U.S. patent application number 16/904793 was filed with the patent office on 2021-09-09 for rsv-specific binding molecule.
The applicant listed for this patent is MedImmune Limited. Invention is credited to Adrianus Q. Bakker, Tim Beaumont, Etsuko Yasuda.
Application Number | 20210277094 16/904793 |
Document ID | / |
Family ID | 1000005795493 |
Filed Date | 2021-09-09 |
United States Patent
Application |
20210277094 |
Kind Code |
A9 |
Beaumont; Tim ; et
al. |
September 9, 2021 |
RSV-SPECIFIC BINDING MOLECULE
Abstract
The invention provides antibodies and functional equivalents
thereof which are capable of specifically binding RSV. Nucleic acid
sequences encoding said antibody, as well as antibody producing
cells and methods for producing said antibody are also
provided.
Inventors: |
Beaumont; Tim; (Ouderkerk
ann de Amstel, NL) ; Bakker; Adrianus Q.; (Hoorn,
NL) ; Yasuda; Etsuko; (Amsterdam, NL) |
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Applicant: |
Name |
City |
State |
Country |
Type |
MedImmune Limited |
Canbridge |
|
GB |
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|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20200317754 A1 |
October 8, 2020 |
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Family ID: |
1000005795493 |
Appl. No.: |
16/904793 |
Filed: |
June 18, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16047524 |
Jul 27, 2018 |
10723786 |
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16904793 |
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15042656 |
Feb 12, 2016 |
10035843 |
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16047524 |
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14039148 |
Sep 27, 2013 |
9283274 |
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15042656 |
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12898325 |
Oct 5, 2010 |
8568726 |
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14039148 |
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61278358 |
Oct 6, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/1027 20130101;
A61K 31/7088 20130101; A61K 2039/505 20130101; A61K 39/42
20130101 |
International
Class: |
C07K 16/10 20060101
C07K016/10; A61K 31/7088 20060101 A61K031/7088; A61K 39/42 20060101
A61K039/42 |
Claims
1. 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 70% identical to the sequence KLSIH, and/or a heavy
chain CDR2 sequence comprising a sequence which is at least 70%
identical to the sequence GYEGEVDEIFYAQKFQH, and/or a heavy chain
CDR3 sequence comprising a sequence which is at least 70% identical
to the sequence LGVTVTEAGLGIDDY, and/or a light chain CDR1 sequence
comprising a sequence which is at least 70% identical to the
sequence RASQIVSRNHLA, and/or a light chain CDR2 sequence
comprising a sequence which is at least 70% identical to the
sequence GASSRAT, and/or a light chain CDR3 sequence comprising a
sequence which is at least 70% identical to the sequence
LSSDSSI.
2. An antibody, functional part, derivative or analogue according
to claim 1, having a heavy chain sequence comprising a sequence
which is at least 70% identical to the sequence
QVQLVQSGAEVKKPGATVKVSCKISGHTLIKLSIHWVRQAPGKGLEWMGGYEGEVDEI
FYAQKFQHRLTVIADTATDTVYMELGRLTSDDTAVYFCGTLGVTVTEAGLGIDDYWGQ GTLVTVSS
and/or having a light chain sequence which is at least 70%
identical to the sequence
EIVLTQSPGTLSLSPGERATLSCRASQIVSRNHLAWYQQKPGQAPRLLIFGASSRATGIPV
RFSGSGSGTDFTLTINGLAPEDFAVYYCLSSDSSIFTFGPGTKVDFK.
3. An antibody, functional part, derivative or analogue according
to claim 1, which is conjugated to PEG or Albumin, and/or which
comprises: a salvage receptor binding epitope, and/or; modified
amino acid residues identified as involved in the interaction
between an Fc and an FcRN receptor, and/or non naturally occurring
amino acid residues.
4. An antibody, functional part, derivative or analogue according
to claim 1, comprising an Fc region which comprises a modification
at one or more positions selected from the group consisting of 234,
235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 247, 251, 252,
254, 255, 256, 262, 263, 264, 265, 266, 267, 268, 269, 279, 280,
284, 292, 296, 297, 298, 299, 305, 313, 316, 325, 326, 327, 328,
329, 330, 331, 332, 333, 334, 339, 341, 343, 370, 373, 378, 392,
416, 419, 421, 440 and 443 as numbered by the EU index as set forth
in Kabat et al, said modification comprising an amino acid
substitution, and/or an amino acid insertion, and/or an amino acid
deletion.
5. An isolated, synthetic or recombinant antibody or a functional
part, derivative and/or analogue thereof which is capable of
specifically binding an epitope that is recognized by an antibody
of claim 1.
6. An antibody, functional part, derivative or analogue according
to claim 5, wherein said antibody, functional part, derivative or
analogue has a dissociation constant (K.sub.d) of less than
1.times.10.sup.-2M, 1.times.10.sup.-3M, 1.times.10.sup.-4M,
1.times.10.sup.-5M, 1.times.10.sup.-6M, 1.times.10.sup.-7M,
1.times.10.sup.-8M, 1.times.10.sup.-9M, 1.times.10.sup.-10 M,
1.times.10.sup.-11M, 1.times.10.sup.-12M, 1.times.10.sup.-13M,
1.times.10.sup.-14M or less than 1.times.10.sup.-15 M.
7-8. (canceled)
9. An isolated, synthetic or recombinant nucleic acid sequence, or
a functional equivalent thereof, comprising a sequence encoding an
amino acid sequence which has at least 70% sequence identity to the
sequence KLSIH, and/or at least 70% sequence identity to the
sequence GYEGEVDEIFYAQKFQH, and/or at least 70% sequence identity
to the sequence LGVTVTEAGLGIDDY, and/or at least 70% sequence
identity to the sequence RASQIVSRNHLA, and/or at least 70% sequence
identity to the sequence GASSRAT, and/or at least 70% sequence
identity to the sequence LSSDSSI, and/or at least 70% sequence
identity to the sequence
QVQLVQSGAEVKKPGATVKVSCKISGHTLIKLSIHWVRQAPGKGLEWMGGYEGEVDEI
FYAQKFQHRLTVIADTATDTVYMELGRLTSDDTAVYFCGTLGVTVTEAGLGIDDYWGQ
GTLVTVSS, and/or at least 70% sequence identity to the sequence
EIVLTQSPGTLSLSPGERATLSCRASQIVSRNHLAWYQQKPGQAPRLLIFGASSRATGIPV
RFSGSGSGTDFTLTINGLAPEDFAVYYCLSSDSSIFTFGPGTKVDFK.
10. An isolated, synthetic or recombinant nucleic acid sequence
with a length of at least 15 nucleotides, or a functional
equivalent thereof, encoding at least one CDR sequence of an
antibody or a functional part, derivative and/or analogue thereof
according to claim 1.
11-12. (canceled)
13. An isolated, synthetic or recombinant nucleic acid sequence, or
a functional equivalent thereof, encoding at least one CDR sequence
capable of specifically binding an epitope that is recognized by an
antibody which comprises: a heavy chain CDR1 sequence comprising
the sequence KLSIH, and/or a heavy chain CDR2 sequence comprising
the sequence GYEGEVDEIFYAQKFQH, and/or a heavy chain CDR3 sequence
comprising the sequence LGVTVTEAGLGIDDY, and/or a light chain CDR1
sequence comprising the sequence RASQIVSRNHLA, and/or a light chain
CDR2 sequence comprising the sequence GASSRAT, and/or a light chain
CDR3 sequence comprising the sequence LSSDSSI.
14-15. (canceled)
16. A vector comprising a nucleic acid sequence or functional
equivalent according to claim 9.
17. An isolated or recombinant cell comprising a nucleic acid
sequence or functional equivalent according to claim 9.
18-19. (canceled)
20. A combination comprising: an antibody, functional part,
derivative or analogue according to claim 1, and a different
RSV-specific agent, preferably an antibody.
21-22. (canceled)
23. A combination according to claim 20 wherein said different
RSV-specific agent is Palivizumab, and/or AM14 and/or AM16, and/or
AM23, and/or D25.
24. Use of an antibody, functional part, derivative or analogue
according to claim 1 for the preparation of a medicament and/or
prophylactic agent for at least in part treating and/or preventing
a RSV-related disorder.
25. A pharmaceutical composition comprising: an antibody,
functional part, derivative or analogue according to claim 1 and a
pharmaceutical acceptable carrier, diluent or excipient.
26. An isolated or recombinant antibody producing cell capable of
producing an antibody, functional part, derivative or analogue
according to claim 1.
27. A method for producing an antibody, functional part, derivative
or analogue, comprising providing a cell with a nucleic acid
sequence or functional equivalent according to claim 9 and allowing
said cell to translate said nucleic acid sequence or functional
equivalent thereby producing said antibody, functional part,
derivative or analogue thereof.
28. A method according to claim 27, further comprising harvesting,
purifying and/or isolating said antibody, functional part,
derivative or analogue thereof.
29. Use of a nucleic acid sequence or functional equivalent
according to claim 9 for the preparation of a medicament and/or
prophylactic agent for at least in part treating and/or preventing
a RSV-related disorder.
30. Use of a combination according to claim 20 for the preparation
of a medicament and/or prophylactic agent for at least in part
treating and/or preventing a RSV-related disorder.
Description
[0001] This application is a continuation patent application of
U.S. patent application Ser. No. 15/042,656, filed on Feb. 12,
2016, which is a continuation patent application of U.S. patent
application Ser. No. 14/039,148, filed on Sep. 27, 2013, which is a
continuation patent application of U.S. patent application Ser. No.
12/898,325, filed on Oct. 5, 2010, which claims priority to U.S.
Provisional Patent Application No. 61/278,358, filed Oct. 6, 2009,
each of which is incorporated herein by reference.
SEQUENCE LISTING
[0002] This application contains a Sequence Listing electronically
submitted to the United States Patent and Trademark Office via
EFS-Web as an ASCII text file entitled
"490-00020104_SequenceListing_ST25.txt" having a size of 16
kilobytes and created on Jul. 26, 2018. Due to the electronic
filing of the Sequence Listing, the electronically submitted
Sequence Listing serves as both the paper copy required by 37 CFR
.sctn. 1.821(c) and the CU required by .sctn. 1.821(c). The
information contained in the Sequence Listing is incorporated by
reference herein.
[0003] The invention relates to the fields of biology and
medicine.
[0004] 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 (F protein)
of RSV. The F protein of RSV is a viral membrane protein and
responsible for fusion of the virion with a host cell after
attachment. In addition, infection of neighboring cells through the
formation of syncytia is promoted by the F protein and its function
is thought to depend on the original oligomeric structure of the
protein. However, palivizumab is not always effective. Therefore,
there is a need in the art for alternative and/or supplementary
antibodies and therapies against RSV.
[0005] It is an object of the invention to provide improved
antibodies against RSV, or functional equivalents of such
antibodies. It is a further object to provide supplementary
antibodies against RSV, which, in combination with existing
RSV-specific antibodies, provide a synergistic effect. It is a
further object of the invention to provide human or humanized
antibodies or functional equivalents against the RSV F protein
which are directed against an epitope that is different from the
epitopes that known RSV-specific antibodies are directed
against.
[0006] Accordingly, the present invention 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:
[0007] a heavy chain CDR1 sequence comprising a sequence which is
at least 70% identical to the sequence KLSIH (SEQ ID NO:4),
and/or
[0008] a heavy chain CDR2 sequence comprising a sequence which is
at least 70% identical to the sequence GYEGEVDEIFYAQKFQH (SEQ ID
NO:8), and/or
[0009] a heavy chain CDR3 sequence comprising a sequence which is
at least 70% identical to the sequence LGVTVTEAGLGIDDY (SEQ ID
NO:12), and/or
[0010] a light chain CDR1 sequence comprising a sequence which is
at least 70% identical to the sequence RASQIVSRNHLA (SEQ ID NO:20),
and/or
[0011] a light chain CDR2 sequence comprising a sequence which is
at least 70% identical to the sequence GASSRAT (SEQ ID NO:24),
and/or
[0012] a light chain CDR3 sequence comprising a sequence which is
at least 70% identical to the sequence LSSDSSI (SEQ ID NO:28).
[0013] The present invention provides an antibody designated
"AM22", which has heavy chain and light chain sequences as depicted
in FIG. 2. The CDR sequences of AM22, which in particular
contribute to the antigen-binding properties of AM22, are also
depicted in FIG. 2. Antibody AM22 is fully human, is capable of
specifically binding RSV (FIG. 3) and is therefore preferred for
prophylactic and/or therapeutic use for human individuals.
[0014] As mentioned above the only available clinically used
anti-RSV antibody is palivizumab. This is a humanized monoclonal
antibody directed against an epitope in the antigenic site of the F
protein of RSV. Humanized antibodies still contain part of a mouse
antibody and although immunogenic properties are diminished as
compared to fully mouse antibodies, side-effects of humanized
antibodies may occur when applied in humans. The present inventors,
however, succeeded in obtaining and culturing human B-cells
producing RSV-specific antibodies, so that human RSV-specific
antibodies have been provided, that have strongly reduced--if at
all--immunogenic activity as a result of the completely human
sequence. As shown in the examples, antibodies according to the
invention have superior characteristics as compared to palivizumab
(FIG. 1 and table 1). The present inventors have shown that Cotton
rats (Sigmodon hispidus) given antibodies according to the
invention by intramuscular injection followed by intranasal
challenge with RSV-X have a lower Pathology Index than Cotton rats
given palivizumab followed by challenge with RSV-X (FIG. 4C and
table 2). The Pathology Index used herein is a sum of scores to
classify three individual markers for lung damage. These three
markers are hyperthropy of bronchus and bronchioli epithelium,
inflammation surrounding bronchus and bronchioli
(peribronch(iol)itis) and inflammation in the alveoli (alveolitis).
In addition, Cotton rats injected intramuscularly with antibodies
according to the invention and subsequent RSV-X challenge had lower
lung virus titers than Cotton rats given palivizumab followed by
challenge with RSV-X (FIG. 4B), which was determined by TCID50 (50%
tissue culture infective dose) assay. Hence, AM22 is preferred over
palivizumab.
[0015] Besides palivizumab, some other RSV-specific antibodies are
known. WO 2008/147196 discloses sequences of RSV binding molecules,
namely antibodies D25, AM14, AM16 and AM23. As described in detail
in example 1 of the current application, RSV specific antibody AM22
was obtained from the same donor as antibodies D25, AM14, AM16 and
AM23. Strikingly however, AM22 recognizes RSV more efficiently than
all other antibodies obtained from the same donor. The IC.sub.50
value of AM22, 1.15 ng ml.sup.-1, is lower than that of
palivizumab, D25, AM14, AM16 or AM23. Therefore, the use of AM22
for treatment and/or prevention of a RSV-related disorder has
advantages over the use of other RSV specific antibodies. Less AM22
antibody is necessary to obtain a similar effect compared to the
other antibodies. Therefore, less AM22 has to be administered to an
individual for treatment and/or prevention of a RSV-related
disorder. Alternatively, with a similar amount of AM22, as compared
to the other antibodies, a more effective treatment and/or
prevention of a RSV-related disorder is achieved.
[0016] Furthermore, an RSV-specific antibody according to the
present invention recognizes a different epitope as the previously
disclosed RSV binding molecules. AM22, similar to the previously
identified antibodies (WO2008/147196), is capable of binding the
RSV F protein (FIG. 3A). However, AM22 does not bind a monomeric
RSV F protein (FIG. 3B left and right panel). Contrary to AM22, the
known antibodies palivizumab and AM16 (disclosed in WO 2008/147196
and FIG. 3B) are capable of binding the monomeric form of the F
protein. Importantly the AM22 B cell line, expressing the antigen
specific B Cell Receptor (BCR) does not recognize a recombinant
form of the F protein (FIG. 3C). Thus AM22 binds a different
epitope of the F protein than palivizumab, D25, AM23 and AM16. When
the recombinant F protein was expressed in a vector containing an
isoleucine zipper trimerization motif with eight HIS-tags
(ILZ-8.times.HIS), then AM22 recognized this trimeric, conformation
dependent structure (FIG. 3D). In contrast AM14 did not recognize
either the monomeric form of the F protein or the ILZ-8.times.HIS F
protein. Thus AM22 binds a different epitope of the F protein than
AM14 as well. Furthermore it was found that AM22 did not interfere
with D25 or palivizumab binding to RSV infected Hep2 cells. Thus,
AM22 binds a different epitope of the F protein as compared to D25,
AM14, AM16, AM23 and palivizumab. Therefore, RSV-specific
antibodies or functional equivalents thereof according to the
present invention are preferably combined with RSV-specific
antibodies that are already known, such as palivizumab, D25, AM14,
AM16 and AM23. By combining an antibody according to the invention
with a known RSV-specific antibody, two or more different epitopes
of RSV are recognized during the same therapy. This way, a stronger
immunogenic response to RSV is obtained. Furthermore, higher
antibody specificity against RSV is reached by combining one of the
known RSV-specific antibodies with an AM22 antibody according to
the invention. With a stronger immunogenic response to and higher
specificity against RSV, such combination will result in more
effective treatment and/or prevention of a RSV-related disorder.
Finally, a lower overall antibody dosage is needed because AM22 has
a stronger binding capacity for the F protein compared to
palivizumab, D25, AM14, AM16 and AM23, as demonstrated by its low
IC.sub.50 value, of about 1.15 ng/ml.
[0017] One embodiment therefore provides an antibody or functional
equivalent according to the invention which has an IC.sub.50 value
less than 1.25 ng/ml in an in vitro neutralization assay wherein
HEp-2 cells are infected with RSV-A2 virus. Said antibody or
functional equivalent preferably has an IC.sub.50 value of less
than 1.2 ng/ml, preferably between 0.5 ng/ml and 1.2 ng/ml.
Additionally, an antibody or functional equivalent according to the
invention preferably has an IC.sub.50 value which is at least
120-fold lower, more preferably at least 130-fold lower, than the
IC.sub.50 value of palivizumab in an in vitro neutralization assay
wherein HEp-2 cells are infected with RSV-A2 virus. Said antibody
or functional equivalent preferably has an IC.sub.50 value of about
1.15 ng/ml. Thus with an RSV-specific antibody or functional
equivalent thereof according to the present invention in
combination with at least one available RSV-specific antibody a
more effective treatment and/or prevention of a RSV-related
disorder is achieved.
[0018] A functional equivalent of an antibody is defined herein as
a functional part, derivative or analogue of an antibody. A
functional equivalent of an antibody is preferably an artificial
binding compound, comprising at least one CDR sequence of an
antibody.
[0019] 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.
[0020] 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.
[0021] A person skilled in the art is well able to generate
analogous compounds of an antibody. This is for instance done using
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.
[0022] An antibody according to the invention is preferably a human
antibody. The use of human antibodies for prophylaxis and therapy
in humans diminishes the chance of side-effects due to an
immunological reaction in a human individual against non-human
sequences. In another embodiment an antibody, functional part,
derivative or analogue according to the invention is a humanized
antibody. Humanized antibodies are made by incorporating non-human
hypervariable domains into human antibodies and therefore
immunogenic properties are diminished as compared to fully
non-human antibodies. In another 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.
[0023] 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.
[0024] 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.
[0025] Now that the present invention provides the insight that the
CDR sequences depicted in FIG. 2 provide desired 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. It is
also possible to alter at least one CDR sequence depicted in FIG. 2
in order to generate a variant antibody, or a functional equivalent
thereof, with at least one altered property as compared to AM22.
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. 2, so that the favorable binding
characteristics of AM22 are at least in part maintained or even
improved. A CDR sequence as depicted in FIG. 2 is preferably
altered such that the resulting antibody or functional equivalent
comprises at least one improved property, such as for instance an
improved stability and/or binding affinity, as compared to AM22.
The binding specificity is preferably maintained (in kind, not
necessarily in amount). 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. 2 are
therefore also 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.
[0026] Measurement of the affinity constant and specificity of
binding between antigen and antibody is preferred in determining
the efficacy of prophylactic, therapeutic, diagnostic and research
methods using anti-RSV antibodies of the invention. "Binding
affinity" generally refers to the strength of the sum total of the
noncovalent interactions between a single binding site of a
molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity can generally be represented by the
equilibrium dissociation constant (Kd), which is calculated as the
ratio k.sub.off/k.sub.on. See, e.g., Chen, Y., et al., (1999) J.
Mol Biol 293:865-881. Affinity can be measured by common methods
known in the art, such as for instance a surface plasmon resonance
(SPR) assay such as BiaCore or IBIS-iSPR instrument at IBIS
Technologies BV (Hengelo, the Netherlands) or solution phase
assays, such as Kinexa.
[0027] According to preferred embodiments, the present anti-RSV
antibodies of the invention have binding affinities for an epitope
on the RSV F protein that include a dissociation constant (K.sub.d)
of less than 1.times.10.sup.-2 M, 1.times.10.sup.-3 M,
1.times.10.sup.-4 M, 1.times.10.sup.-5 M, 1.times.10.sup.-6 M,
1.times.10.sup.-7 M, 1.times.10.sup.-8 M, 1.times.10.sup.-9 M,
1.times.10.sup.-10 M, 1.times.10.sup.-11 M, 1.times.10.sup.-12 M,
1.times.10.sup.-13 M, 1.times.10.sup.-14 M or less than
1.times.10.sup.-15 M. In one embodiment, the anti-RSV antibodies
have a K.sub.d of less than 10.sup.-7 M, less than
5.times.10.sup.-8 M, less than 10.sup.-8 M, less than
5.times.10.sup.-9 M, less than 10.sup.-9 M, less than
5.times.10.sup.-10 M, less than 10.sup.-10 M, less than
5.times.10.sup.-11 M, less than 10.sup.-11 M, less than
5.times.10.sup.-12 M, less than 10.sup.-12 M, less than
5.times.10.sup.-13 M, less than 10.sup.-13 M, less than
5.times.10.sup.-14 M, less than 10.sup.-14 M, less than
5.times.10.sup.-15 M, or less than 10.sup.-15 M.
[0028] The invention further provides an isolated, synthetic or
recombinant antibody or a functional part, derivative and/or
analogue thereof, which comprises:
[0029] a heavy chain CDR1 sequence comprising a sequence which has
at least 70% sequence identity to the sequence KLSIH (SEQ ID NO:
4), and/or
[0030] a heavy chain CDK2 sequence comprising a sequence which has
at least 70% sequence identity to the sequence GYEGEVDEIFYAQKFQH
(SEQ ID NO: 8), and/or
[0031] a heavy chain CDR3 sequence comprising a sequence which has
at least 70% sequence identity to the sequence LGVTVTEAGLGIDDY (SEQ
ID NO: 12) and/or
[0032] a light chain CDR1 sequence comprising a sequence which is
at least 70% identical to the sequence RASQIVSRNHLA (SEQ ID NO:
20), and/or
[0033] a light chain CDR2 sequence comprising a sequence which is
at least 70% identical to the sequence GASSRAT (SEQ ID NO: 24),
and/or
[0034] a light chain CDR3 sequence comprising a sequence which is
at least 70% identical to the sequence LSSDSSI (SEQ ID NO: 28).
[0035] Preferably, an antibody or 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. 2. Most preferably, an antibody or
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. 2. The particularly preferred antibody
AM22, described above, comprises CDR sequences which consist of the
CDR sequences depicted in FIG. 2. 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 RSV and which
comprises:
[0036] a heavy chain CDR1 sequence comprising the sequence KLSIH
(SEQ ID NO: 4), and/or
[0037] a heavy chain CDR2 sequence comprising the sequence
GYEGEVDEIFYAQKFQH (SEQ ID NO: 8), and/or
[0038] a heavy chain CDR3 sequence comprising the sequence
LGVTVTEAGLGIDDY (SEQ ID NO: 12), and/or
[0039] a light chain CDR1 sequence comprising the sequence
RASQIVSRNHLA (SEQ ID NO: 20), and/or
[0040] a light chain CDR2 sequence comprising the sequence GASSRAT
(SEQ ID NO: 24), and/or
[0041] a light chain CDR3 sequence comprising the sequence LSSDSSI
(SEQ ID NO: 28).
[0042] In one embodiment an antibody or functional equivalent is
provided which comprises the heavy chain CDR1 and CDR2 sequences
and the light chain CDR1 and CDR2 sequences as depicted in FIG. 2,
or sequences that are at least 70%, preferably at least 75%, more
preferably at least 80%, more preferably at least 85% identical
thereto. Further provided is therefore an isolated, synthetic or
recombinant antibody or a functional part, derivative and/or
analogue thereof which comprises a heavy chain CDR1 sequence
comprising a sequence which is at least 70% identical to the
sequence KLSIH (SEQ ID NO: 4) and a heavy chain CDR2 sequence
comprising a sequence which is at least 70% identical to the
sequence GYEGEVDEIFYAQKFQH (SEQ ID NO: 8) and a light chain CDR1
sequence comprising a sequence which is at least 70% identical to
the sequence RASQIVSRNHLA (SEQ ID NO: 20) and a light chain CDR2
sequence comprising a sequence which is at least 70% identical to
the sequence GASSRAT (SEQ ID NO: 24). 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
light chain CDR sequences. Preferably, said antibody or functional
equivalent also comprises a heavy chain CDR3 sequence comprising a
sequence which is at least 70% identical to the sequence
LGVTVTEAGLGIDDY (SEQ ID NO: 12), and/or a light chain CDR3 sequence
comprising a sequence which is at least 70% identical to the
sequence LSSDSSI (SEQ ID NO: 28). 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.
[0043] Optionally, said at least one human CDR sequence is
optimized, preferably in order to improve binding efficacy or
stability. This is for instance done by mutagenesis experiments
where after the stability and/or binding efficacy of the resulting
compounds are preferably tested and an improved antibody or
functional equivalent is selected.
[0044] Besides optimizing CDR sequences, it is often advantageous
to optimize at least one sequence in at least one of the frame work
regions. This is preferably done in order to improve binding
efficacy or stability. Frame work sequences are for instance
optimized by mutating a nucleic acid molecule encoding such frame
work sequence where after the characteristics of the resulting
antibody--or functional part--is preferably tested. This way, it is
possible to obtain improved antibodies or functional parts.
Isolated, synthetic or recombinant antibodies or functional parts,
derivatives and/or analogues thereof comprising a heavy chain amino
acid sequence which has at least 70% sequence identity to the heavy
chain sequence as depicted in FIG. 2 are therefore also provided.
Such heavy chain sequence provides desired binding properties, as
evidenced by antibody AM22. Moreover, light chain amino acid
sequences which have at Least 70% sequence identity to the light
chain sequence as depicted in FIG. 2 also provide desired binding
properties, as evidenced by antibody AM22. Further provided is
therefore an isolated, synthetic or recombinant antibody or a
functional part, derivative and/or analogue thereof according to
the invention, having a heavy chain sequence comprising a sequence
which has at least 70% sequence identity to the sequence
QVQLVQSGAEVKKPGAIVKVSCKISGHTLIKXjSIHWVRQAPGKGLEWMGG
YEGEVDEIFYAQKFQHRKLTVIADTATDTVYMELGKLTSDDTAVYFCGTLGV
TVTEAGLGIDDYWGQGTLVTVSS (SEQ ID NO: 16) and/or having a light chain
sequence which has at least 70% sequence identity to the sequence
EIVLTQSPGTLSLSPGERATLSCRASQIVSRNHLAWYQQKPGQAPRLLIFGA
SSRATGIPVRFSGSGSGTDFTLTINGLAPEDFAVYYCLSSDSSIFTFGPGTKV DFK (SEQ ID
NO: 32).
[0045] An isolated, synthetic or recombinant antibody or a
functional part, derivative and/or analogue thereof according to
the invention preferably comprises a heavy chain sequence and/or a
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. 2. The
higher the homology, the more closely said antibody or functional
equivalent, resembles antibody AM22. An isolated, synthetic or
recombinant antibody or a functional part, derivative and/or
analogue thereof according to the invention preferably comprises a
heavy chain as well as a light chain which resemble the heavy and
light chain of AM22. Further provided is therefore an isolated,
synthetic or recombinant antibody or a functional part, derivative
and/or analogue thereof 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, respectively, as depicted in
FIG. 2. In one embodiment ail antibody or functional equivalent is
provided which has a heavy chain sequence as depicted in FIG. 2 and
a light chain sequence as depicted in FIG. 2.
[0046] One embodiment provides an isolated, synthetic or
recombinant antibody or a functional part, derivative and/or
analogue thereof comprising a heavy chain sequence consisting of
the heavy chain sequence as depicted in FIG. 2, and/or comprising a
light chain sequence consisting of the light chain sequence as
depicted in FIG. 2. 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 or a single domain antibody or a single chain antibody or
a nanobody or an unibody or a scFv fragment based on a heavy chain
sequence or light chain sequence depicted in FIG. 2 is produced. A
functional part of an antibody comprising at least a functional
part of a sequence as depicted in FIG. 2 is therefore also
provided. Said functional part has a length of at least 20 amino
acids and comprises at least one sequence selected from the group
consisting of a sequence which is at least 70% identical to the
heavy chain CDR1 sequence depicted in FIG. 2 and a sequence which
has at least 70% sequence identity to the heavy chain CDR2 sequence
depicted in FIG. 2 and a sequence which has at least 70% sequence
identity to the heavy chain CDR3 sequence depicted in FIG. 2 and a
sequence which has at least 70% sequence identity to the light
chain CDR1 sequence depicted in FIG. 2 and a sequence which has at
least 70% sequence identity to the light chain CDR2 sequence
depicted in FIG. 2 and a sequence which has at least 70% sequence
identity to the light chain CDR3 sequence depicted in FIG. 2.
[0047] As said before, antibodies and functional equivalents
according to the present invention recognize a unique epitope of an
RSV F protein trimer. Hence, antibodies and functional equivalents
are provided that specifically recognize this epitope. Antibodies
or functional equivalents thereof that specifically recognize said
unique epitope are preferably combined with RSV-specific antibodies
that are already known, such as palivizumab, D25, AM14, AM16 and
AM23. By combining an antibody or functional equivalent according
to the invention that specifically recognizes said unique epitope
with a known RSV-specific antibody, two or more different epitopes
of RSV are recognized during the same therapy. This way, a stronger
immunogenic response to RSV and/or a higher antibody specificity
against RSV is reached With a stronger immunogenic response to and
higher specificity against RSV, such combination will result in
more effective treatment and/or prevention of a RSV-related
disorder.
[0048] Therefore, the invention provides an isolated, synthetic or
recombinant antibody or a functional part, derivative and/or
analogue thereof which is capable of specifically binding an
epitope that is recognized by an antibody which comprises:
[0049] a heavy chain CDR1 sequence comprising the sequence KLSIH
(SEQ ID NO: 4), and/or
[0050] a heavy chain CDR2 sequence comprising the sequence
GYEGEVDEIFYAQKFQH (SEQ ID NO: 8), and/or
[0051] a heavy chain CDR3 sequence comprising the sequence
LGVTVTEAGLGIDDY (SEQ ID NO: 12), and/or
[0052] a light chain CDR1 sequence comprising the sequence
RASQIVSRNHLA (SEQ ID NO: 20), and/or
[0053] a light chain CDR2 sequence comprising the sequence GASSRAT
(SEQ ID NO: 24), and/or
[0054] a light chain CDR3 sequence comprising the sequence LSSDSSI
(SEQ ID NO: 28).
[0055] In a particularly preferred embodiment the invention
provides an isolated, synthetic or recombinant antibody or a
functional part, derivative and/or analogue thereof which is
capable of specifically binding an epitope that is recognized by an
AM22 antibody which comprises:
[0056] a heavy chain CDR1 sequence comprising the sequence KLSIH
(SEQ ID NO: 4), and
[0057] a heavy chain CDR2 sequence comprising the sequence
GYEGEVDEIFYAQKFQH (SEQ ID NO: 8), and
[0058] a heavy chain CDR3 sequence comprising the sequence
LGVTVTEAGLGIDDY (SEQ ID NO: 12), and
[0059] a light chain CDR1 sequence comprising the sequence
RASQIVSRNHLA (SEQ ID NO: 20), and
[0060] a light chain CDR2 sequence comprising the sequence GASSRAT
(SEQ ID NO: 24), and
[0061] a light chain CDR3 sequence comprising the sequence LSSDSSI
(SEQ ID NO: 28).
[0062] A further embodiment of the invention contemplates certain
antibody constant region (Fc) modifications to alter effector
functions. For example, the serum half-life of proteins comprising
Fc regions is increased by increasing the binding affinity of the
Fc region for FcRn. The term "antibody half-life" as used herein
means a pharmacokinetic properly of an antibody that is a measure
of the mean survival time of antibody molecules following their
administration. Antibody half-life can be expressed as the time
required to eliminate 50 percent of a known quantity of
immunoglobulin from the patient's body (or other mammal) or a
specific compartment thereof, for example, as measured in serum,
i.e., circulating half-life, or in other tissues. Half-life may
vary from one immunoglobulin or class of immunoglobulin to another.
In general, an increase in antibody half-life results in an
increase in mean residence time (MRT) in circulation for the
antibody administered.
The increase in half-life allows for the reduction in amount of
drug given to a patient as well as reducing the frequency of
administration. To increase the serum half life of an antibody
according to the invention, one may incorporate a salvage receptor
binding epitope into the antibody (especially an antibody fragment)
as described in U.S. Pat. No. 5,739,277, for example. As used
herein, the term "salvage receptor binding epitope" refers to an
epitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2,
IgG3, or IgG4) that is responsible for increasing the in vivo serum
half-life of the IgG molecule. Alternatively, antibodies of the
invention with increased half-lives may be generated by modifying
amino acid residues identified as involved in the interaction
between the Fc and an FcRn receptor (see, for examples, U.S. Pat.
Nos. 6,321,505 and 7,083,784). In addition, the half-life of
antibodies of the invention may be increased by conjugation to PEG
or Albumin by techniques widely utilized in the art. In some
embodiments antibodies comprising Fc variant regions of the
invention have an increased half-life of about 5%, about 10%, about
15%, about 20%, about 25%, about 30%, about 35%, about 40%, about
45%, about 50%, about 60%, about 65%, about 70%, about 80%, about
85%, about 90%, about 95%, about 100%, about 125%, about 150% or
more as compared to an antibody comprising a native Fc region. In
some embodiments antibodies comprising Fc variant regions have an
increased half-life of about 2 fold, about 3 fold, about 4 fold,
about 5 fold, about 10 fold, about 20 fold, about 50 fold or more,
or is between 2 fold and 10 fold, or between 5 fold and 25 fold, or
between 15 fold and 50 fold, as compared to an antibody comprising
a native Fc region. The invention therefore provides an antibody,
functional part, derivative or analogue according the invention,
comprising a salvage receptor binding epitope, and/or modified
amino add residues identified as involved in the interaction
between the Fc and an FcRN receptor, and/or non naturally occurring
amino acid residues. Another preferred embodiment provides an
antibody or functional equivalent according to the invention which
is conjugated to PEG or Albumin.
[0063] In one embodiment, the present invention provides Fc
variants according to the invention, wherein the Fc region
comprises a modification (e.g., amino acid substitution, amino acid
insertion, amino acid deletion) at one or more positions selected
from the group consisting of 234, 235, 236, 237, 238, 239, 240,
241, 243, 244, 245, 247, 251, 252, 254, 250, 256, 262, 263, 264,
265, 266, 267, 268, 269, 279, 280, 284, 292, 296, 297, 298, 299,
305, 313, 316, 325, 326, 827, 328, 329, 330, 331, 332, 333, 334,
339, 341, 343, 370, 373, 378, 392, 416, 419, 421, 440 and 443 as
numbered by the EU index as set forth in Kabat et al (J Immunol.
1991; 147(5): 1709-19). Optionally, the Fc region comprises a non
naturally occurring amino acid residue at additional and/or
alternative positions known to one skilled in the art (see. e.g.,
U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; 7,083,784;
7,317,091; 7,217,797; 7,276,585; 7,355,008; 2002/0147311;
2004/0002587; 2005/0215768; 2007/0135620; 2007/0224188;
2008/0089892; WO 94/29351; and WO 99/58572).
[0064] In a specific embodiment, the present invention provides an
Fc variant antibody according to the invention, wherein the Fc
region comprises at least one non-naturally occurring amino acid at
one or more positions selected from the group consisting of 252,
254, and 256. In one embodiment, the non-naturally occurring amino
acids are selected from the group consisting of 252Y, 254T and
256E.
[0065] The present invention provides RSV-specific antibodies and
functional equivalents thereof having improved properties as
compared to prior art antibodies. The inventors have succeeded in
generating RSV-specific antibodies with the lowest IC.sub.50 value
currently known. 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
therefore provides an antibody which has an IC.sub.50 value of less
than 1.25 ng/ml, preferably less than 1.2 ng/mL, more preferably
less that 0.1.19 ng/ml, more preferably less than 1.18 ng/ml, and
most preferably between 1.1 ng/ml and 1.17 as determined in the in
vitro neutralization assay described in the examples (see FIG.
1).
[0066] The invention further provides an isolated, synthetic or
recombinant nucleic, acid sequence or a functional equivalent
thereof with a length of at least 15 nucleotides, preferably at
least 30 nucleotides, more preferably at least 60 nucleotides, more
preferably at least 75 nucleotides, encoding at least an
antigen-binding part of 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. A preferred embodiment provides a
nucleic acid sequence comprising a sequence which has at least 70%
sequence identity to at least 15 nucleotides of a nucleic acid
sequence as depicted in FIG. 2. A nucleic acid sequence according
to the invention preferably comprises a sequence which has at least
75%, more preferably at least 80%, more preferably at least 85%,
more preferably at least 90%, most preferably at least 95% sequence
identity to at least 15 nucleotides of a nucleic acid sequence as
depicted in FIG. 2. Preferably, said nucleic acid sequence as
depicted in FIG. 2 comprises at least one CDR encoding
sequence.
[0067] One preferred embodiment provides an isolated, synthetic or
recombinant nucleic acid sequence with a length of at least 15
nucleotides, or a functional equivalent thereof, encoding at least
one CDR sequence of an antibody or functional equivalent according
to the invention. Said nucleic acid sequence preferably encodes at
least one CDR sequence which has at least 70% sequence identity to
a CDR region of antibody AM22. Nucleic acid sequences encoding AM22
CDR regions are depicted in FIG. 2. Further provided is therefore
an isolated, synthetic or recombinant nucleic acid sequence, or a
functional equivalent thereof, comprising a sequence which has at
least 70% sequence identity to a sequence selected from the group
consisting of aaattatccatteac (SEQ ID NO: 3),
ggttatgagggtgaggtcgatgagattttctacgcacagaagttccagcac (SEQ ID NO: 7),
ctaggtgtgacagtgactgaggctggactggggatcgatgactac (SEQ ID NO: 11),
agggccagtcagattgttagcaggaaccacttagcc (SEQ ID NO: 19),
ggtgcgtccagtcgggccact (SEQ ID NO: 23) and ctgtcctctgattcctccata
(SEQ ID NO: 27).
[0068] Said nucleic acid sequence or functional equivalent
preferably comprises a sequence which has at least 75%, more
preferably at least 80%, more preferably at least 85%, more
preferably at least 90% sequence identity to any of the above
mentioned nucleic acid sequences. Further provided is a nucleic
acid sequence or functional equivalent thereof comprising a
sequence which has at least 70% sequence identity to at least part
of a nucleotide sequence as depicted in FIG. 2, said part having at
least 15 nucleotides and encoding at least one CDR region as
depicted in FIG. 2.
[0069] A nucleic acid sequence or functional equivalent thereof
according to the present invention preferably encodes a region
which has at least 70% sequence identity to an AM22 CDR region, an
AM22 heavy chain and/or an AM22 light chain. One embodiment thus
provides an isolated, synthetic or recombinant nucleic acid
sequence, or a functional equivalent, thereof, comprising a
sequence encoding an amino acid sequence which has at least 70%
sequence identity to the sequence KLSIH (SEQ ID NO: 4), and/or at
least 70% sequence identity to the sequence GYEGEVDEIFYAQKFQH (SEQ
ID NO: 8), and/or at least 70% sequence identity to the sequence
LGVTVTEAGLGIDDY (SEQ ID NO: 12), and/or at least 70% sequence
identity to the sequence RASQIVSRNHLA (SEQ ID NO: 20), and/or at
least 70% sequence identity to the sequence GASSRAT (SEQ ID NO:
24), and/or at least 70% sequence identity to the sequence LSSDSSI
(SEQ ID NO: 28), and/or at least 70% sequence identity to the
sequence QVQLVQSGAEVKKPGATVKVSCKISGHTLIKLSIHWVRQAPGKGLEWMGG
YEGEVDEIFYAQKFQHRLTVIADTATDTVYMELGRLTSDDTAVYFCGTLGV
TVTEAGLGIDDYWGQGTLVTVSS (SEQ ID NO: 16), and/or at least 70%
sequence identity to the sequence
EIVLTQSPGTLSLSPGERATLSCRASQIVSRNHLAWYQQKPGQAPRLLIFGA
SSRATGIPVRFSGSGSGTDFTLTINGLAPEDFAVYYCLSSDSSIFTFGPGTKV DFK (SEQ ID
NO: 32).
[0070] As said before, an antibody or functional equivalent
according to the invention is capable of recognizing a unique
epitope present on trimeric RSV F proteins. A nucleic acid sequence
according to the invention thus preferably encodes a CDR sequence
capable of specifically binding this unique epitope. Also provided
by the invention is therefore an isolated, synthetic or recombinant
nucleic acid sequence or a functional equivalent thereof, encoding
at least one CDR sequence capable of specifically binding an
epitope that is recognized by an antibody which comprises:
[0071] a heavy chain CDR1 sequence comprising the sequence KLSIH
(SEQ ID NO: 4), and/or
[0072] a heavy chain CDR2 sequence comprising the sequence
GYEGEVDEIFYAQKFQH (SEQ ID NO: 8), and/or
[0073] a heavy chain CDR3 sequence comprising the sequence
LGVTVTEAGLGIDDY (SEQ ID NO: 12), and/or
[0074] a light chain CDR1 sequence comprising the sequence
RASQIVSRNHLA (SEQ ID NO: 20), and/or
[0075] a light chain CDR2 sequence comprising the sequence GASSRAT
(SEQ ID NO: 24), and/or
[0076] a light chain CDR3 sequence comprising the sequence LSSDSSI
(SEQ ID NO: 28).
[0077] Preferably, said nucleic acid sequence encodes a whole
antibody or functional equivalent (for instance comprising a heavy
chain or light chain) according to the invention. Further provided
is therefore an isolated, synthetic or recombinant nucleic acid
sequence, or a functional equivalent thereof, encoding an antibody
or functional equivalent thereof capable of specifically binding an
epitope that is recognized by an antibody which comprises:
[0078] a heavy chain CDR1 sequence comprising the sequence KLSIH
(SEQ ID NO: 4), and/or
[0079] a heavy chain CDR2 sequence comprising the sequence
GYEGEVDEIFYAQKFQH (SEQ ID NO: 8), and/or
[0080] a heavy chain CDR3 sequence comprising the sequence
LGVTVTEAGLGIDDY (SEQ ID NO: 12), and/or
[0081] a light chain CDR1 sequence comprising the sequence
RASQIVSRNHLA (SEQ ID NO: 20), and/or
[0082] a light chain CDR2 sequence comprising the sequence GASSRAT
(SEQ ID NO: 24), and/or
[0083] a light chain CDR3 sequence comprising the sequence LSSDSSI
(SEQ ID NO: 28).
[0084] In one embodiment of the invention a nucleic acid sequence
or functional equivalent encodes an antibody or a functional part,
derivative and/or analogue thereof capable of specifically binding
an epitope that is recognized by an AM22 antibody, which comprises
a heavy chain CDR L sequence comprising the sequence KLSIH (SEQ ID
NO: 4), and a heavy chain CDR2 sequence comprising the sequence
GYEGEVDEIFYAQKFQH (SEQ II) NO: 8), and a heavy chain CDR3 sequence
comprising the sequence LGVTVTEAGLGIDDY (SEQ ID NO: 12), and a
light chain CDR1 sequence comprising the sequence RASQIVSRNHLA (SEQ
ID NO: 20), and a light chain CDR2 sequence comprising the sequence
GASSRAT (SEQ ID NO: 24), and a light chain CDR3 sequence comprising
the sequence LSSDSSI (SEQ ID NO: 28).
[0085] A nucleic acid sequence or functional equivalent, according
to the invention preferably encodes an antibody or functional
equivalent thereof that has a dissociation constant (K.sub.d) of
less than 1.times.10.sup.-2 M, 1.times.10.sup.-3 M,
1.times.10.sup.-4 M, 1.times.10.sup.-5 M, 1.times.10.sup.-6 M,
1.times.10.sup.-7 M, 1.times.10.sup.-8 M, 1.times.10.sup.-9 M,
1.times.10.sup.-10 M, 1.times.10.sup.-11 M, 1.times.10.sup.-12 M,
1.times.10.sup.-13 M, 1.times.10.sup.-14 M or less than
1.times.10.sup.-15 M.
[0086] Further provided is a vector comprising a nucleic acid
sequence according to the invention. Such vector is suitable for a
variety of applications. For instance, a vector of the invention
comprising a therapeutically beneficial nucleic acid sequence is
suitable for prophylactic or therapeutic applications.
Administration of such vector to an individual in need thereof
results in expression of said prophylactic or therapeutic nucleic
acid sequence in vivo. Said vector can also be used in applications
involving in vitro expression of a nucleic acid sequence of
interest, for instance for (commercial) production of antibodies or
functional equivalents according to the invention. Methods for
constructing a vector with a nucleic acid sequence according to the
invention are well known in the art. Non-limiting examples of
vectors suitable for generating a vector of the invention are
retroviral and lentiviral vectors.
[0087] The term "% sequence identity" is defined herein as the
percentage of residues in a candidate amino acid sequence or
candidate nucleic acid sequence that is identical with the residues
in a reference sequence after aligning the two sequences and
introducing gaps, if necessary, to achieve the maximum percent
identity. Methods and computer programs for the alignment are well
known in the art.
[0088] As used herein, a nucleic acid molecule or nucleic acid
sequence of the invention preferably comprises a chain of
nucleotides, more preferably DNA and/or RNA. In other embodiments a
nucleic acid molecule or nucleic acid sequence of the invention
comprises other kinds of nucleic acid structures such as for
instance a DNA/RNA helix, peptide nucleic acid (PNA), locked
nucleic acid (LNA) and/or a ribozyme. Such other nucleic acid
structures are referred to as functional equivalents of a nucleic
acid sequence. The term "functional equivalent of a nucleic acid
sequence" also encompasses a chain comprising non-natural
nucleotides, modified nucleotides and/or non-nucleotide building
blocks which exhibit the same function as natural nucleotides.
[0089] A nucleic acid sequence or vector according to the present
invention is particularly useful, for generating antibodies or
functional equivalents which are specific for RSV. This is for
instance done by introducing such nucleic acid sequence or vector
into a cell so that the cell's nucleic acid translation machinery
will produce the encoded antibody or functional equivalent. In one
embodiment, a nucleic acid sequence or vector encoding a heavy
and/or light chain according to the invention is expressed in so
called producer cells, such as for instance cells of a Chinese
hamster ovary (CHO), NSO (a mouse myeloma) or 293(T) cell line,
some of which are adapted to commercial antibody production.
Proliferation of said producer cells results in a producer cell
line capable of producing antibodies or functional equivalents
thereof according to the present invention. Preferably, said
producer cell line is suitable for producing antibodies for use in
humans. Hence, said producer cell line is preferably free of
pathogenic agents such as pathogenic micro-organisms. Most
preferably, antibodies or functional equivalents consisting of
human sequences are generated using at least one nucleic acid
sequence or vector according to the invention.
[0090] An isolated or recombinant antibody producing cell capable
of producing an antibody or a functional part, derivative and/or
analogue thereof according to the invention is therefore also
provided, as well as a method for producing an isolated, synthetic
or recombinant antibody or functional part, derivative and/or
analogue according to the invention, comprising providing a cell
with a nucleic acid sequence or functional equivalent or vector
according to the invention and allowing said cell to translate said
nucleic acid sequence or functional equivalent or vector, thereby
producing said antibody or functional part, derivative and/or
analogue thereof.
[0091] An antibody producing cell is defined herein as a cell which
is capable of producing and/or secreting antibody or a functional
equivalent, thereof, and/or which is capable of developing into a
cell which is capable of producing and/or secreting antibody or a
functional equivalent thereof. An antibody producing cell according
to the invention is preferably a producer cell which is adapted to
commercial antibody production. Preferably, said producer cell is
suitable for producing antibodies for use in humans.
[0092] A method according to the invention preferably further
comprises a step of harvesting, purifying and/or isolating said
antibody or functional part, derivative and/or analogue thereof
according to the invention. Obtained antibodies or functional
equivalents according to the invention are preferably used in human
therapy, optionally after additional, purifying, isolation or
processing steps.
[0093] Now that improved Respiratory Syncytial Virus-specific
antibodies or functional equivalents according to the invention and
nucleic acid sequences and vectors coding therefore have been
provided, including human antibodies or functional equivalents,
improved prophylactic and/or therapeutic applications have become
available. RSV is counteracted by antibodies or functional
equivalents according to the invention. An antibody or functional
equivalent according to the invention is therefore particularly
suitable for use as a medicine or prophylactic agent, optionally in
combination with at least one other RSV-specific antibody known in
the art. Preferably, antibodies or functional equivalents are used
which consist of human sequences, or which have at most 5% of
non-human sequences, in order to reduce the chance of adverse side
effects when human individuals are treated. An isolated, synthetic
or recombinant antibody or a functional part, derivative and/or
analogue thereof or a nucleic acid sequence or functional
equivalent thereof or a vector or a cell according to the invention
for use as a medicament and/or prophylactic agent is therefore also
herewith provided. When a nucleic acid or functional equivalent or
vector according to the invention is administered, it will be
translated in situ into an antibody or functional equivalent
according to the invention. In a particularly preferred embodiment
said antibody comprises antibody AM22, or a functional equivalent
thereof. Said medicament or prophylactic agent is preferably used
for counteracting or at least in part preventing an infection by
RSV. Further provided is therefore an isolated, synthetic or
recombinant antibody or a functional part, derivative and/or
analogue thereof or a nucleic acid sequence or functional
equivalent thereof or vector or cell according to the invention for
use as a medicament and/or prophylactic agent for at least in part
treating and/or preventing a disorder related to RSV. A medicament
that comprises AM22 in combination with at least one other
RSV-specific agent, preferably an antibody, known in the art, is
particularly advantageous because with such combination a stronger
immunogenic response to RSV is obtained and/or higher antibody
specificity against RSV is reached. Further provided is therefore a
combination of an isolated, synthetic or recombinant antibody or a
functional part, derivative and/or analogue thereof or a nucleic
acid sequence or functional equivalent thereof or vector or cell
according to the invention and another, different RSV-specific
agent, preferably an antibody or functional equivalent thereof, for
use as a medicament and/or prophylactic agent. A combination
according to the invention preferably comprises AM22 and an
antibody selected from the group consisting of palivizumab, D25,
AM14, AM16 and AM23. As said before, such combination is
particularly suitable for at least in part treating or preventing a
RSV-related disorder. Further provided is therefore a use of a
combination according to the invention for the preparation of a
medicament and/or prophylactic agent, for at least in part treating
and/or preventing a disorder related to RSV. A use of an isolated,
synthetic or recombinant antibody or a functional part, derivative
and/or analogue thereof or a nucleic acid sequence or functional
equivalent thereof or vector or cell according to the invention for
the preparation of a medicament and/or prophylactic agent for at
least in part treating and/or preventing an RSV-related disorder is
therefore also provided, as well as a method for at least in part
treating or preventing a RSV-related disorder, the method
comprising administering to an individual in need thereof a
therapeutically effective amount, of an isolated, synthetic or
recombinant antibody or a functional part, derivative and/or
analogue thereof according to the invention. In one preferred
embodiment, a combination with at least one other RSV-specific
agent, preferable another RSV specific antibody, is used. Said
individual has preferably been diagnosed to be infected by RSV
before treatment.
[0094] Said antibody preferably comprises antibody AM22, or a
functional part thereof. Said at least one other RSV-specific
antibody is preferably palivizumab. D25, AM14, AM16 or AM23. Moat
preferably a combination of AM22 and D25 is used.
[0095] In order to at least in part treat or prevent a disorder
related to Respiratory Syncytial Virus, an antibody or functional
equivalent according to the invention is preferably administered to
an individual before an infection has taken place. Alternatively,
an antibody or functional equivalent according to the invention is
administered when an individual is already infected. Said antibody
or functional equivalent is preferably administered to individuals
with an increased risk of complications, such as for instance
hospitalized individuals and/or individuals with compromised
immunity. Also elderly people have an increased risk of RSV
infection. Antibodies or functional equivalents according to the
invention are preferably administered via one or more injections.
Dose ranges of antibodies or functional equivalents according to
the invention to be used in the prophylactic or 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 prophylactic or therapeutic
application antibodies or functional equivalents according to the
invention are typically combined with a pharmaceutically acceptable
carrier, diluent and/or excipient. Examples of suitable carriers
for instance comprise keyhole limpet haemocyanin (KLH), serum
albumin (e.g. BSA or RSA) and ovalbumin. In one preferred
embodiment said suitable carrier comprises a solution like for
example saline.
[0096] In yet another embodiment a nucleic acid or a vector
encoding an antibody or functional equivalent according to the
invention is used. As already described, upon administration of
such nucleic acid or vector, antibodies or functional equivalents
are produced by the host's machinery. Produced antibodies or
functional equivalents are capable of at least in part preventing
and/or counteracting Respiratory Syncytial Virus infection and/or
the adverse effects of such infection. A nucleic acid sequence or
functional equivalent or a vector according to the invention for
use as a medicament a rid/or prophylactic agent is therefore also
herewith provided. Further provided is a use of a nucleic acid
sequence or functional equivalent or vector 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.
[0097] Further provided is a pharmaceutical composition comprising
an isolated, synthetic or recombinant antibody or a functional
part, derivative and/or analogue thereof or a nucleic acid sequence
or functional equivalent thereof or vector or cell according to the
invention and a pharmaceutical acceptable carrier, diluent or
excipient. Said pharmaceutical composition is preferably suitable
for human use. In one preferred embodiment said antibody is AM22.
In a further preferred embodiment said nucleic acid encodes AM22 or
a functional equivalent thereof. In one embodiment said
pharmaceutical composition further comprises at least one other
RSV-specific antibody, preferably palivizumab, D25, AM14, AM16
and/or AM23.
[0098] 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.
REFERENCES
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acid sequences and segments of sequences in antibodies of different
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Lukens M V, Van Bleek G M, Widjojoatmodjo M N, Bogers W M J M, Mei
H, Radbruch A, Scheeren F A, Spits H and Beaumont T. Generation of
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Brummelkamp T R, Scheeren F, Koh E, Daley G Q, Spits H, Bernards R.
A senescence rescue screen identifies BCL6 as an inhibitor of
anti-proliferative p19(ARF)-p53 signaling. Genes Dev. 2002;
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Nagasawa M, Wijnands E, Gimeno R, Vyth-Dreese F A, Blom B, Spits H.
STAT5 regulates the self-renewal capacity and differentiation of
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US 2008/0089892 [0123] WO 94/2935.1 [0124] WO 99/58572 [0125] WO
2008/147196
BRIEF DESCRIPTION OF THE DRAWINGS
[0126] FIG. 1. AM22, a novel fully human monoclonal antibody,
neutralizes the RSV A2 virus very efficiently on Hep2 cells
compared to palivizumab.
[0127] FIGS. 2A and 2B. FIG. 2A shows AM22 heavy chain nucleotide
sequences and amino acid sequences. FIG. 2B shows light chain
nucleotide sequences and amino acid sequences.
[0128] FIGS. 3A-3D. Human anti-RSV monoclonal antibodies recognize
conformational epitopes on the Fusion (F) protein of RSV as
determined by ELISA and FACS staining. FIG. 3A shows binding of the
antibodies to EL-4 cells infected with Vesicular Stomatitis Virus
(VSV) which was pseudotyped with the RSV F or RSV G protein. FIG.
3B shows binding of the anti-RSV F antibodies to ELISA plates that
were coated with a lysate of RSV infected HEp2 (left panel) or with
Ni-NTA HisSorp Plates (Qiagen) coated with recombinant HIS tagged F
protein (right panel). Antibody binding to the F protein is
detected with HRP-conjugated IgG detection antibody (dilutions
1:2500, Jackson). FIG. 3C shows binding of the recombinant RSV F
Long strain containing a poly HIS tag to the original B cell
clones, binding to the BCR is detected with an anti-pentaHIS
antibody. FIG. 3D shows the intracellular binding of the human
anti-RSV antibodies to 293T cells transfected with a recombinant
RSV F construct containing a trimerization domain (ILZ domain).
[0129] FIGS. 4A-4C. RSV challenge in Cotton rats prophylactically
treated with human immunoglobulins. FIG. 4A shows the retrieval of
human IgG1 from serum at day 1 and day 5 after intramuscular
administration of indicated doses of antibodies in Cotton rats.
FIG. 4B shows the RSV load in the lungs of Cotton rats treated with
the indicated antibodies 24 hours before infection with RSV-X. RSV
load was determined 5 days after infection with TCID.sub.50
culture. Experiments were performed twice with four to six
individual animals per treatment group. Lung pathology was studied
in the same animal groups (FIG. 4C), indicated is the average lung
pathology of AM22 and palivizumab, see also table 2.
EXAMPLES
Example 1. B Cell Culture, Immortalization and Selection
Methods
[0130] B cells were immortalized and cultured as described before
(Scheeren F A, et al. (2005) Nat Immunol 6: 303-313; Diehl S A, et
al. (2008) J Immunol 180: 4805-4815 and Kwakkenbos M J, et al.
(2009) Nat Med in press). In brief we isolated B cells from
peripheral blood (Sanquin, Amsterdam, The Netherlands) by Ficoll
separation, CD22 MACS microbeads (Miltenyi Biotech) and
subsequently cell sorting for
CD19.sup.+CD3.sup.-CD27.sup.+IgM.sup.-IgA.sup.- (IgG memory cells)
on a FACSAria (Becton Dickinson). The use of these tissues was
approved by the medical ethical committees of the institution and
was contingent on informed consent. Retroviral transduced B cells,
were maintained at 2.times.10.sup.5 cells ml.sup.-1 in IMDM
supplemented with recombinant mouse IL-21 (25 ng ml.sup.-1, R&D
systems) and co-cultured with .gamma.-irradiated (50Gy) mouse L
cell fibroblasts stably expressing CD40L (CD40L-L cells, 10.sup.5
cells ml.sup.-1) for 30 hours. The BCL6 and Bcl-xL retroviral
constructs were described previously (Shvarts A, et al. (2002)
Genes Dev 16: 681-686 and Jaleco A C, et al. (1999) Blood 94:
2637-2646)) and were cloned into the LZRS retroviral vector and
transfected in Phoenix packaging cells as described before and
added to the stimulated B cells (Shvarts A, et al (2002) Genes Dev
16; 681-686 and Scheeren F A, et al. (2005) Nat Immunol 6:
303-313). Transduced B cells were maintained in IMDM, in the
presence of recombinant IL-21 and CD40L-L cells for prolonged
periods of time. Given the relatively high amounts of secreted
antibodies by BCL6+Bcl-xL transduced B cells we examined whether we
could select antigen-specific B cells on the basis of secretion of
specific antibody. BCL6+Bcl-xL transduced memory B cells of a
healthy donor were seeded at 100 cells/well and expanded with
CD40L-L cells and IL-21. After 2 weeks of culture, supernatants
were harvested and screened for the presence of RSV-neutralizing
antibodies in a microneutralization experiment. Of 384 cultures
(100 cell s/well), 31 prevented RSV A2 infection, of HEp2 cells.
Besides the four microcultures from which D25, AM14, AM16, and AM23
were subcloned by limiting dilution, we next obtained AM22. AM22
has a median half maximum inhibitory concentrations (IC.sub.50)
against the RSV-A2 virus of 1.15 ng ml.sup.-1 (FIG. 1).
[0131] To obtain the sequence of the heavy and Light, chain of the
immunoglobulin locus of AM22, we isolated total RNA with the
RNeasy.RTM. mini kit (Qiagen), generated cDNA, performed PCR and
cloned the heavy and light chain variable regions into the pCR2.1
TA cloning vector (Invitrogen), To rule out reverse transcriptase
or DNA polymerase induced mutations, we performed several
independent cloning experiments. To produce recombinant AM22 mAb we
cloned AM22 heavy and light variable regions in frame with human
IgG1 and Kappa constant regions into a pcDNA3.1 (Invitrogen) based
vector and transiently transfected 293T cells. We purified
recombinant AM22 from the culture supernatant with Protein A.
Results
[0132] Previously we developed four potent, conformational
dependent anti-RSV antibodies, named AM14, AM16, AM23 and D25.
These antibodies have been described in WO 2008/147196 and by
Kwakkenbos M J et al. (2009) Nat Med in press. From the same donor
we also discovered AM22, which recognized the RSV virus even more
efficiently compared to the other antibodies as is evident from an
IC.sub.50 of 1.15 ng ml.sup.-1 against the RSV A2 virus (table 1
and FIG. 1). The amino acid sequence of the VH and VL chain of AM22
revealed that this antibody is different from the other antibodies
(FIG. 2).
Example 2. In Vitro Binding Experiments
[0133] To further determine the antigen specificity of the AM22
antibody we performed in vitro binding experiments to reveal
whether the protein recognized the RSV F or G protein and in what
type of conformation.
Methods
(1) FACS Staining of RSV G or F Protein
[0134] Virus stock of wild type and recombinant Vesicular
Stomatitis Virus (VSV) expressing RSV-G protein (VSV-G) or RSV-F
protein (VSV-F) (experiments were performed by M. Lukens at WKZ,
Utrecht and VSV viruses were kindly provided by J. S. Kahn and J. K
Rose, Yale University School, of Medicine) were prepared on BHK
cells grown in DMEM containing 5% FCS, penicillin/streptomycin and
50 .mu.M 2-mercapto-ethanol. VSV infection was performed on EL-4
cells that were cultured in Iscove's Modified Dulbecco's medium
(IMDM, Gibco. Invitrogen) supplemented with 5% FCS,
penicillin/streptomycin and 50 .mu.M 2-mercapto-ethanol. EL-4 cells
infected with the VSV virus variants were incubated with
recombinant antibody and subsequently stained with mouse-anti-human
PE (FIG. 3A).
(2) RSV ELISA
[0135] Plates were coated with lysate of RSV infected HEp-2 cells
in PBS for 1 hour at 37.degree. C. or o/n at 4.degree. C. and
washed in ELISA wash buffer (PBS, 0.5% Tween-20). Plates were
blocked by incubation with 4% milk in PBS, before the anti RSV
antibodies or polyclonal goat anti-RSV (Biodesign) in combination
with enzyme-conjugated anti-IgG antibodies were added (dilutions
1:2500 for HRP-conjugated anti-IgG (Jackson). TMB substrate/stop
solution (Biosource) was used for development of the ELISAs (FIG.
3B left panel). In addition to the lysate of RSV-A2 infected HEp-2
cells, HIS-tagged F protein from RSV Long strain (kindly provided
by Frank Coenjaerts, UMCU, Utrecht based on Ternette N, et al.
(2007) Vaccine 25: 7271-7279) was used to coat Ni-NTA HisSorp
Plates (Qiagen) (FIG. 3B right panel). The binding of the RSV F
antibodies was detected with MRP-conjugated IgG detection antibody
(dilutions 1:2500, Jackson). In another setting the original B cell
clones were used for FACS analysis (FIG. 3C). The B cells were
incubated with the recombinant HIS tagged F protein and binding to
the BCR was detected with a labeled anti-penta HIS antibody ALEXA
fluor 647 (Qiagen).
(3) RSV Trimers
[0136] In addition to the recombinant RSV long strain derived F
protein we created RSV A2 F trimers by inserting the open reading
frame of F into a construct, which is fused to an isoleucine zipper
domain followed by 8 HIS repeats (ILZ-8.times.HIS), Protein
constructs were transiently expressed in 293T cells and detected by
using an intracellular staining protocol using the Fix Perm kit of
BD (FIG. 3D).
Results
[0137] All antibodies recognized the RSV-F protein when expressed
by recombinant VSV (FIG. 3A). Furthermore, except for AM16,
recognition was dependent on the presence of conformational
epitopes in the RSV-F protein since they did not recognize a lysate
of RSV infected HEp-2 cells (FIG. 3B left panel). Also purified
HIS-tagged recombinant RSV-F Long strain protein was not recognized
by D25, AM14, AM22 and AM23 when tested in a direct ELISA (FIG. 3B
right panel). However when the original stable BCR expressing B
cell lines were incubated with this non-purified culture
supernatant of the HIS tagged RSV-F protein we did observe binding
to the B cell clones AM16, AM23 and less strong to D25 (FIG. 3C).
The protein in this non purified culture supernatant possibly
contains a fraction of RSV F trimers that do bind the BCR but are
only captured as monomers on the HisSorp Plates. However still no
RSV-F protein capture was observed to the BCRs of the AM14 and the
AM22 B cell lines (FIG. 3C). Thus AMU and AM22 bind to different
epitopes. Possibly a low percentage of protein F homotrimers or
dimers was present in the untreated culture supernatant of the F
protein producing cell line. These more native F conformations may
express the epitopes recognized by AM23 and D25, which were lost in
the denaturizing conditions of the ELISA procedures. Interestingly
when we performed an intracellular staining on 293T cells
transfected with the RSV trimerization construct containing the
ILZ-8.times.HIS sequence we found that, AM22 did recognize RSV F
(FIG. 3D), however AM14 still did not recognize this protein
structure. Thus AM22 is unique since it recognizes a conformation
of the RSV-F protein that is not present in denaturing conditions
and thus is not present in the monomeric form of the protein. Only
when the RSV F proteins are forced to stay together and form
trimers then we can detect F binding by the AM22 antibody, a
conformation that is present on virus particles and thus explain
the high neutralizing potency of AM22.
Example 3. In Vivo Potency Experiments
[0138] To study the in vivo potency of the AM22 monoclonal antibody
we performed Cotton rat experiments.
Methods
[0139] Pathogen free 7-9 week-old Cotton rats (Sigmodon hispidus,
Harlan Laboratories, Nederland) were anesthetized with isoflurane
and given 0.1 ml of purified antibody by intramuscular (i.m.)
injection at doses of 2.0 or 0.4 mg kg.sup.-1 for the control
antibody, palivizuinab, AM22, AM23 and D25, while AM14 was
administered at 0.4 and 0.1 mg kg.sup.-1. Twenty-four hours later,
animals were anesthetized, bled for serum hIgG determination and
challenged by intranasal instillation of 10.sup.6 TCID.sub.50 RSV-X
(100 .mu.l). Five days later animals were sacrificed and their
lungs were harvested. Lung virus titers were determined and the
lowest limit of detection was 2.1 log.sub.10 TCID.sub.50 g.sup.-1.
The Animal Experiment Committee of the Netherlands Vaccine
Institute approved all procedures involving Cotton rats.
Results
[0140] The anti-RSV antibody panel, except AM16, was tested in
Cotton rats. Animals were prophylactically treated with 2.0 or 0.4
mg kg.sup.-1 of monoclonal antibodies before RSV-X, a primary RSV A
isolate, was intra-nasally administered. Due to relatively low
antibody production, the AM14 antibody was administered at 0.4 and
0.1 mg kg.sup.-1. The level of recovered human IgG from Cotton rat
sera at day 1 (day of virus inoculation) and day 5 (day of
sacrifice) was in the same range for all antibodies and the decline
of antibodies in time was comparable (FIG. 4A).
[0141] The retrieval of RSV virus from the lungs of sacrificed
animals was strongly reduced in all animal groups treated with 2.0
mg/kg of immunoglobulin compared to the control group (FIG. 4B).
Animals treated with 0.4 mg kg.sup.-1 palivizumab and AM23 showed
significant virus replication, while in the AM14 and D25 groups one
out of 6 animals showed detectable virus replication. No virus
could be retrieved from animals treated with AM22. These results
demonstrate that AM22, that specifically recognized conformational
epitopes on the RSV F protein, harbors strong in vivo neutralizing
capacities.
Analysis of Lung Pathology of RSV Challenged Cotton Rats
[0142] From each Cotton rat at day 5 after i.n. RSV infection the
left lung was removed and fixed with formalin. Lung damage was
classified between 0-6 for three individual markers: 1) hypertrophy
of bronchus and bronchioli epithelia, 2) inflammation surrounding
bronchus and bronchioli (peribronchiolitis) and 3) inflammation in
the alveoli (alveolitis). The average sum of the scores of all
animals in one group created the pathology index (maximum score 15)
(FIG. 4c, table 2). Lung pathology after RSV infection was
significantly reduced in animal groups treated with high doses of
immunoglobulin (2 mg kg.sup.-1)(table 3). However only in the AM14,
AM22 and AM23 groups the pathology was significantly reduced at
lower concentrations (0.4 mg kg.sup.-1). While a complete absence
of pathology was seen in 3 out of 5 animals treated with AM22 and
AM23 at 2 mg kg.sup.-1, at 0.4 mg kg.sup.-1 complete protection was
detected in 2 or 1 out of 5 animals in the AM14 and AM22 groups
respectively (table 3).
[0143] When combining the results of examples 1, 2 and 3 it is
concluded that AM22 has certain advantages. AM22 demonstrates the
lowest IC.sub.50 value, meaning that with a lower amount of AM22
similar prophylactic or therapeutic effects are achieved as
compared with other antibodies. AM22 recognizes a different epitope
of the RSV F protein than the other antibodies and therefore can be
used in combination with one of the other antibodies to achieve a
stronger immunogenic response to RSV and higher antibody
specificity against RSV. Finally, treatment of Cotton rats with
AM22 resulted in nearly complete inhibition of virus replication
with potential complete absence of pathology in these Cotton
rats.
Example 4: Affinity of AM22 for the RSV F Protein
[0144] Determining the affinity constant and specificity of binding
between the RSV F protein and AM22 is preferred to establish the
prophylactic, therapeutic and diagnostic value of the antibody.
This is a challenging feature since the antibody affinity has to be
determined for an oligomeric protein structure. Usually affinity
constants are determined against immobilized agents that are
captured on a chip. However protein F captured on a chip would not
be recognized by the AM14, AM22, AM23 and D25 antibodies.
Method
[0145] "Binding affinity" generally refers to the strength of the
sum total of the noncovalent interactions between a single binding
site of a molecule (e.g., an antibody) and its binding partner
(e.g., an antigen). The affinity of a molecule X for its partner Y
can generally be represented by the equilibrium dissociation
constant (Kd), which is calculated as the ratio k.sub.off/k.sub.on.
Affinity can be measured by common methods known in the art, such
as a surface plasmon resonance (SPR) assay. Affinities (KD),
on-rates (ka) and off-rates (kd) will be measured by SPR analysis
with the IBIS-iSPR instrument at IBIS Technologies BV (Hengelo, the
Netherlands). Briefly, anti-RSV antibodies are immobilized and
purified RSV F protein (containing penta-HIS) is diluted and rate
and affinity constants are measured by injection of at least three
serial dilutions of the protein.
[0146] Another set up in the IMIS-iSPR machine is to immobilize 1)
an anti-penta HIS antibody on which the F-penta HIS protein is
coupled or 2) F-penta HIS protein is directly immobilized on the
chip and then samples on the chip are incubated with the AIMM
antibodies to determine the affinity constants.
TABLE-US-00001 TABLE 1 RSV neutralizing activity of purified IgGs.
RSV A2.sup.a palivizumab 152 D25 1.28 AM14 2.09 AM16 78.7 AM22 1.15
AM23 4.18
[0147] The IC.sub.50 (ng/ml) values of the selected anti-RSV IgGs
were determined with standard TCID.sub.50 culture assay on HEp2
cells with the RSV A2 virus.
TABLE-US-00002 TABLE 2 Cumulative pathology score in Cotton rats.
Pathology score antibody mg/kg (SEM) P values Palivizumab 2.0 3.20
(0.4) 0.0005 Palivizumab 0.4 5.40 (0.8) <0.05 D25 2.0 3.40 (0.4)
0.0005 D25 0.4 5.83 (1.0) 0.05 AM14 0.4 4.20 (0.8) 0.05 AM14 0.1
3.67 (1.2) 0.05 AM22 2.0 2.75 (0.8) 0.0005 AM22 0.4 4.00 (0.9)
<0.05 AM23 2.0 2.40 (0.2) 0.0005 AM23 0.4 3.50 (0.9) <0.05
Ctrl IgG1 2.0 8.80 (1.1) N.A. MOCK N.A. 1.80 (0.4) 0.0005
[0148] Cumulative pathology score of the lungs of Cotton rats,
treated 24 hour before RSV infection with the indicated antibodies.
Lung specimens obtained Ft days after infection were evaluated by a
pathologist randomly. Lungpathology was classified with scores
between 0-5 for three individual markers: 1) hyperthropy of
bronchus and bronchioli epithelia. 2) peribronchiolitis and 3)
alveolitis. Average pathology score (maximum score 15) with
standard error of the mean SEM) and statistical differences in
relation to Ctrl IgG1 group was calculated with the 2-sided
Wilcoxon test. Experiments were performed twice with four to six
individual animals per treatment group. N.A. not applicable.
TABLE-US-00003 TABLE 3 Prevention of pathology in Cotton rats.
Significant reduction antibody mg/kg in pathology No pathology
Palivizumab 2 5/5 1/5 Palivizumab 0.4 3/5 0/6 D25 2 5/5 0/5 D25 0.4
2/6 0/6 AM14 0.4 5/6 2/6 AM14 0.1 4/5 0/5 AM22 2 5/5 3/5 AM22 0.4
5/6 1/6 AM23 2 5/5 3/5 AM23 0.4 5/6 0/6
[0149] Number of animals with significant reduction in/or absence
of lung pathology at day 5 during RSV-X infection. Experiments were
performed twice with four to six individual animals per treatment
group.
Sequence CWU 1
1
32190DNAArtificialanti-RSV antibody heavy chain Fw1CDS(1)..(90)
1cag gtc cag ctg gta cag tct ggg gct gag gtg aag aag ccc ggg gcc
48Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15aca gtg aaa gtc tcc tgc aag att tcc gga cac acc ctc att
90Thr Val Lys Val Ser Cys Lys Ile Ser Gly His Thr Leu Ile 20 25
30230PRTArtificialSynthetic Construct 2Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Thr Val Lys Val Ser Cys
Lys Ile Ser Gly His Thr Leu Ile 20 25 30315DNAArtificialanti-RSC
antibody heavy chain CDR1CDS(1)..(15) 3aaa tta tcc att cac 15Lys
Leu Ser Ile His1 545PRTArtificialSynthetic Construct 4Lys Leu Ser
Ile His1 5542DNAArtificialanti-RSV antibody heavy chain
Fw2CDS(1)..(42) 5tgg gtg cga cag gct cct gga aag ggg ctt gag tgg
atg gga 42Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met Gly1
5 10614PRTArtificialSynthetic Construct 6Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Met Gly1 5 10751DNAArtificialanti-RSV
antibody heavy chain CDR2CDS(1)..(51) 7ggt tat gag ggt gag gtc gat
gag att ttc tac gca cag aag ttc cag 48Gly Tyr Glu Gly Glu Val Asp
Glu Ile Phe Tyr Ala Gln Lys Phe Gln1 5 10 15cac
51His817PRTArtificialSynthetic Construct 8Gly Tyr Glu Gly Glu Val
Asp Glu Ile Phe Tyr Ala Gln Lys Phe Gln1 5 10
15His996DNAArtificialanti-RSV antibody heavy chain Fw3CDS(1)..(96)
9aga ctc acc gtg atc gcc gac aca gcg aca gac aca gtc tac atg gaa
48Arg Leu Thr Val Ile Ala Asp Thr Ala Thr Asp Thr Val Tyr Met Glu1
5 10 15ctg ggc agg ctc acc tct gac gac acg gcc gtc tat ttc tgt gga
aca 96Leu Gly Arg Leu Thr Ser Asp Asp Thr Ala Val Tyr Phe Cys Gly
Thr 20 25 301032PRTArtificialSynthetic Construct 10Arg Leu Thr Val
Ile Ala Asp Thr Ala Thr Asp Thr Val Tyr Met Glu1 5 10 15Leu Gly Arg
Leu Thr Ser Asp Asp Thr Ala Val Tyr Phe Cys Gly Thr 20 25
301145DNAArtificialanti-RSV antibody heavy chain CDR3CDS(1)..(45)
11cta ggt gtg aca gtg act gag gct gga ctg ggg atc gat gac tac 45Leu
Gly Val Thr Val Thr Glu Ala Gly Leu Gly Ile Asp Asp Tyr1 5 10
151215PRTArtificialSynthetic Construct 12Leu Gly Val Thr Val Thr
Glu Ala Gly Leu Gly Ile Asp Asp Tyr1 5 10
151333DNAArtificialanti-RSV antibody heavy chain Fw4CDS(1)..(33)
13tgg ggc cag gga acc ctg gtc acc gtc tcc tca 33Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser1 5 101411PRTArtificialSynthetic Construct
14Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser1 5
1015372DNAArtificialAnti-RSV antibody heavy chainCDS(1)..(372)
15cag gtc cag ctg gta cag tct ggg gct gag gtg aag aag ccc ggg gcc
48Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15aca gtg aaa gtc tcc tgc aag att tcc gga cac acc ctc att aaa
tta 96Thr Val Lys Val Ser Cys Lys Ile Ser Gly His Thr Leu Ile Lys
Leu 20 25 30tcc att cac tgg gtg cga cag gct cct gga aag ggg ctt gag
tgg atg 144Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Met 35 40 45gga ggt tat gag ggt gag gtc gat gag att ttc tac gca
cag aag ttc 192Gly Gly Tyr Glu Gly Glu Val Asp Glu Ile Phe Tyr Ala
Gln Lys Phe 50 55 60cag cac aga ctc acc gtg atc gcc gac aca gcg aca
gac aca gtc tac 240Gln His Arg Leu Thr Val Ile Ala Asp Thr Ala Thr
Asp Thr Val Tyr65 70 75 80atg gaa ctg ggc agg ctc acc tct gac gac
acg gcc gtc tat ttc tgt 288Met Glu Leu Gly Arg Leu Thr Ser Asp Asp
Thr Ala Val Tyr Phe Cys 85 90 95gga aca cta ggt gtg aca gtg act gag
gct gga ctg ggg atc gat gac 336Gly Thr Leu Gly Val Thr Val Thr Glu
Ala Gly Leu Gly Ile Asp Asp 100 105 110tac tgg ggc cag gga acc ctg
gtc acc gtc tcc tca 372Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 12016124PRTArtificialSynthetic Construct 16Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Thr Val Lys
Val Ser Cys Lys Ile Ser Gly His Thr Leu Ile Lys Leu 20 25 30Ser Ile
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly
Gly Tyr Glu Gly Glu Val Asp Glu Ile Phe Tyr Ala Gln Lys Phe 50 55
60Gln His Arg Leu Thr Val Ile Ala Asp Thr Ala Thr Asp Thr Val Tyr65
70 75 80Met Glu Leu Gly Arg Leu Thr Ser Asp Asp Thr Ala Val Tyr Phe
Cys 85 90 95Gly Thr Leu Gly Val Thr Val Thr Glu Ala Gly Leu Gly Ile
Asp Asp 100 105 110Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 1201769DNAArtificialanti-RSV antibody light chain
Fw1CDS(1)..(69) 17gaa att gtg ttg aca cag tct cca ggc acc ctg tct
ttg tct cca gga 48Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser
Leu Ser Pro Gly1 5 10 15gaa aga gcc acc ctc tcc tgc 69Glu Arg Ala
Thr Leu Ser Cys 201823PRTArtificialSynthetic Construct 18Glu Ile
Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu
Arg Ala Thr Leu Ser Cys 201936DNAArtificialAnti-RSV antibody light
chain CDR1CDS(1)..(36) 19agg gcc agt cag att gtt agc agg aac cac
tta gcc 36Arg Ala Ser Gln Ile Val Ser Arg Asn His Leu Ala1 5
102012PRTArtificialSynthetic Construct 20Arg Ala Ser Gln Ile Val
Ser Arg Asn His Leu Ala1 5 102145DNAArtificialanti-RSV antibody
light chain Fw2CDS(1)..(45) 21tgg tac cag caa aaa cct ggc cag gct
ccc agg ctc ctc atc ttt 45Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Arg Leu Leu Ile Phe1 5 10 152215PRTArtificialSynthetic Construct
22Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Phe1 5 10
152321DNAArtificialanti-RSV antibody light chain CDR2CDS(1)..(21)
23ggt gcg tcc agt cgg gcc act 21Gly Ala Ser Ser Arg Ala Thr1
5247PRTArtificialSynthetic Construct 24Gly Ala Ser Ser Arg Ala Thr1
52596DNAArtificialAnti-RSV antibody light chain Fw3CDS(1)..(96)
25ggc atc cca gtc cgg ttc agt ggc agt ggg tct ggg aca gac ttc act
48Gly Ile Pro Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr1
5 10 15ctc acc atc aac gga ctg gcg cct gaa gat ttt gca gtt tac tac
tgt 96Leu Thr Ile Asn Gly Leu Ala Pro Glu Asp Phe Ala Val Tyr Tyr
Cys 20 25 302632PRTArtificialSynthetic Construct 26Gly Ile Pro Val
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr1 5 10 15Leu Thr Ile
Asn Gly Leu Ala Pro Glu Asp Phe Ala Val Tyr Tyr Cys 20 25
302721DNAArtificialAnti-RSV antibody light chain CDR3CDS(1)..(21)
27ctg tcc tct gat tcc tcc ata 21Leu Ser Ser Asp Ser Ser Ile1
5287PRTArtificialSynthetic Construct 28Leu Ser Ser Asp Ser Ser Ile1
52936DNAArtificialAnti-RSV antibody light chain Fw4CDS(1)..(36)
29ttc aca ttc ggc cct ggg acc aag gtg gat ttc aaa 36Phe Thr Phe Gly
Pro Gly Thr Lys Val Asp Phe Lys1 5 103012PRTArtificialSynthetic
Construct 30Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Phe Lys1 5
1031324DNAArtificialAnti-RSV antibody light chainCDS(1)..(324)
31gaa att gtg ttg aca cag tct cca ggc acc ctg tct ttg tct cca gga
48Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1
5 10 15gaa aga gcc acc ctc tcc tgc agg gcc agt cag att gtt agc agg
aac 96Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ile Val Ser Arg
Asn 20 25 30cac tta gcc tgg tac cag caa aaa cct ggc cag gct ccc agg
ctc ctc 144His Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu 35 40 45atc ttt ggt gcg tcc agt cgg gcc act ggc atc cca gtc
cgg ttc agt 192Ile Phe Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Val
Arg Phe Ser 50 55 60ggc agt ggg tct ggg aca gac ttc act ctc acc atc
aac gga ctg gcg 240Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Asn Gly Leu Ala65 70 75 80cct gaa gat ttt gca gtt tac tac tgt ctg
tcc tct gat tcc tcc ata 288Pro Glu Asp Phe Ala Val Tyr Tyr Cys Leu
Ser Ser Asp Ser Ser Ile 85 90 95ttc aca ttc ggc cct ggg acc aag gtg
gat ttc aaa 324Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Phe Lys 100
10532108PRTArtificialSynthetic Construct 32Glu Ile Val Leu Thr Gln
Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ile Val Ser Arg Asn 20 25 30His Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Phe Gly
Ala Ser Ser Arg Ala Thr Gly Ile Pro Val Arg Phe Ser 50 55 60Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Gly Leu Ala65 70 75
80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Leu Ser Ser Asp Ser Ser Ile
85 90 95Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Phe Lys 100 105
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