) in complete medium. The next day medium is
replaced for 1 h at RT with the mixture of RSV virus and cell
culture supernatant which have been pre-incubated for 30 min at
37.degree. C. The total volume is 25 .mu.l and the RSV end
concentration is 0.1 MOI. After 1 h the virus supernatant mixture
is 9 times diluted with PBS and replaced with 100 .mu.l IMDM/5%
FCS. After 2 days cells are fixed with 80% acetone and stained with
polyclonal anti-RSV-HRP (Biodesign). Using H.sub.2O.sub.2 and AEC
cells infected with RSV develop a red stain. Using light microscopy
infected cells can be observed and counted if necessary. As a
control for RSV neutralization a goat polyclonal anti-RSV (Abeam,
Cambridge, Mass.) is used.
RT-PCR and Cloning of VH and VL Regions
[0197] Total RNA was isolated from .about.5.times.10 B cells with
the RNeasy.RTM. mini kit (Qiagen, Venlo, The Netherlands). 250 ng
of total RNA was reverse transcribed in a volume of 20 .mu.l
containing 1.times. first strand buffer, 500 .mu.M dNTP, 250 ng
random hexamers, 5 mM DTT, 40 U RNasin (Promega) and 200 U
SuperScript III RT (Invitrogen). The cDNA was diluted 10.times. in
Ultrapure water and 2.5 .mu.l of cDNA was subjected to PCR in a 50
.mu.l solution containing 20 mM Tris-HCL, 50 mM KCL, 2.5 mM MgCl2,
250 .mu.M dNTP, 1 U AmpliTaq Gold DNA polymerase (Applied
Biosystems Inc.), and 25 pmol of each primer. PCR conditions were
as follows: 8 min denaturing step at 96.degree. C. followed by 35
cycles of 30 sec at 96.degree. C., 30 sec at 60.degree. C., 1 min
at 72.degree. C., and a final 10 min extension at 72.degree. C.
[0198] PCR products were run on agarose gels, purified and cloned
into the pCR2.1 TA cloning vector according to manufacturers'
recommendations. Sequence analysis was performed using BigDye
Terminator chemistry (Applied Biosystems Inc.) and Vector-NTI
software (Invitrogen).
[0199] To rule out reverse transcriptase and/or DNA polymerase
induced mutations, several independent cDNA conversions and PCR
reactions were performed and individually cloned and sequence
analyzed. Consensus sequences were determined with Vector-NTI
Contig Express software.
[0200] For recombinant protein antibody expression 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 VH region of clone D25 introducing a 5'-NheI site and a 3'-XhoI
site. The IgG1 constant region (CH1-hinge-CH2-CH3) was amplified
from the same cDNA while introducing a 5'-XhoI and a 3'-NotI site.
The full length heavy chain expression vector was obtained by three
point ligation into NheI/NotI digested pcDNA3.1(+)Zeo. The full
length light chain expression construct was generated by PCR
amplification of the light chain leader sequence, VL region and
light chain constant region with primers introducing a 5'-NheI and
3'-NotI site. The latter product was cloned into NheI/NotI digested
pcDNA3.1(+)Zeo to obtain a full length light chain expression
vector.
[0201] Sequence analysis was performed to confirm correctness of
the expression constructs.
[0202] Transient double transfection (Fugene-6, Roche, Germany or
Lipofectamine LTX, Invitrogen) of 293T cells with both heavy and
light chain expression vectors was performed to produce recombinant
monoclonal antibody. A FACS staining with the resulting culture
supernatant (48 hours) on RSV infected Hep2 cells was performed to
show functional binding of the antibody to the RSV F-protein.
The oligonucleotides used for PCR amplifications were:
TABLE-US-00001 VH regions: VH1-For (SEQ ID NO: 11) 5'
-AAATCGATACCACCATGGACTGGACCTGGAGG-3' VH1B-For (SEQ ID NO: 12) 5'
-AAATCGATACCACCATGGACTGGACCTGGAGM-3' VH2A-For (SEQ ID NO: 13) 5'
-AAATCGATACCACCATGGACACACTTTGCTMCAC-3' VH2B-For (SEQ ID NO: 14) 5'
-AAATCGATACCACCATGGACATACTTTGTTCCAAC- 3' VH3-For (SEQ ID NO: 15) 5'
-AAATCGATACCACCATGGAGTTTGGGCTGAGC-3' VH3B-For (SEQ ID NO: 16) 5'
-AAATCGATACCACCATGGARYTKKGRCTBHGC-3' VH4-For (SEQ ID NO: 17) 5'
-AAATCGATACCACCATGAAACACCTGTGGTTCTT-3' VH5-For (SEQ ID NO: 18) 5'
-AAATCGATACCACCATGGGGTCAACCGCCATC-3' VH6- For (SEQ ID NO: 19) 5'
-AAATCGATACCACCATGTCTGTCTCCTTCCTC-3' Cgamma-Rev (SEQ ID NO: 20) 5'
-GGGTCTAGACAGGCAGCCCAGGGCCGCTGTGC-3' Vkappa regions: Vk1-For (SEQ
ID NO: 21) 5' -AAATCGATACCACCATGGACATGAGGGTCCCY-3' Vk1B-For (SEQ ID
NO: 22) 5'-AAATCGATACCACCATGGACATGAGRGTCCYY-3' Vk2-For (SEQ ID NO:
23) 5' -AAATCGATACCACCATGAGGCTCCCTGCTCAG-3' Vk3-For (SEQ ID NO: 24)
5' -AAATCGATACCACCATGGAARCCCCAGCGCA-3' Vk4-For (SEQ ID NO: 25) 5'
-AAATCGATACCACCATGGTGTTGCAGACCCAG-3' Ck-Rev (SEQ ID NO: 26) 5'
-GATCGCGGCCGCTTATCAACACTCTCCCCTGTTGAAGCTCTT-3' Vlambda regions:
V11aecb (SEQ ID NO: 27) 5' -AAATCGATACCACCATGGCCTGGTCCCCTCTCCTCC-3'
V11g (SEQ ID NO: 28) 5' -AAATCGATACCACCATGGCCGGCTTCCCTCTCCTCC-3'
V12/10 (SEQ ID NO: 29) 5' -AAATCGATACCACCATGGCCTGGGCTCTGCTCCTCC-3'
V13jpah (SEQ ID NO: 30) 5' -AAATCGATACCACCATGGCCTGGACCGCTCTCCTGC-3'
V15/7 (SEQ ID NO: 31) 5' -AAATCGATACCACCATGGCCTGGACTCCTCTCCTTC-3'
V16/9 (SEQ ID NO: 32) 5' -AAATCGATACCACCATGGCCTGGGCTCCTCTCCTTC-3'
V13rm (SEQ ID NO: 33) 5' -AAATCGATACCACCATGGCCTGGATCCCTCTCCTCC- 3'
V131 (SEQ ID NO: 34) 5' -AAATCGATACCACCATGGCCTGGACCCCTCTCTGGC-3'
V13e (SEQ ID NO: 35) 5' -AAATCGATACCACCATGGCCTGGGCCACACTCCTGC-3'
V14c (SEQ ID NO: 36) 5' -AAATCGATACCACCATGGCCTGGGTCTCCTTCTACC-3'
V18a (SEQ ID NO: 37) 5' -AAATCGATACCACCATGGCCTGGATGATGCTTCTCC-3'
C12/7 (SEQ ID NO: 38) 5'
-GATCGCGGCCGCTTATCAWGARCATTCTGYAGGGGCCACTG-3'
The oligonucleotides used for expression vector constructions
were:
TABLE-US-00002 Heavy chain expression vector: VH1-L-NheI: (SEQ ID
NO: 39) 5' -GCGGCTAGCCACCATGGACTGGACCTGGAGG-3' JH4/5-XhoI: (SEQ ID
NO: 40) 5' -GCGCTCGAGACGGTGACCAGGGTTCCCTG-3' CHfw-XhoI: (SEQ ID NO:
41) 5' -CGCGCTCGAGTGCCTCCACCAAGGGCCCATCGGTC-3' CHrev-NotI: (SEQ ID
NO: 42) 5' -GATCGCGGCCGCTTATCATTTACCCGGRGACAGGGAGAGGC-3' Light
chain expression vector: VKl-L-NheI: (SEQ ID NO: 43) 5'
-GCGGCTAGCCACCATGGACATGAGGGTCCCY-3' CK-NotI: (SEQ ID NO: 44) 5'
-GATCGCGGCCGCTTATCAACACTCTCCCCTGTTGAAGCTCTT-3'
EBV RT-PCR
[0203] To test if the strong proliferative response was related to
the presence of EBV, an EBV RT-PCR was performed. The RT procedure
is described above. The PCR conditions were as follows: a 7-minute
denaturing step at 94.degree. C. followed by 30 cycles of 30 s at
94.degree. C., 30 s at 62.degree. C. (HPRT1), 52.degree. C. (LMP-1)
and 58.degree. C. (EBNA1/2) and 30 s at 72.degree. C., and a final
7-minute extension at 72.degree. C. The oligonucleotides used for
RT-PCR were as follows: HPRT1 forward
(5'-TATGGACAGGACTGAACGTCTTGC-3') (SEQ ID NO: 45) and HPRT1 reverse
(5'-GACACAAACATGATTCAAATCCCTGA-3') (SEQ ID NO: 46); LMP-I forward:
(5'-GCGACTCTGCTGGAAATGAT-3') (SEQ ID NO: 47) and LMP-I reverse (5'
GACATGGTAATGCCTAGAAG-3') (SEQ ID NO: 48); EBNA1/2 forward
(5'-AGCAAGAAGAGGAGGTGGTAAG-3') (SEQ ID NO: 49) and EBNA1/2 reverse
(5'-GGCTCAAAGTGGTCTCTAATGC-3') (SEQ ID NO: 50).
[0204] In addition to the RT-PCR we performed a PCR directly on
cell pellet and supernatant DNA that was isolated using the QIAmp
isolation kit (Qiagen).
Example 1
Results
B Cell Phenotype
[0205] The use of human memory B cells as the platform to isolate
therapeutics medicines relies on the ability to grow and test these
cells for a relative long period of time. Human B cells can be
cultured and maintained in a laboratory setting however not long
enough to expand, select and clone single B cell lines against an
antigen of interest. We developed immortalization techniques based
on genetic modifications of human B cells. We studied downstream
targets of STAT5. One target besides others is Bcl-6. Bcl-6
inhibits differentiation of B cells to plasma cells that are
arrested in proliferation. Overexpression of Bcl-6 keeps BLIMP1 in
balance, a transcription factor which expression is strongly
enhanced by stimulating B cells with IL-21 (works via STAT3).
BLIMP1 is necessary to induce the development of 1 g producing
cells (CD20-CD38+) whereas Bcl-6 can prevent this (cells maintain
CD20 expression, the so-called germinal center phenotype).
[0206] To study the possible skewing of certain cell populations
within the B cell compartment, CFSE labeling prior to stimulation
of fresh memory and naive human B cells revealed that all cells
start dividing and that all populations of B cells are equally
transduced (FIG. 2). Shown are memory B cells transduced with Bcl-6
and cultured in the presence of IL-21 and IL-4. Naive B cells were
transduced at a lower level and division rates were lower at 36 hrs
but were identical to memory B cells after another 3 days of
culture (data not shown).
[0207] Next we show that Bcl-6, together with Bcl-XL
(anti-apoptotic downstream target of STAT5), CD40L signaling and in
the presence of IL-21, maintain human IgG memory B cells in the
CD20+CD38dull phenotype for long periods of time (>3 months)
(FIG. 3). In addition, the Bcl-6 Bcl-XL B cells have a phenotype
corresponding to activated B cells (see Table 1, exemplified by
FACS staining of 3 TT+ B cell clones), since 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
TABLE-US-00003 staining result CD2 neg CD5 neg CD7 neg CD10 pos
CD20 pos CD21 pos CD22 pos CD23 neg/5% pos CD24 neg CD25 pos CD27
neg/low CD28 neg CD30 pos(56-74%) CD38 pos/intermediate CD40 pos
CD44 pos CD45 pos CD45RA pos/high CD69 neg CD70 pos CD71 pos CD73
neg CD80 pos/high CD86 pos CD95 pos/high CD126 neg CD132 (common
gamma) pos CD138 neg/2% pos CD154 (CD40L) 8% pos ICOSL pos IgM neg
IgG pos HLA-DR pos(high) Kappa pos/neg Lambda pos/neg IL21-R
pos
Antibody Membrane Expression
[0208] The Bcl-6 Bcl-XL transduced, EBV negative cells remained BCR
expression positive as determined by antigen binding or Kappa and
Lambda staining (FIGS. 3 and 4). Hence, such cells are particularly
suitable for isolating and/or screening after a long period of
culture for a desired specificity, for instance using labeled
antigen, because such cells will bind said labeled antigen with
their BCR. This was confirmed by single cell sorting of Bcl-6 and
Bcl-XL double transduced B cells that bind PE labeled TT using the
FACSAria. After three weeks single cell sorted clones were stained
with appropriate markers and TT-PE in 96 well plates and measured
for binding in the FACS Canto (BD) (FIG. 4). In conclusion, in
cases where the presence of a B cell receptor on B cells is
desired, such as for instance in screening assays, the B cells are
preferably transduced with Bcl-6 and Bcl-XL and not infected with
EBV.
Cell Division and Growth Curves
[0209] Bcl-6 Bcl-XL transduced B cells divide on average 0.6 times
per day. Division rate varies between donors and cell density of
the cultures (FIG. 5a). The anti-RSV clone D25 had a division rate
of 0.47 times per day (FIG. 5b). Cells can be grown at densities
below 1 cell/96 well for cloning purposes.
Antibody Secretion of Bcl-6 Bcl-XL B Cells
[0210] The Bcl-6 Bcl-XL transduced B cells secrete on average one
.mu.g/ml of antibodies, which is enough to grow quantities
necessary for pre-clinical tests (FIG. 6). Surprisingly the D25
anti-RSV clone produced three times more antibodies compared to the
other cell lines tested.
Determine EBV Content
[0211] EBV RT-PCR on mRNA of Bcl-6 Bcl-XL cell lines that were
cultured with IL-21 and CD40L signaling. In the cell lines obtained
with this immortalization technique no EBV gene transcript have
ever been detected (data not shown).
Selection Procedure
[0212] Due to the stability in growth and expression of the BCR,
these cells are well suited to isolate antigen-specific B cells. It
gave us the opportunity to use several different selection and
cloning procedures. One is to immediately obtain antigen specific
cells after introduction of Bcl-6 and Bcl-XL by FACS or Magnetic
Bead sorting using labeled antigen of interest thereby enhancing
the probability of generating multiple antigen-specific B cell
clones. Another option is to grow purified, bulk Bcl-6 Bcl-XL
transduced memory (or any other) B cells at low cell densities (for
example 100 cells/well). Supernatants from these 100 c/w cultures
can be collected and tested for their specificity. 100 cell/well
cultures that are found positive for antigen recognition, are then
subcloned by limiting dilution cultures to obtain monoclonal cell
lines. Using both methods we could isolate over 40 Tetanus Toxoid
(TT) recognizing B cell clones. Thus these clones were either
selected on TT binding to the BCR on the FACSAria or they were
selected by ELISA screening of series of cultures till the single
anti-TT monoclonal cell line was isolated (not shown).
Selection of RSV Neutralizing Antibodies
[0213] From donor B63, 25 100 cell/well cultures completely blocked
RSV infection and replication. D10, one of the neutralizing 100
cell/well cultures produced a strong anti-RSV antibody which we
cloned by limiting dilution culture. One of the monoclonal
antibodies, D25 was used to continue studies. D25, a monoclonal
antibody with an IgG1 heavy chain, as determined by commercial
ELISA (Sanquin, Amsterdam, not shown) and a Kappa light chain (FIG.
7), very efficiently blocked RSV infection with an IC.sub.50 value
of between 0.5 and 1.5 ng/ml (.+-.10 pM) whereas the IC.sub.50 of
the standard anti-RSV antibody used in the clinic (palivizumab
developed by Medimmune) is 0.453 .mu.g/ml (3.02 nM) (H. Wu et al.
2005 J. MoI. Biol, and A. Mejias et al. 2005 Antimicrob. Agents
Chemother.) (FIG. 8).
Antigen Recognition
[0214] In addition to the neutralization experiments, the binding
of D25 to RSV infected HEp2 cells was determined. HEp2 cell were
infected using the regular virus production protocol. HEp2 cells
infected with RSV were trypsinized and incubated with 25-50 .mu.l
culture supernatant. Cells were washed and stained with
mouse-anti-human IgG-PE (BD or Jackson) to detect binding of the
D25 antibody to the infected cells. The r-Biopharm ELISA control
antibody was used as an internal control. Shown in FIG. 9a is the
binding of D25 to intact, RSV infected HEp2 cells.
[0215] Since the RSV envelope (membrane) proteins exist of two
proteins namely the G and F-protein, the binding of D25 was tested
against cells infected with the VSV virus pseudotyped with either
no or the RSV F or RSV G protein (kindly provided by John K Rose).
As shown in FIG. 9b, D25 bound strongly to EL-4 cells infected with
the VSV-F protein. In an attempt to study the epitope recognized by
D25 versus palivizumab, VSV-F protein infected EL-4 cells were
incubated with increasing amounts of D25 or palivizumab. Cells were
washed and stained with a mixture of 3 mouse-anti-RSV-F antibodies
(Dako). In contrast to Palivizumab that showed competition for the
binding to infected VSV-F cells with the mouse-anti-RSV-F antibody,
D25 binding was not affected (data not shown).
[0216] FIG. 9c shows the binding of Palivizumab (Synagis) and D25
in a concentration dependent manner to infected HEp2 cells. Since
both antibodies bind 1 to 1 to their target protein there is no
difference in binding to infected HEp2 cells.
Frequency of RSV Antigen Binding Vs Neutralizing Clones
[0217] We calculated that the frequency 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 donor B63. D25
binds to a conformational epitope that is different than the
epitope recognized by palivizumab. This is illustrated in FIG. 10
in which D25 does not bind to denatured, linear epitopes presented
by lysed RSV infected cell lysate coated on ELISA plates while
palivizumab does bind to denatured (F) protein.
Isolation and Purification of Antibody Fragments
[0218] From several B cell lines including the highly RSV
neutralizing clone D25 we were able to grow volumes as much as 500
ml. These culture supernatants contain at least 2 .mu.g/ml,
therefore we should be able to obtain enough purified antibody to
perform pre-clinical (animal) studies. The purification is
performed using Montage Antigen Purification Kit (Millipore,
Billerica, Mass., USA) and HiTrap Protein A HP columns (GE
Healthcare, Diegem, Belgium).
[0219] In addition, 293T cells were transfected with the heavy and
light chain of D25 that were subcloned in pCDA3.1 protein
expression vectors using lipofectamine LTX (Invitrogen). The amount
of IgG that were present in the supernatant was approximately 22
.mu.g/ml (total volume 50 ml). This antibody derived from the
cloned nucleotide sequence of the antibody expressed by the D25 B
cell line did also recognized infected HEp2 cells (data not
shown).
Antibody Sequence
[0220] FIG. 11a shows the heavy and light chain nucleotide and
amino acid sequence of the B63D10-D25 clone. By using standard
RT-PCR and antibody specific primers, the heavy (Vhl-69) and light
(VkI O8/O18) chain sequences were determined. The whole antibody
sequence was cloned by using TOPO vectors and after sequence
control, subcloned into the pcDNA3.1 mammalian protein expression
vector (Invitrogen). FIGS. 11b and 11c depict the VH and VL4 chain
of the clone, Asterisks indicate mutations compared to the germline
sequence of the Vhl-69 that must have occurred during affinity
maturation and further B cell selection.
[0221] To summarize, we here show the isolation, characterization
and long-term culture of human memory B cells using the transgenes
Bcl-6 and Bcl-XL. They give us the tool necessary to isolate
antibodies with unique properties, like the anti-RSV monoclonal
antibody B63D10-B25. Since the B cells are from a human origin,
they can readily be deployed as a therapeutic medicine.
Example 2
[0222] The D25 heavy and light chain were cloned into standard
expression vectors as described before (p44 `antibody sequence`).
To create an expression construct that allows for maximum protein
expression the D25 heavy and light chain sequences were codon
optimized by GENEART (Regensburg, Germany). In this procedure
additional restriction sites were created to simplify future
cloning procedures but most importantly nucleotide codons that
translate into amino acid sequences were optimized for maximum
translation into protein. Thus the nucleotide sequence was
optimized but the amino acid sequence remained unchanged. Shown in
EXAMPLE 4 is the neutralizing capacity of purified B cell
supernatant derived D25, recombinant D25 and GENEART optimized D25.
All efficiently neutralize RSV.
[0223] The GENEART modifications compared to the original D25
sequence are depicted in FIG. 12.
Example 3
[0224] Next to the in vitro RSV neutralization experiments we
tested the D25 monoclonal antibody in in vivo models. The models
that have been described for in vivo anti-RSV tests are BALB/c mice
and cotton rats (Sigmodon hispidus) (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 since the cotton rats are difficult
to get and maintain, we first set up D25 tests in BALB/c mice.
Protocol: RSV Specific Antibodies in BALB/c, Day 5
[0225] Experimental design: Day -1. LP. injection 100 .mu.l
antibodies Day 0. I.N infection 1.times.10.sup.7 pfu RSV A2 in 50
.mu.l Day 1 to 5, check general well being and weigh mice Day 5,
autopsy, collect BAL, blood and lungs Draw blood via vena puncture
Collect 2.0 ml BAL via trachea canule Collect lungs Immediately
start TCID.sub.50 on BAL material (1 ml) Freeze 1 ml BAL material
(ELISA cytokine/RT-PCR) -80 C Perform TCID.sub.50 on prepared long
material (1 ml) Freeze 1 ml long material (ELISA cytokine/RT-PCR)
-80 C Collect/spin blood for hIgG ELISA on serum en store at -80 C
The results are shown in FIG. 13:
[0226] (A) One day before RSV challenge (1.times.10.sup.7 RSV-A2
particles) by nasal spray, animals were IP injected with different
amounts of Synagis (MedImmune), purified D25 or an IgG1 ctrl
antibody (Eureka) (Table 3). (FIG. 13B) Human IgG levels were
determined in mice sera from day 5 and the drop in antibody serum
levels in 5 days; Table 4 shows an overview of the half-life
values. FIG. 13D depicts virus titers found in lung lavages (BAL)
at day 5 in treated and untreated animals whereas FIG. 13E depicts
T and B cell numbers in peripheral blood of treated and untreated
mice. FIG. 13F shows the histology of the lungs with bronchi and
infiltration of (normally mainly eosinophils) untreated and treated
animals.
Conclusion/Result:
[0227] An estimate of the D25 half-live is 5 to 9 days based on the
(linear) calculation that 60 and 30 .mu.g of antibody was injected
on day 0 (2 and 1 mg/kg respectively) and at day 5 33 or 16 .mu.g
was detected (total volume of mice 1.5). When we started with 0.5
mg/kg injection per animal on d0 then Ig levels drop from 15 .mu.g
to 11 .mu.g on day 5, which would indicate a 9 day half life (Table
4).
TABLE-US-00004 TABLE 4 total administered detected on half-life
mg/kg d 0 (.mu.g) d 5 (.mu.g) (days) 2.0 60 33 5.6 1.0 30 16 5.4
0.5 15 11 9.4
[0228] Virus titer as determined TCID50 assay shows that in control
animals 1.times.10.sup.4 PFU can be detected whereas no virus was
detected in the Synagis (2 mg/kg) or D25 (2, 1 and 0.5 mg/kg)
treated animals.
[0229] Animals treated with Synagis or D25 maintain higher % of
peripheral CD4 T cells and B220 B cells. Animals treated with
Synagis (2 mg/kg) have lower % of CD4 T cells compared to D25
treated animals. Although this may not be significant it is
important to note that animals treat with a low dose of D25 (1 and
0.5 mg/kg) maintain high levels of B and T cells when compared to
control treated animals.
[0230] Although the histology data (FIG. 13F) are not quantitative
it is clear that Synagis and D25 reduce influx of immune cells into
the lungs and around the bronchi compared to control. When D25 and
Synagis are compared, then D25 treated animals seem to have less
cellular infiltration into the lungs and around the bronchi.
[0231] To test D25 in the Cotton rats, experiments are set up to
compare animals pre-treated with Synagis and D25 before challenge
with the RSV-X virus at the NVI (Bilthoven, Netherlands).
Example 4
[0232] In addition to B63-D10-D25, we isolated three new potent RSV
neutralizing antibodies (AM14, AM16 and AM23) from the same donor
(B63). 100 cell per well bulk B cell cultures that were originally
selected for RSV neutralization and were frozen and stored in
liquid nitrogen, were thawed and culture supernatant was tested for
binding to RSV infected. HEp2 cells. We tested for binding to
infected Hep2 cells since that is a marker for antibody recognition
of native, oligomeric RSV membrane proteins like F and G protein
and may serve as a good predictor for neutralization. When binding
was detected, cells were single cell cultured and screened for
binding to obtain clones. All three antibodies were cloned into the
GENEART vector that was originally constructed for D25. In addition
like D25 all recognize the RSV-F protein (not shown). After cloning
and expression in 293T cells recombinant protein was purified
(nucleotide and amino acid sequences are depicted in FIGS. 14A, B
and C). Antibodies were tested for neutralization against several
primary RSV isolates on Vero and HEp2 cells (FIG. 15). All three
antibodies are of the IgG1 isotype. AM14 has a Kappa light chain,
while AM16 and AM23 have a Lambda light chain. All three
antibodies, like D25, contain somatic hypermutations in their
antibody variable domains suggesting that they in vivo have
undergone affinity maturation during a germinal center reaction, a
process that creates unique antibody sequences.
[0233] The results are shown in FIGS. 15-I and 15-II: RS virus
neutralization assay with purified B cell line supernatant derived
D25 (sD25), recombinant purified D25 (rD25), recombinant GENEART
codon optimized D25 (rD25 GA), AM14, AM16, AM23 (all purified
recombinant protein) and Synagis. Virus antibody neutralization was
tested on two different cell lines (FIG. 15-I) Vero and (FIG.
15-II) Hep2 cells with different antibodies: A2 (A), X (B) and
2006/1 (C) are RSV subtype A while virus Z (D) and 2007-2 (E) are
subtype B. 100TCID50 of each virus was added to serial antibody
dilutions in DMEM/1% FCS and incubated for 1 hour at 37 degree
before 100 ul Vero or HEp2 cells (1.times.10.sup.6/ml) were added.
Virus antibody mixture was not washed away. After three days
supernatant was removed and cells were fixed with 80% acetone for
10' at RT. After removal of the acetone, the fixed cell layer was
dried and kept at 4.degree. C. or frozen at -20.degree. C. To stain
RSV infected HEp2 cells, plates were first blocked with 5%
milkpower in PBS 0.1% Tween 20, then plates were washed 3 times
before being incubated for 3-5 hours at 37.degree. C. with
polyclonal goat anti-RSV-HRP (1:500, Biodesign, Saco, Me., US) and
washed extensively. Subsequently all wells were incubated with AEC
substrate for 30' at RT. Infected foci stain red and can be
observed by eye using a light microscope and can be counted.
Result/Conclusion
[0234] All antibodies neutralize the RSV A and B strains (Table 5).
In general the different D25 antibodies neutralize the RSV viruses
efficiently, although minor inter-experimental variations can be
seen. AM14 is just as potent as D25 while AM16 is just as potent as
Synagis. AM23 however does neutralize the RSV A strains very
efficient, while it is less potent in neutralizing RSV B strains,
although still comparable to Synagis.
TABLE-US-00005 TABLE 5 IC50 values (ng/ml) Cell line RSV rD25 used
subtype sD25 rD25 GA AM14 AM16 AM23 Vero A 3.4 1.6 3.2 15.2 304.3
19.4 Vero B 9.0 0.3 1.2 1.1 126.4 168.8 HEp2 A 3.3 2.1 5.3 21.5
285.6 25.0 HEp2 B 14.3 1.9 1.3 6.7 124.8 190.7 The IC50 value for
each antibody on RS virus subtype A on Vero or HEp2 cells was
calculated as the average 50% neutralization on three virus strains
(A2, X and 2006-1). The IC50 value for each antibody on RS virus
subtype B on Vero or HEp2 cells was calculated as the average 50%
neutralization on two virus strains (2007-2 and Z). Each of the
neutralizations assays was performed in riplo and repeated twice
(also shown in FIG. 15A and B). sD25 = purified B cell derived
culture supernatant rD25 = purified recombinant D25 rD25 GA =
supernatant of 293T cells with GENEART codon optimized recombinant
D25
Example 5
Synergistic and Blocking Effects of Anti-RSV Antibodies.
[0235] To analyze whether D25, Synagis or the new AM antibody set
interfere with each other for recognition of the RSV F protein, we
pre-incubated RSV infected HEp2 cells with increasing
concentrations of unlabeled antibodies till they reached the
plateau of maximum binding. We determine for each antibody the
plateau phase in which no increase in binding was detected when the
amount of Ig was increased, (not shown). After washing, samples
were incubated with a standard dose (3 pmol) of PE labeled D25 or
APC labeled Synagis. This dose gives also maximum binding.
Result
[0236] As shown in FIG. 16 labeled Synagis and D25 show a reduced
binding to RSV infected HEp2 cells when these cells were
pre-incubated with either unlabeled Synagis or D25. Synagis shows
furthermore a slight reduction in binding induced by AM16. D25
binding is strongly blocked by AM23 but on the contrary D25 binding
is strongly enhanced after pre-incubation with AM14. That indicates
that the epitope recognized by D25 is normally not even fully
exposed but exposure is enhanced after binding of AM14 to its
native epitope. That demonstrates that these two antibodies can
work together and enhance neutralization.
BRIEF DESCRIPTION OF THE DRAWINGS
[0237] FIG. 1.
[0238] Isolation of human, IgG positive, memory B cells. PBMC
isolated from buffy coat using Ficoll density separation (Amersham)
were incubated with anti-CD22 magnetic beads before being isolated
using MACS columns (Miltenyi). CD22 positive cells were then
incubated with antibodies against human CD19, CD27, IgM, IgD and
IgA (BD). Cells negative for IgM, IgD and IgA and positive for CD
19 and CD27 were sorted using high speed single cell sorting
(FACSAria, BD).
[0239] FIG. 2
[0240] CFSE staining. Fresh human memory B cells were isolated,
labeled with CSFE and stimulated for 36 h with IL-21 before being
transduced with Bcl-6-IRES-NGFR. Cells were kept an additional 3
days on IL-21 before CFSE content was determined. The CFSE dye is
diluted with every cell division.
[0241] FIG. 3
[0242] An example of human B cells transduced with Bcl-6 and Bcl-XL
or Bcl-XL only. Cells were maintained on irradiated L cells
expressing CD40L and the cytokine IL-21. Shown on the left is the
BCR expression as determined by kappa and lambda staining (93% of
the kappa lambda positive cells are of the IgG isotype, not shown).
On the right is shown the CD38 expression on the X-axes and CD20
expression on the Y-axes. The CD38.sup.dullCD20.sup.+ staining
indicates memory or germinal center B cells; the
CD38.sup.+CD20.sup.- staining indicate plasmablasts.
[0243] FIG. 4
[0244] Isolation of immortalized, antigen specific human B cells.
Human memory B cells were isolated as described in FIG. 1 and
subsequently transduced with Bcl-6-IRES-NGFR and Bcl-XL-IRES-GFP.
Cells expressing NGFR, GFP and were binding to PE-labeled Tetanus
Toxin were isolated using the FACSAria. Cells were single cell
cultured in 96 well flat bottom plates in the presence of
irradiated L cells and IL-21 before being selected based on TT-PE
binding using the FACS Canto (BD).
[0245] FIG. 5
[0246] Cumulative cell growth and division rate of 6XL B cell
clones. B cells from (A) two anti-TT clones and (B) one anti-RSV
clone (B63D10-D25) were cultured in the presence of IL-21 and
irradiated L cells.
[0247] FIG. 6
[0248] Fresh cultures were started with 200,000 cell/24 welkin 1.0
ml IMDM with 8% FCS and pen/strep. The FCS used was either normal
(HyClone) or Ultralow Bovine IgG FCS (Gibco). After 3 days the
culture supernatant was replaced and cell numbers were adjusted to
200,000 cell/ml. Shown is the average IgG production in 3 days
measured in 3 consecutive time points the difference was not
significant (p value 0.2).
[0249] FIG. 7
[0250] To determine the light chain phenotype of the D25 anti-RSV
clone, the D25 B cell line was stained with either
kappa-phycoerythrin or lambda-phycoerythrin (BD) antibodies. Only
the kappa-phycoerythrin antibodies bound to the cell line, showing
this antibody has a kappa light chain.
[0251] FIG. 8
[0252] From donor B63, 100 cell/well cultures were grown using
Bcl-6 Bcl-XL positive human memory B cells. One of those cultures,
D10 showed strong neutralization. LD derived monoclonal cell lines
were made, one D25 neutralized the RSV A-2 virus efficiently. Shown
here is D25 compared to palivizumab (synagis) and a polyclonal goat
ant-RSV. Not shown are irrelevant culture supernatants of Bcl6
Bcl-XL transduced B cell clones cultured with IL-21 and CD40L
signaling that produce high levels of antibodies but did not block
RSV infection. The D25 clone was used for further
characterization.
[0253] FIG. 9
[0254] In FIG. 9a: HEp2 cell were seeded at 10-12e6 cells per T175
flask (Nunc) in IMDM/5% FCS. The next day the medium was replaced
with 5 ml of medium with RSV virus (1.0 MOI) and incubated for 45'
at RT before 20 ml of fresh medium was added and the cells were
cultured o/n at 37.degree. C. The next day the medium was replaced
with IMDM/1% FCS and cultured o/n with a closed lid at 37.degree.
C. The next day cells were washed with PBS and treated with
trypsin. To stain infected cells the primary incubation was
performed with culture supernatant. The secondary incubation was
done with anti-human IgG-PE (BD). Cells were analyzed using the
LSRII (BD). As a positive control the positive control of the
commercial ELISA KIT from r-Biopharm was used.
[0255] In FIG. 9b: EL-4 cells were infected with VSV virus
pseudotyped with RSV F or G protein (kindly provided by John Rose)
and incubated with D25 culture supernatant. Cells were washed and
incubated with anti-human-IgG-PE (Jackson) to detect binding of D25
to the infected cells. Only binding of D25 to the VSV virus
infected cells pseudotyped with the RSV F protein was detected.
FIG. 9c shows the binding of Palivizumab (Synagis) and D25 in a
concentration dependent manner to infected HEp2 cells. Shown is the
mean fluorescence intensity (MFI).
[0256] FIG. 10
[0257] Binding of polyclonal goat anti-RSV (pos ctrl), palivizumab
(synagis) and D25 to coated HEp2 infected cell lysate.
[0258] FIG. 11
[0259] Sequence analysis of the D25 clone. 11a shows nucleotide and
predicted amino acid sequence of the variable heavy and light chain
domains (SEQ ID NOs: 7-10), 11b/c show the D25 heavy and light
chain sequence compared to predicted germline. Asterisks indicate
mutations that probably occurred during selection and affinity
maturation of the B cell clone in vivo. (SEQ ID NOs: 7-8, 55 and
57).
[0260] FIG. 12
[0261] Cloning and expression of recombinant human antibodies from
BCL6 BCL-xL transduced B cell lines. This has already been
described for the D25 antibody (FIG. 11). Here are depicted the
GENEART nucleotide modifications compared to the original D25
sequence, note that these mutations do not change the amino acid
composition of the D25 antibody (SEQ ID NOs: 139-142),
[0262] FIG. 13
[0263] BALB/c mice challenge with purified, B cell supernatant
derived D25 and Synagis. (A) One day before RSV challenge
(1.times.10.sup.7 RSV-A2 particles) by nasal spray, animals were IP
injected with different amounts of Synagis (MedImmune), purified
D25 or an IgG1 ctrl antibody (Eureka) (table 3). (B) human IgG
levels were determined in mice sera from day 5 and the drop in
antibody serum levels in 5 days (C); table 4 shows an overview of
the half-life values. FIG. 13D depicts virus titers found in lung
lavages (BAL) at day 5 in treated and untreated animals whereas
FIG. 13E depicts T and B cell numbers in peripheral blood of
treated and untreated mice. (F) shows the histology of the lungs
with bronchi and infiltration of (normally mainly eosinophils)
untreated and treated animals.
[0264] FIG. 14
[0265] Nucleotide and amino acid sequences of three new potent RSV
neutralizing antibodies (A) AM14, (B) AM16 and (C) AM23 (SEQ ID
NOs: 73-138, 147-148, 153-154 and 159-160).
[0266] FIG. 15
[0267] RS virus neutralization assay with purified B cell line
supernatant derived D25 (sD25), recombinant purified D25 (rD25),
recombinant GENEART codon optimized D25 (rD25 GA), AM14, AM16, AM23
(all purified recombinant protein) and Synagis. Virus antibody
neutralization was tested on two different cell lines (FIG. 15-I)
Vero and (FIG. 15-II) Hep2 cells with different antibodies A2 (A),
X (B) and 2006/1 (C) are RSV subtype A while virus Z (D) and 2007-2
(E) are subtype B. 100TCID50 of each virus was added to serial
antibody dilutions in DMEM/1% FCS and incubated for 1 hour at 37
degree before 100 ul Vero or HEp2 cells (1.times.10.sup.6/ml) were
added.
[0268] FIG. 16
[0269] Relative binding of a fixed amount (3 pmol) of APC-labeled
Synagis and PE-labeled rD25 to RSV infected HEp2 cells that were
pre-incubated with increasing concentrations of the indicated
unlabeled antibodies.
REFERENCES
[0270] Banchereau, J., de Paoli, P., Valle, A., Garcia, E.,
Rousset, F., (1991). Long term human B cell lines dependent on
interleukin-4 and antibody to CD40, Science 251, 70-2.
[0271] Boise, L. H., M. Gonzalez-Garcia, C. E. Postema, L. Ding, T.
Lindsten, L. A. Turka, X. Mao, G. Nunez, and C. B. Thompson.
(1993). Bcl-x, a bcl-2-related gene that functions as a dominant
regulator of apoptotic cell death. Cell 74:597, [0272] Dadgostar,
H., Zarnegar, B., Hoffmann, A., Qin, X. F., Truong, U., Rao, G.,
Baltimore, D., and Cheng, G. (2002). Cooperation of multiple
signaling pathways in CD40-regulated gene expression in B
lymphocytes. Proc. Natl. Acad. Sci USA 99, 1497-1502, [0273]
Heemskerk et al, 1997: J. Exp. Med. Vol. 186, page 1597-1602 [0274]
Heemskerk et al, 1999: Cell Immunol. Vol. 195, page 10-17 [0275]
Kinsella and Nolan, 1996: Hum. Gene Ther. Vol. 7 page 1405-1413
[0276] Malisan, F., Briere, F., Bridon, J. M., Harindranath, N.,
Mills, F. C., Max, E. E., Banchereau, J., Martinez-Valdez, H.
(1996). Interleukin-10 induces immunoglobulin G isotype switch
recombination in human CD40-activated naive B lymphocytes, J. Exp.
Med. 183, 937-47. [0277] Mathas S, Janz M, Hummel F, Hummel M,
Wollert-Wulf B, Lusatis S, Anagnostopoulos I, Lietz A, Sigvardsson
M, Jundt F, Johrens K, Bommert K, Stein H, Dorken B (2006).
Intrinsic inhibition of transcription factor E2A by HLH proteins
ABF-I and Id2 mediates reprogramming of neoplastic B cells in
Hodgkin lymphoma, Nat. Immunol. 7, 207-215. [0278] Mejias A et al.,
Antimicrobial Agents and chemotherapy 2004; p1811, Johnson S et
al., JID 1997; pl215 Wu H et al., JMB 2007:p652 [0279] Shvarts A.
et al, 2002: Genes Dev. Vol. 16, page 681-686 [0280] Traggiai, E.,
Becker, S., Subbarao, K., Kolesnikova, L., Uematsu, Y., Gismondo,
M. R., Murphy, B. R., Rappuoli, R., Lanzavecchia, A. (2004). An
efficient method to make human monoclonal antibodies from memory B
cells: potent neutralization of SARS coronavirus. Nature Medicine
Volume 10, No. 8, 871-875. [0281] Ye, B. H., Cattoretti, G., Shen,
Q., Zhang, J., Hawe, N., de Waard, R., Leung, C., Nouri-Shirazi,
M., Orazi, A., Chaganti, R. S., et al. (1997). The BCL-6
proto-oncogene controls germinal-centre formation and Th2-type
inflammation. Nat Genet. 16, 161-170.
Sequence CWU 1
1
16015PRTHomo sapiens 1Asn Tyr Ile Ile Asn 1 5 217PRTHomo sapiens
2Gly Ile Ile Pro Val Leu Gly Thr Val His Tyr Ala Pro Lys Phe Gln 1
5 10 15 Gly 317PRTHomo sapiens 3Glu Thr Ala Leu Val Val Ser Thr Thr
Tyr Leu Pro His Tyr Phe Asp 1 5 10 15 Asn 411PRTHomo sapiens 4Gln
Ala Ser Gln Asp Ile Val Asn Tyr Leu Asn 1 5 10 57PRTHomo sapiens
5Val Ala Ser Asn Leu Glu Thr 1 5 67PRTHomo sapiens 6Gln Gln Tyr Asp
Asn Leu Pro 1 5 7126PRTHomo sapiens 7Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Met Val Ser
Cys Gln Ala Ser Gly Gly Pro Leu Arg Asn Tyr 20 25 30 Ile Ile Asn
Trp Leu Arg Gln Ala Pro Gly Gln Gly Pro Glu Trp Met 35 40 45 Gly
Gly Ile Ile Pro Val Leu Gly Thr Val His Tyr Ala Pro Lys Phe 50 55
60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Asp Thr Ala Tyr
65 70 75 80 Ile His Leu Ile Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr
Tyr Cys 85 90 95 Ala Thr Glu Thr Ala Leu Val Val Ser Thr Thr Tyr
Leu Pro His Tyr 100 105 110 Phe Asp Asn Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 115 120 125 8110PRTHomo sapiens 8Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ala Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Val Asn Tyr 20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Val Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr
Asp Asn Leu Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys Arg Thr Val 100 105 110 9379DNAHomo sapiens 9caggtgcagc
tggtacagtc tggggctgaa gtgaagaagc ctgggtcctc ggtgatggtc 60tcctgccagg
cctctggagg ccccctcaga aactatatta tcaactggct acgacaggcc
120cctggacaag gccctgagtg gatgggaggg atcattcctg tcttgggtac
agtacactac 180gcaccgaagt tccagggcag agtcacgatt accgcggacg
aatccacaga cacagcctac 240atccatctga tcagcctgag atctgaggac
acggccatgt attactgtgc gacggaaaca 300gctctggttg tatctactac
ctacctacca cactactttg acaactgggg ccagggaacc 360ctggtcaccg tctcctcag
37910324DNAHomo sapiens 10gacatccaga tgacccagtc tccatcctcc
ctgtctgcag ctgtaggaga cagagtcacc 60atcacttgcc aggcgagtca ggacattgtc
aactatttaa attggtatca acagaaacca 120gggaaagccc ctaagctcct
gatctacgtt gcatccaatt tggagacagg ggtcccatca 180aggttcagtg
gaagtggatc tgggacagat tttagtctca ccatcagcag cctgcagcct
240gaagatgttg caacatatta ttgtcaacaa tatgataatc tcccactcac
attcggcgga 300gggaccaagg ttgagatcaa aaga 3241132DNAartificial
sequencePrimer VH1 11aaatcgatac caccatggac tggacctgga gg
321232DNAartificial sequencePrimer VH1B 12aaatcgatac caccatggac
tggacctgga gm 321334DNAartificial sequencePrimer VH2A 13aaatcgatac
caccatggac acactttgct mcac 341435DNAartificial sequencePrimer VH2B
14aaatcgatac caccatggac atactttgtt ccaac 351532DNAartificial
sequencePrimer VH3 15aaatcgatac caccatggag tttgggctga gc
321632DNAartificial sequencePrimer VH3B 16aaatcgatac caccatggar
ytkkgrctbh gc 321734DNAartificial sequencePrimer VH4 17aaatcgatac
caccatgaaa cacctgtggt tctt 341832DNAartificial sequencePrimer VH5
18aaatcgatac caccatgggg tcaaccgcca tc 321932DNAartificial
sequencePrimer VH6 19aaatcgatac caccatgtct gtctccttcc tc
322032DNAartificial sequencePrimer Cgamma-Rev 20gggtctagac
aggcagccca gggccgctgt gc 322132DNAartificial sequencePrimer Vk1
21aaatcgatac caccatggac atgagggtcc cy 322232DNAartificial
sequencePrimer Vk1B 22aaatcgatac caccatggac atgagrgtcc yy
322332DNAartificial sequencePrimer Vk2 23aaatcgatac caccatgagg
ctccctgctc ag 322431DNAartificial sequencePrimer Vk3 24aaatcgatac
caccatggaa rccccagcgc a 312532DNAartificial sequencePrimer Vk4
25aaatcgatac caccatggtg ttgcagaccc ag 322642DNAartificial
sequencePrimer Ck-Rev 26gatcgcggcc gcttatcaac actctcccct gttgaagctc
tt 422736DNAartificial sequencePrimer V11aecb 27aaatcgatac
caccatggcc tggtcccctc tcctcc 362836DNAartificial sequencePrimer
V11g 28aaatcgatac caccatggcc ggcttccctc tcctcc 362936DNAartificial
sequencePrimer V12/10 29aaatcgatac caccatggcc tgggctctgc tcctcc
363036DNAartificial sequencePrimer V13jpah 30aaatcgatac caccatggcc
tggaccgctc tcctgc 363136DNAartificial sequencePrimer V15/7
31aaatcgatac caccatggcc tggactcctc tccttc 363236DNAartificial
sequencePrimer V16/9 32aaatcgatac caccatggcc tgggctcctc tccttc
363336DNAartificial sequencePrimer V13rm 33aaatcgatac caccatggcc
tggatccctc tcctcc 363436DNAartificial sequencePrimer V131
34aaatcgatac caccatggcc tggacccctc tctggc 363536DNAartificial
sequencePrimer V13e 35aaatcgatac caccatggcc tgggccacac tcctgc
363636DNAartificial sequencePrimer V14c 36aaatcgatac caccatggcc
tgggtctcct tctacc 363736DNAartificial sequencePrimer V18a
37aaatcgatac caccatggcc tggatgatgc ttctcc 363841DNAartificial
sequencePrimer C12/7 38gatcgcggcc gcttatcawg arcattctgy aggggccact
g 413931DNAartificial sequencePrimer VH1-L-NheI 39gcggctagcc
accatggact ggacctggag g 314029DNAartificial sequencePrimer
JH4/5-XhoI 40gcgctcgaga cggtgaccag ggttccctg 294135DNAartificial
sequencePrimer CHfw-XhoI 41cgcgctcgag tgcctccacc aagggcccat cggtc
354241DNAartificial sequencePrimer CHrev-NotI 42gatcgcggcc
gcttatcatt tacccggrga cagggagagg c 414331DNAartificial
sequencePrimer VK1-L-NheI 43gcggctagcc accatggaca tgagggtccc y
314442DNAartificial sequencePrimer CK-NotI 44gatcgcggcc gcttatcaac
actctcccct gttgaagctc tt 424524DNAartificial sequencePrimer HPRT1
forward 45tatggacagg actgaacgtc ttgc 244626DNAartificial
sequencePrimer HPRT1 reverse 46gacacaaaca tgattcaaat ccctga
264720DNAartificial sequencePrimer LMP-1 forward 47gcgactctgc
tggaaatgat 204820DNAartificial sequencePrimer LMP-1 reverse
48gacatggtaa tgcctagaag 204922DNAartificial sequencePrimer EBNA1/2
forward 49agcaagaaga ggaggtggta ag 225022DNAartificial
sequencePrimer EBNA1/2 reverse 50ggctcaaagt ggtctctaat gc
2251379DNAartificial sequenceAnti-RSV clone B63D10-D25 51cag gtg
cag ctg gta cag tct ggg gct gaa gtg aag aag cct ggg tcc 48Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15
tcg gtg atg gtc tcc tgc cag gcc tct gga ggc ccc ctc aga aac tat
96Ser Val Met Val Ser Cys Gln Ala Ser Gly Gly Pro Leu Arg Asn Tyr
20 25 30 att atc aac tgg cta cga cag gcc cct gga caa ggc cct gag
tgg atg 144Ile Ile Asn Trp Leu Arg Gln Ala Pro Gly Gln Gly Pro Glu
Trp Met 35 40 45 gga ggg atc att cct gtc ttg ggt aca gta cac tac
gca ccg aag ttc 192Gly Gly Ile Ile Pro Val Leu Gly Thr Val His Tyr
Ala Pro Lys Phe 50 55 60 cag ggc aga gtc acg att acc gcg gac gaa
tcc aca gac aca gcc tac 240Gln Gly Arg Val Thr Ile Thr Ala Asp Glu
Ser Thr Asp Thr Ala Tyr 65 70 75 80 atc cat ctg atc agc ctg aga tct
gag gac acg gcc atg tat tac tgt 288Ile His Leu Ile Ser Leu Arg Ser
Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 gcg acg gaa aca gct ctg
gtt gta tct act acc tac cta cca cac tac 336Ala Thr Glu Thr Ala Leu
Val Val Ser Thr Thr Tyr Leu Pro His Tyr 100 105 110 ttt gac aac tgg
ggc cag gga acc ctg gtc acc gtc tcc tca g 379Phe Asp Asn Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 52126PRTartificial
sequenceSynthetic Construct 52Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Met Val Ser Cys Gln
Ala Ser Gly Gly Pro Leu Arg Asn Tyr 20 25 30 Ile Ile Asn Trp Leu
Arg Gln Ala Pro Gly Gln Gly Pro Glu Trp Met 35 40 45 Gly Gly Ile
Ile Pro Val Leu Gly Thr Val His Tyr Ala Pro Lys Phe 50 55 60 Gln
Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Asp Thr Ala Tyr 65 70
75 80 Ile His Leu Ile Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95 Ala Thr Glu Thr Ala Leu Val Val Ser Thr Thr Tyr Leu
Pro His Tyr 100 105 110 Phe Asp Asn Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120 125 53324DNAartificial sequenceVL region 53gac
atc cag atg acc cag tct cca tcc tcc ctg tct gca gct gta gga 48Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ala Val Gly 1 5 10
15 gac aga gtc acc atc act tgc cag gcg agt cag gac att gtc aac tat
96Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Val Asn Tyr
20 25 30 tta aat tgg tat caa cag aaa cca ggg aaa gcc cct aag ctc
ctg atc 144Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 tac gtt gca tcc aat ttg gag aca ggg gtc cca tca
agg ttc agt gga 192Tyr Val Ala Ser Asn Leu Glu Thr Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 agt gga tct ggg aca gat ttt agt ctc acc
atc agc agc ctg cag cct 240Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr
Ile Ser Ser Leu Gln Pro 65 70 75 80 gaa gat gtt gca aca tat tat tgt
caa caa tat gat aat ctc cca ctc 288Glu Asp Val Ala Thr Tyr Tyr Cys
Gln Gln Tyr Asp Asn Leu Pro Leu 85 90 95 aca ttc ggc gga ggg acc
aag gtt gag atc aaa aga 324Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys Arg 100 105 54108PRTartificial sequenceSynthetic Construct
54Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ala Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Val Asn
Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Val Ala Ser Asn Leu Glu Thr Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Ser Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr
Cys Gln Gln Tyr Asp Asn Leu Pro Leu 85 90 95 Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys Arg 100 105 5598PRTartificial
sequenceVH1-69 germl 55Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro
Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg
Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg 56126PRTartificial sequenceB63D10-D25 56Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val
Met Val Ser Cys Gln Ala Ser Gly Gly Pro Leu Arg Asn Tyr 20 25 30
Ile Ile Asn Trp Leu Arg Gln Ala Pro Gly Gln Gly Pro Glu Trp Met 35
40 45 Gly Gly Ile Ile Pro Val Leu Gly Thr Val His Tyr Ala Pro Lys
Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Asp
Thr Ala Tyr 65 70 75 80 Ile His Leu Ile Ser Leu Arg Ser Glu Asp Thr
Ala Met Tyr Tyr Cys 85 90 95 Ala Thr Glu Thr Ala Leu Val Val Ser
Thr Thr Tyr Leu Pro His Tyr 100 105 110 Phe Asp Asn Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 115 120 125 5795PRTartificial
sequenceVkI O8/O18 57Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala
Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn
Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro 85 90 95
58110PRTartificial sequenceB63D10-D25 58Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ala Val Gly 1 5 10 15 Asp Arg Val Thr Ile
Thr Cys Gln Ala Ser Gln Asp Ile Val Asn Tyr 20 25 30 Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr
Val Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu
Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg
Thr Val 100 105 110 5991DNAartificial sequenceFR1 VHeavy region D25
59caggtgcagc tggtacagtc tggggctgaa gtgaagaagc ctgggtcctc ggtgatggtc
60tcctgccagg cctctggagg ccccctcaga a 916014DNAartificial
sequenceCDR1 VHeavy region D25 60actatattat caac
146142DNAartificial sequenceFR2 VHeavy region D25 61tggctacgac
aggcccctgg acaaggccct gagtggatgg ga 426251DNAartificial
sequenceCDR2 VHeavy region D25 62gggatcattc ctgtcttggg tacagtacac
tacgcaccga agttccaggg c 516396DNAartificial sequenceFR3 VHeavy
region D25 63agagtcacga ttaccgcgga cgaatccaca gacacagcct acatccatct
gatcagcctg 60agatctgagg acacggccat gtattactgt gcgacg
966451DNAartificial sequenceCDR3 VHeavy region D25 64gaaacagctc
tggttgtatc tactacctac ctaccacact actttgacaa c 516534DNAartificial
sequenceFR4
VHeavy region D25 65tggggccagg gaaccctggt caccgtctcc tcag
346669DNAartificial sequenceFR1 VLight region D25 66gacatccaga
tgacccagtc tccatcctcc ctgtctgcag ctgtaggaga cagagtcacc 60atcacttgc
696733DNAartificial sequenceCDR1 VLight region D25 67caggcgagtc
aggacattgt caactattta aat 336845DNAartificial sequenceFR2 VLight
region D25 68tggtatcaac agaaaccagg gaaagcccct aagctcctga tctac
456921DNAartificial sequenceCRD2 VLight region D25 69gttgcatcca
atttggagac a 217096DNAartificial sequenceFR3 VLight region D25
70ggggtcccat caaggttcag tggaagtgga tctgggacag attttagtct caccatcagc
60agcctgcagc ctgaagatgt tgcaacatat tattgt 967121DNAartificial
sequenceCDR3 VLight region D25 71caacaatatg ataatctccc a
217239DNAartificial sequenceFR4 VLight region D25 72ctcacattcg
gcggagggac caaggttgag atcaaaaga 39738PRTHomo sapiens 73Gly Phe Ser
Phe Ser His Tyr Ala 1 5 748PRTHomo sapiens 74Ile Ser Tyr Asp Gly
Glu Asn Thr 1 5 7516PRTHomo sapiens 75Ala Arg Asp Arg Ile Val Asp
Asp Tyr Tyr Tyr Tyr Gly Met Asp Val 1 5 10 15 766PRTHomo sapiens
76Gln Asp Ile Lys Lys Tyr 1 5 7710PRTHomo sapiens 77Gln Gln Tyr Asp
Asn Leu Pro Pro Leu Thr 1 5 10 78123PRTHomo sapiens 78Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser His Tyr 20 25
30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ala Val Ile Ser Tyr Asp Gly Glu Asn Thr Tyr Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ser Lys
Asn Thr Val Ser 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp
Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Ile Val Asp Asp
Tyr Tyr Tyr Tyr Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Ala Thr
Val Thr Val Ser Ser 115 120 79111PRTHomo sapiens 79Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Lys Lys Tyr 20 25 30
Leu Asn Trp Tyr His Gln Lys Pro Gly Lys Val Pro Glu Leu Leu Met 35
40 45 His Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Arg Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75 80 Glu Asp Ile Gly Thr Tyr Tyr Cys Gln Gln Tyr
Asp Asn Leu Pro Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys Arg Thr Val 100 105 110 808PRTHomo sapiens 80Gly Phe
Thr Phe Ser Ser Tyr Asn 1 5 818PRTHomo sapiens 81Ile Ser Ala Gly
Ser Ser Tyr Ile 1 5 8218PRTHomo sapiens 82Ala Arg Glu Asp Tyr Gly
Pro Gly Asn Tyr Tyr Ser Pro Asn Trp Phe 1 5 10 15 Asp Pro
839PRTHomo sapiens 83Ser Ser Asn Ile Gly Ala Gly Tyr Asp 1 5
849PRTHomo sapiens 84His Ser Tyr Asp Arg Ser Leu Ser Gly 1 5
85125PRTHomo sapiens 85Glu Val Gln Leu Val Glu Thr Gly Gly Gly Leu
Ala Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Asn Met Asn Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Ser Ala
Gly Ser Ser Tyr Ile Tyr Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Val Ser Arg Asp Asn Val Arg Asn Ser Val Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Glu Asp Tyr Gly Pro Gly Asn Tyr Tyr Ser Pro Asn Trp Phe
100 105 110 Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 125 86110PRTHomo sapiens 86Gln Ser Val Val Thr Gln Pro Pro Ser
Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr
Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp
Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr
Gly Asn Thr Asn Arg Pro Ser Gly Val Ser Asp Arg Phe 50 55 60 Ser
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70
75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys His Ser Tyr Asp Arg
Ser 85 90 95 Leu Ser Gly Ser Val Phe Gly Gly Gly Thr Lys Leu Thr
Val 100 105 110 878PRTHomo sapiens 87Gly Phe Asn Phe His Asn Tyr
Gly 1 5 888PRTHomo sapiens 88Val Trp Tyr Asp Gly Ser Lys Lys 1 5
8913PRTHomo sapiens 89Val Arg Asp Lys Val Gly Pro Thr Pro Tyr Phe
Asp Ser 1 5 10 906PRTHomo sapiens 90Asn Ile Gly Ser Glu Thr 1 5
9111PRTHomo sapiens 91Gln Val Trp Asp Arg Ser Asn Tyr His Gln Val 1
5 10 92120PRTHomo sapiens 92Glu Val Gln Leu Val Glu Ser Gly Gly Asn
Val Val Lys Pro Gly Thr 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Thr Gly Phe Asn Phe His Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Val Trp
Tyr Asp Gly Ser Lys Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Thr Gly
Arg Phe Ala Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Val Arg Asp Lys Val Gly Pro Thr Pro Tyr Phe Asp Ser Trp Gly
Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120
93107PRTHomo sapiens 93Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser
Leu Ala Pro Gly Gly 1 5 10 15 Thr Ala Ala Ile Thr Cys Gly Arg Asn
Asn Ile Gly Ser Glu Thr Val 20 25 30 His Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Asp Asp Arg
Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly
Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp
Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Arg Ser Asn Tyr His 85 90
95 Gln Val Phe Gly Gly Gly Thr Lys Leu Thr Val 100 105 9475DNAHomo
sapiens 94gaggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc
cctgagactc 60tcctgtgcgg cctct 759524DNAHomo sapiens 95ggattcagct
tcagtcacta tgcc 249651DNAHomo sapiens 96atgcactggg tccgccaggc
tccaggcaag ggactggagt gggtggcagt t 519724DNAHomo sapiens
97atatcttatg atggagaaaa taca 2498114DNAHomo sapiens 98tattacgcag
actccgtgaa gggccgattc tccatctcca gagacaattc caagaacaca 60gtgtctctgc
aaatgaacag cctgagacct gaggacacgg ctctatatta ctgt 1149948DNAHomo
sapiens 99gcgagagacc gcatagtgga cgactactac tactacggta tggacgtc
4810034DNAHomo sapiens 100tggggccaag gggccacggt caccgtctcc tcag
34101369DNAHomo sapiens 101gaggtgcagc tggtggagtc tgggggaggc
gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcgg cctctggatt cagcttcagt
cactatgcca tgcactgggt ccgccaggct 120ccaggcaagg gactggagtg
ggtggcagtt atatcttatg atggagaaaa tacatattac 180gcagactccg
tgaagggccg attctccatc tccagagaca attccaagaa cacagtgtct
240ctgcaaatga acagcctgag acctgaggac acggctctat attactgtgc
gagagaccgc 300atagtggacg actactacta ctacggtatg gacgtctggg
gccaaggggc cacggtcacc 360gtctcctca 36910278DNAHomo sapiens
102gacatccaga tgacccagtc tccatcttcc ctgtctgcat ctgtaggaga
cagagtcacc 60atcacttgcc aggcgagt 7810318DNAHomo sapiens
103caggacatta agaagtat 1810451DNAHomo sapiens 104ttaaattggt
atcatcagaa accagggaaa gtccctgagc tcctgatgca c 51105108DNAHomo
sapiens 105aatttggaaa caggggtccc atcaaggttc agtggcaggg gatctgggac
agattttact 60ctcaccatta gcagcctgca gcctgaagat attggaacat attactgt
10810630DNAHomo sapiens 106caacagtatg ataatctgcc tccgctcact
3010731DNAHomo sapiens 107ttcggcggag ggaccaaggt ggagatcaaa c
31108333DNAHomo sapiens 108gacatccaga tgacccagtc tccatcttcc
ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc aggcgagtca ggacattaag
aagtatttaa attggtatca tcagaaacca 120gggaaagtcc ctgagctcct
gatgcacgat gcatccaatt tggaaacagg ggtcccatca 180aggttcagtg
gcaggggatc tgggacagat tttactctca ccattagcag cctgcagcct
240gaagatattg gaacatatta ctgtcaacag tatgataatc tgcctccgct
cactttcggc 300ggagggacca aggtggagat caaacgaact gtg 33310975DNAHomo
sapiens 109gaggtgcagc tggtggagac cgggggaggc ctggcccagc ctggggggtc
cctgagactc 60tcctgtgcag cctct 7511024DNAHomo sapiens 110ggattcacat
tcagtagtta taac 2411151DNAHomo sapiens 111atgaactggg tccgccaggc
tccagggaag gggctggagt gggtctcaca c 5111224DNAHomo sapiens
112attagtgcgg gtagtagtta cata 24113114DNAHomo sapiens 113tactactcag
actcagtgaa gggccgattc accgtctcca gagacaacgt caggaactca 60gtatatctgc
aaatgaacag cctgagagcc gctgacacgg ctgtgtatta ctgt 11411454DNAHomo
sapiens 114gcgagagagg attatggtcc gggaaattat tatagtccta actggttcga
cccc 5411534DNAHomo sapiens 115tggggccagg gaaccctggt caccgtctcc
tcag 34116375DNAHomo sapiens 116gaggtgcagc tggtggagac cgggggaggc
ctggcccagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacattcagt
agttataaca tgaactgggt ccgccaggct 120ccagggaagg ggctggagtg
ggtctcacac attagtgcgg gtagtagtta catatactac 180tcagactcag
tgaagggccg attcaccgtc tccagagaca acgtcaggaa ctcagtatat
240ctgcaaatga acagcctgag agccgctgac acggctgtgt attactgtgc
gagagaggat 300tatggtccgg gaaattatta tagtcctaac tggttcgacc
cctggggcca gggaaccctg 360gtcaccgtct cctca 37511775DNAHomo sapiens
117cagtctgtcg tgacgcagcc gccctcagtg tctggggccc cagggcagag
agtcaccatc 60tcctgcactg ggagc 7511827DNAHomo sapiens 118agctccaaca
tcggggcagg ttatgat 2711951DNAHomo sapiens 119gtacactggt accagcagct
tccaggaaca gcccccaaac tcctcatcta t 51120108DNAHomo sapiens
120aatcggccct caggggtctc cgaccgattc tctggctcca agtctggcac
ctcagcctcc 60ctggccatca ctggactcca ggctgaggat gaggctgatt attactgc
10812127DNAHomo sapiens 121cactcctatg acagaagcct gagtggt
2712237DNAHomo sapiens 122tcagtattcg gcggagggac caagctgacc gtcctag
37123330DNAHomo sapiens 123cagtctgtcg tgacgcagcc gccctcagtg
tctggggccc cagggcagag agtcaccatc 60tcctgcactg ggagcagctc caacatcggg
gcaggttatg atgtacactg gtaccagcag 120cttccaggaa cagcccccaa
actcctcatc tatggcaaca ctaatcggcc ctcaggggtc 180tccgaccgat
tctctggctc caagtctggc acctcagcct ccctggccat cactggactc
240caggctgagg atgaggctga ttattactgc cactcctatg acagaagcct
gagtggttca 300gtattcggcg gagggaccaa gctgaccgtc 33012475DNAHomo
sapiens 124caggtgcaac tggtggagtc tgggggaaat gtggtcaagc ctgggacgtc
cctgagactg 60tcctgtgcag cgact 7512524DNAHomo sapiens 125ggattcaact
tccataacta cggc 2412651DNAHomo sapiens 126atgaactggg tccgccaggc
tccaggcaag gggctggagt gggtggcggt t 5112724DNAHomo sapiens
127gtttggtatg atggaagtaa gaaa 24128114DNAHomo sapiens 128tactatgcag
actccgtgac gggccgattc gccatctcca gagacaattc caagaacact 60ctgtatctgc
aaatgaacag cctgagagtc gaggacacgg ctgtttatta ttgt 11412939DNAHomo
sapiens 129gtgagagata aagtgggacc gactccctac tttgactcc
3913034DNAHomo sapiens 130tggggccagg gaaccctggt caccgtatcc tcag
34131360DNAHomo sapiens 131gaggtgcagc tggtggagtc tgggggaaat
gtggtcaagc ctgggacgtc cctgagactg 60tcctgtgcag cgactggatt caacttccat
aactacggca tgaactgggt ccgccaggct 120ccaggcaagg ggctggagtg
ggtggcggtt gtttggtatg atggaagtaa gaaatactat 180gcagactccg
tgacgggccg attcgccatc tccagagaca attccaagaa cactctgtat
240ctgcaaatga acagcctgag agtcgaggac acggctgttt attattgtgt
gagagataaa 300gtgggaccga ctccctactt tgactcctgg ggccagggaa
ccctggtcac cgtctcgagt 36013275DNAHomo sapiens 132tcctatgtgc
tgactcagcc accctcggtg tcactggccc caggagggac ggccgcgatc 60acctgtggaa
gaaac 7513318DNAHomo sapiens 133aacattggaa gtgaaact 1813451DNAHomo
sapiens 134gtgcactggt accagcagaa gccaggccag gcccctgtgc tggtcgtcta t
51135108DNAHomo sapiens 135gaccggccct cagggatccc tgagcgattc
tctggctcca actctgggaa cacggccacc 60ctgaccatca gcagggtcga ggccggggat
gaggccgact attactgt 10813633DNAHomo sapiens 136caggtgtggg
ataggagtaa ttatcatcag gta 3313731DNAHomo sapiens 137ttcggcggag
ggaccaagtt gaccgtccta g 31138321DNAHomo sapiens 138tcctatgtgc
tgactcagcc cccctcggtg tcactggccc caggagggac ggccgcgatc 60acctgtggaa
gaaacaacat tggaagtgaa actgtgcact ggtaccagca gaagccaggc
120caggcccctg tgctggtcgt ctatgatgat gacgaccggc cctcagggat
ccctgagcga 180ttctctggct ccaactctgg gaacacggcc accctgacca
tcagcagggt cgaggccggg 240gatgaggccg actattactg tcaggtgtgg
gataggagta attatcatca ggtattcggc 300ggagggacca agctgaccgt c
321139378DNAHomo sapiens 139caggtgcagc tggtacagtc tggggctgaa
gtgaagaagc ctgggtcctc ggtgatggtc 60tcctgccagg cctctggagg ccccctcaga
aactatatta tcaactggct acgacaggcc 120cctggacaag gccctgagtg
gatgggaggg atcattcctg tcttgggtac agtacactac 180gcaccgaagt
tccagggcag agtcacgatt accgcggacg aatccacgga cacagcctac
240atccatctga tcagcctgag atctgaggac acggccatgt attactgtgc
gacggaaaca 300gctctggttg tatctactac ctacctacca cactactttg
acaactgggg ccagggaacc 360ctggtcaccg tctcctca
378140378DNAArtificialRSV#D25 VH codon optimized 140caggtgcagc
tggtgcagag cggagccgag gtgaagaaac ccggcagcag cgtgatggtg 60tcctgccagg
ccagcggcgg acccctgcgg aactacatca tcaactggct gcggcaggcc
120ccaggccagg gccctgagtg gatgggcggc atcatccccg tgctgggcac
cgtgcactac 180gcccccaagt tccagggccg ggtgaccatc accgccgacg
agagcaccga caccgcctac 240atccacctga tcagcctgcg gagcgaggac
accgccatgt actactgcgc caccgagacc 300gccctggtgg tgtccaccac
ctacctgccc cactacttcg acaactgggg ccagggcacc 360ctggtgacag tctcgagt
378141326DNAHomo sapiens 141gacatccaga tgacccagtc tccatcctcc
ctgtctgcag ctgtaggaga cagagtcacc 60atcacttgcc aggcgagtca ggacattgtc
aactatttaa attggtatca acagaaacca 120gggaaagccc ctaagctcct
gatctacgtt gcatccaatt tggagacagg ggtcccatca 180aggttcagtg
gaagtggatc tgggacagat tttagtctca ccatcagcag cctgcagcct
240gaagatgttg caacatatta ttgtcaacaa tatgataatc tcccactcac
attcggcgga 300gggaccaagg ttgagatcaa aagaac
326142326DNAArtificialRSV#D25 VL codon optimized 142gacatccaga
tgacccagag ccccagcagc ctgtctgccg ccgtgggcga ccgggtgacc 60atcacctgcc
aggccagcca ggacatcgtg aactacctga actggtatca gcagaagccc
120ggcaaggccc ccaagctgct gatctacgtg gccagcaacc tggaaaccgg
cgtgcccagc 180cggtttagcg gcagcggctc cggcaccgac ttcagcctga
ccatcagcag cctgcagccc 240gaggacgtgg ccacctacta ctgccagcag
tacgacaacc tgcccctgac ctttggcggc 300ggaacaaagg tggagatcaa gcggac
326143369DNAHomo sapiensCDS(1)..(369) 143gag gtg cag ctg gtg gag
tct ggg gga ggc gtg gtc cag cct ggg agg 48Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 tcc ctg aga ctc
tcc tgt gcg gcc tct gga ttc agc
ttc agt cac tat 96Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser
Phe Ser His Tyr 20 25 30 gcc atg cac tgg gtc cgc cag gct cca ggc
aag gga ctg gag tgg gtg 144Ala Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 gca gtt ata tct tat gat gga gaa
aat aca tat tac gca gac tcc gtg 192Ala Val Ile Ser Tyr Asp Gly Glu
Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 aag ggc cga ttc tcc atc
tcc aga gac aat tcc aag aac aca gtg tct 240Lys Gly Arg Phe Ser Ile
Ser Arg Asp Asn Ser Lys Asn Thr Val Ser 65 70 75 80 ctg caa atg aac
agc ctg aga cct gag gac acg gct cta tat tac tgt 288Leu Gln Met Asn
Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 gcg aga
gac cgc ata gtg gac gac tac tac tac tac ggt atg gac gtc 336Ala Arg
Asp Arg Ile Val Asp Asp Tyr Tyr Tyr Tyr Gly Met Asp Val 100 105 110
tgg ggc caa ggg gcc acg gtc acc gtc tcc tca 369Trp Gly Gln Gly Ala
Thr Val Thr Val Ser Ser 115 120 144123PRTHomo sapiens 144Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser His Tyr 20
25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Glu Asn Thr Tyr Tyr Ala
Asp Ser Val 50 55 60 Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ser
Lys Asn Thr Val Ser 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu
Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Ile Val Asp
Asp Tyr Tyr Tyr Tyr Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Ala
Thr Val Thr Val Ser Ser 115 120 145333DNAHomo sapiensCDS(1)..(333)
145gac atc cag atg acc cag tct cca tct tcc ctg tct gca tct gta gga
48Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 gac aga gtc acc atc act tgc cag gcg agt cag gac att aag aag
tat 96Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Lys Lys
Tyr 20 25 30 tta aat tgg tat cat cag aaa cca ggg aaa gtc cct gag
ctc ctg atg 144Leu Asn Trp Tyr His Gln Lys Pro Gly Lys Val Pro Glu
Leu Leu Met 35 40 45 cac gat gca tcc aat ttg gaa aca ggg gtc cca
tca agg ttc agt ggc 192His Asp Ala Ser Asn Leu Glu Thr Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 agg gga tct ggg aca gat ttt act ctc
acc att agc agc ctg cag cct 240Arg Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 gaa gat att gga aca tat tac
tgt caa cag tat gat aat ctg cct ccg 288Glu Asp Ile Gly Thr Tyr Tyr
Cys Gln Gln Tyr Asp Asn Leu Pro Pro 85 90 95 ctc act ttc ggc gga
ggg acc aag gtg gag atc aaa cga act gtg 333Leu Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 146111PRTHomo
sapiens 146Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp
Ile Lys Lys Tyr 20 25 30 Leu Asn Trp Tyr His Gln Lys Pro Gly Lys
Val Pro Glu Leu Leu Met 35 40 45 His Asp Ala Ser Asn Leu Glu Thr
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Arg Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Gly
Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro Pro 85 90 95 Leu Thr
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110
14798PRTArtificialIGHV#-30 germl. 147Gln Val Gln Leu Val Glu Ser
Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg 14898PRTArtificialIGKV1-33 germl. 148Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr
Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys
Gln Gln Tyr Asp Asn Leu Pro Pro 85 90 95 Leu Thr 149375DNAHomo
sapiensCDS(1)..(375) 149gag gtg cag ctg gtg gag acc ggg gga ggc ctg
gcc cag cct ggg ggg 48Glu Val Gln Leu Val Glu Thr Gly Gly Gly Leu
Ala Gln Pro Gly Gly 1 5 10 15 tcc ctg aga ctc tcc tgt gca gcc tct
gga ttc aca ttc agt agt tat 96Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30 aac atg aac tgg gtc cgc cag
gct cca ggg aag ggg ctg gag tgg gtc 144Asn Met Asn Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 tca cac att agt gcg
ggt agt agt tac ata tac tac tca gac tca gtg 192Ser His Ile Ser Ala
Gly Ser Ser Tyr Ile Tyr Tyr Ser Asp Ser Val 50 55 60 aag ggc cga
ttc acc gtc tcc aga gac aac gtc agg aac tca gta tat 240Lys Gly Arg
Phe Thr Val Ser Arg Asp Asn Val Arg Asn Ser Val Tyr 65 70 75 80 ctg
caa atg aac agc ctg aga gcc gct gac acg gct gtg tat tac tgt 288Leu
Gln Met Asn Ser Leu Arg Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90
95 gcg aga gag gat tat ggt ccg gga aat tat tat agt cct aac tgg ttc
336Ala Arg Glu Asp Tyr Gly Pro Gly Asn Tyr Tyr Ser Pro Asn Trp Phe
100 105 110 gac ccc tgg ggc cag gga acc ctg gtc acc gtc tcc tca
375Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125
150125PRTHomo sapiens 150Glu Val Gln Leu Val Glu Thr Gly Gly Gly
Leu Ala Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Asn Met Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Ser
Ala Gly Ser Ser Tyr Ile Tyr Tyr Ser Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Val Ser Arg Asp Asn Val Arg Asn Ser Val Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Glu Asp Tyr Gly Pro Gly Asn Tyr Tyr Ser Pro Asn Trp
Phe 100 105 110 Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125 151330DNAHomo sapiensCDS(1)..(330) 151cag tct gtc gtg
acg cag ccg ccc tca gtg tct ggg gcc cca ggg cag 48Gln Ser Val Val
Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 aga gtc
acc atc tcc tgc act ggg agc agc tcc aac atc ggg gca ggt 96Arg Val
Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30
tat gat gta cac tgg tac cag cag ctt cca gga aca gcc ccc aaa ctc
144Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
35 40 45 ctc atc tat ggc aac act aat cgg ccc tca ggg gtc tcc gac
cga ttc 192Leu Ile Tyr Gly Asn Thr Asn Arg Pro Ser Gly Val Ser Asp
Arg Phe 50 55 60 tct ggc tcc aag tct ggc acc tca gcc tcc ctg gcc
atc act gga ctc 240Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala
Ile Thr Gly Leu 65 70 75 80 cag gct gag gat gag gct gat tat tac tgc
cac tcc tat gac aga agc 288Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys
His Ser Tyr Asp Arg Ser 85 90 95 ctg agt ggt tca gta ttc ggc gga
ggg acc aag ctg acc gtc 330Leu Ser Gly Ser Val Phe Gly Gly Gly Thr
Lys Leu Thr Val 100 105 110 152110PRTHomo sapiens 152Gln Ser Val
Val Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg
Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25
30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
35 40 45 Leu Ile Tyr Gly Asn Thr Asn Arg Pro Ser Gly Val Ser Asp
Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala
Ile Thr Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys
His Ser Tyr Asp Arg Ser 85 90 95 Leu Ser Gly Ser Val Phe Gly Gly
Gly Thr Lys Leu Thr Val 100 105 110 15373PRTArtificialIGHV3-21
germl. 153Gly Phe Thr Phe Ser Ser Tyr Ser Met Asn Trp Val Arg Gln
Ala Pro 1 5 10 15 Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Ser
Ser Ser Ser Tyr 20 25 30 Ile Tyr Tyr Ala Asp Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp 35 40 45 Asn Ala Lys Asn Ser Leu Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu 50 55 60 Asp Thr Ala Val Tyr Tyr
Cys Ala Arg 65 70 15499PRTArtificialIGLV1-40 germl. 154Gln Ser Val
Val Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg
Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25
30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
35 40 45 Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp
Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala
Ile Thr Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys
Gln Ser Tyr Asp Ser Ser 85 90 95 Leu Ser Gly 155360DNAHomo
sapiensCDS(1)..(360) 155gag gtg cag ctg gtg gag tct ggg gga aat gtg
gtc aag cct ggg acg 48Glu Val Gln Leu Val Glu Ser Gly Gly Asn Val
Val Lys Pro Gly Thr 1 5 10 15 tcc ctg aga ctg tcc tgt gca gcg act
gga ttc aac ttc cat aac tac 96Ser Leu Arg Leu Ser Cys Ala Ala Thr
Gly Phe Asn Phe His Asn Tyr 20 25 30 ggc atg aac tgg gtc cgc cag
gct cca ggc aag ggg ctg gag tgg gtg 144Gly Met Asn Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 gcg gtt gtt tgg tat
gat gga agt aag aaa tac tat gca gac tcc gtg 192Ala Val Val Trp Tyr
Asp Gly Ser Lys Lys Tyr Tyr Ala Asp Ser Val 50 55 60 acg ggc cga
ttc gcc atc tcc aga gac aat tcc aag aac act ctg tat 240Thr Gly Arg
Phe Ala Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 ctg
caa atg aac agc ctg aga gtc gag gac acg gct gtt tat tat tgt 288Leu
Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 gtg aga gat aaa gtg gga ccg act ccc tac ttt gac tcc tgg ggc cag
336Val Arg Asp Lys Val Gly Pro Thr Pro Tyr Phe Asp Ser Trp Gly Gln
100 105 110 gga acc ctg gtc acc gtc tcg agt 360Gly Thr Leu Val Thr
Val Ser Ser 115 120 156120PRTHomo sapiens 156Glu Val Gln Leu Val
Glu Ser Gly Gly Asn Val Val Lys Pro Gly Thr 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Thr Gly Phe Asn Phe His Asn Tyr 20 25 30 Gly
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ala Val Val Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Ala Asp Ser Val
50 55 60 Thr Gly Arg Phe Ala Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Val Arg Asp Lys Val Gly Pro Thr Pro Tyr
Phe Asp Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser
115 120 157321DNAHomo sapiensCDS(1)..(321) 157tcc tat gtg ctg act
cag ccc ccc tcg gtg tca ctg gcc cca gga ggg 48Ser Tyr Val Leu Thr
Gln Pro Pro Ser Val Ser Leu Ala Pro Gly Gly 1 5 10 15 acg gcc gcg
atc acc tgt gga aga aac aac att gga agt gaa act gtg 96Thr Ala Ala
Ile Thr Cys Gly Arg Asn Asn Ile Gly Ser Glu Thr Val 20 25 30 cac
tgg tac cag cag aag cca ggc cag gcc cct gtg ctg gtc gtc tat 144His
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40
45 gat gat gac gac cgg ccc tca ggg atc cct gag cga ttc tct ggc tcc
192Asp Asp Asp Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60 aac tct ggg aac acg gcc acc ctg acc atc agc agg gtc gag
gcc ggg 240Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu
Ala Gly 65 70 75 80 gat gag gcc gac tat tac tgt cag gtg tgg gat agg
agt aat tat cat 288Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Arg
Ser Asn Tyr His 85 90 95 cag gta ttc ggc gga ggg acc aag ctg acc
gtc 321Gln Val Phe Gly Gly Gly Thr Lys Leu Thr Val 100 105
158107PRTHomo sapiens 158Ser Tyr Val Leu Thr Gln Pro Pro Ser Val
Ser Leu Ala Pro Gly Gly 1 5 10 15 Thr Ala Ala Ile Thr Cys Gly Arg
Asn Asn Ile Gly Ser Glu Thr Val 20 25 30 His Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Asp Asp
Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser
Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70
75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Arg Ser Asn Tyr
His 85 90 95 Gln Val Phe Gly Gly Gly Thr Lys Leu Thr Val 100 105
15998PRTArtificialIGHV3-33 germl. 159Gln Val Gln Leu Val Glu Ser
Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg 16098PRTArtificialIGLV3-21 germl. 160Ser
Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10
15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val
20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val
Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg
Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile
Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln
Val Trp Asp Ser Ser Ser Asp His 85 90 95 Gln Val
* * * * *