Note: Descriptions are shown in the official language in which they were submitted.
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BINDING MOLECULES CAPABLE OF NEUTRALIZING RABIES VIRUS AND USES
THEREOF
This application is a divisional application of co-pending
application Serial No. 2,568,162, filed May 26, 2005.
FIELD OF THE INVENTION
The invention relates to medicine. In particular the
invention relates to rabies virus neutralizing binding
molecules. The binding molecules are useful in the
postexposure prophylaxis of rabies.
BACKGROUND OF THE INVENTION
Rabies is a viral infection with nearly worldwide
distribution that affects principally wild and domestic
animals but also involves humans, resulting in a devastating,
almost invariable fatal encephalitis. Annually, more than
70,000 human fatalities are estimated, and millions of others
require postexposure treatment.
The rabies virus is a bullet-shaped, enveloped, single-
stranded RNA virus classified in the rhabdovirus family and
Lyssavirus genus. The genome of rabies virus codes for five
viral proteins: RNA-dependent RNA polymerase (L); a
nucleoprotein (N); a phosphorylated protein (P); a matrix
protein (M) located on the inner side of the viral protein
envelope; and an external surface glycoprotein (G).
The G protein (62-67 kDa) is a type-I glycoprotein
composed of 505 amino acids that has two to four potential N-
glycosylation sites, of which only one or two are glycosylated
depending on the virus strains. The G protein forms the
protrusions that cover the outer surface of the virion
envelope and is known to induce virus-neutralizing antibodies.
Rabies can be treated or prevented by both passive and
active immunizations. Rabies postexposure prophylaxis includes
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prompt local wound care and administration of both passive
(anti-rabies immunoglobulins) and active (vaccines)
immunizations.
Currently, the anti-rabies immunoglobulins (RIG) are
prepared from the serum samples of either rabies virus-immune
humans (HRIG) or rabies virus-immune horses (ERIG). A
disadvantage of ERIG as well as HRIG is that they are not
available in sufficient amounts and, in case of HRIG, are too
expensive. In addition, the use of ERIG might lead to adverse
reactions such as anaphylactic shock. The possibility of
contamination by known or unknown pathogens is an additional
concern associated with HRIG. To overcome these disadvantages
it has been suggested to use monoclonal antibodies capable of
neutralizing rabies virus in postexposure prophylaxis: Rabies-
virus neutralizing murine monoclonal antibodies are known in
the art (see Schumacher et al., 1989). However, the use of
murine antibodies in vivo is limited due to problems
associated with administration of..murine antibodies to humans,
such as short serum half life, an inability to trigger certain
human effector functions and elicitation of an unwanted
dramatic immune response against the murine antibody in a
human (the "human anti-mouse antibody" (HAMA) reaction).
Recently, human rabies virus-neutralizing monoclonal
antibodies have been described (see Dietzschold et al., 1990,
Champion et al., 2000, and Hanlon et al., 2001). For human
anti-rabies monoclonal antibodies to be as effective as HRIG
in postexposure prophylaxis a mixture of monoclonal antibodies
should be used. In such a mixture each antibody should bind to
a different epitope or site on the virus to prevent the escape
of resistant variants of the virus.
Currently, there is still a significant need for new
human rabies virus-neutralizing monoclonal antibodies having
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improved postexposure prophylactic potential, particularly
antibodies having different epitope-recognition specificities.
The present invention provides such human monoclonal
antibodies that offer the potential to be used in mixtures
useful in the postexposure prophylaxis of a wide range of
rabies viruses and neutralization-resistant variants thereof.
SUMMARY
There is provided herein an isolated binding molecule having
rabies virus neutralizing activity, wherein the binding
molecule comprises: a heavy chain variable region comprising
the amino acid sequence of SEQ ID NO:39 or a sequence that is
at least 80% homologous thereto, and wherein the binding
molecule comprises a light chain variable region comprising
the amino acid sequence of SEQ ID NO:63 or a sequence that is
at least 80% homologous thereto.
There is provided herein a vector, and a host cell comprising
this isolated binding molecule.
There is provided a pharmaceutical composition comprising the
above-noted binding molecule, together with at least one
pharmaceutically acceptable excipient, comprising at least one
additional binding molecule capable of reacting with a
different, non-competing epitope of the rabies virus.
Further, there is described herein use of the above-noted
binding molecule for diagnosing, preventing, treating, or a
combination thereof, exposure to or infection by a rabies
virus in a subject.
Additionally, there is described an isolated binding molecule
having rabies virus neutralizing activity, the binding
molecule comprising: a heavy chain variable region comprising
SEQ ID NO:39, and a light chain variable region comprising SEQ
ID NO:63. Further, there is provided herein a vector, and a
host cell comprising this isolated binding molecule.
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3a
There is provided an isolated antibody having rabies virus
neutralizing activity, wherein the antibody comprises: a heavy
chain variable region comprising the CDR1, CDR2 and CDR3
regions of SEQ ID NO:39, and a light chain variable region
comprising the CDR1, CDR2 and CDR3 regions of SEQ ID NO:63.
Further, there is provided use of the above-noted
pharmaceutical composition for treating or preventing
conditions resulting from exposure to rabies virus in a
subject that has been exposed or potentially exposed to a
rabies virus.
Additionally, there is provided an isolated binding molecule
having rabies virus neutralizing activity, wherein the binding
molecule comprises: a heavy chain variable region comprising
the amino acid sequence of SEQ ID NO:39 or a sequence having
at least 90% sequence identity with SEQ ID NO:39, and a light
chain variable region comprising the amino acid sequence of
SEQ ID NO:63 or a sequence having at least 90% sequence
identity with SEQ ID NO:63.
Further, there is provided an isolated human binding molecule
having rabies virus neutralizing activity, wherein the binding
molecule comprises: a heavy chain variable region comprising
the amino acid sequence of SEQ ID NO:39 or a sequence having
at least 80% sequence identity with SEQ ID NO:39, and a light
chain variable region comprising the amino acid sequence of
SEQ ID NO:63 or a sequence having at least 80% sequence
identity with SEQ ID NO:63, and wherein the antibody does not
have neutralizing activity against a rabies virus with
mutation asparagine (N) to aspartic acid (D) at amino acid
position 336 of the mature rabies virus glycoprotein.
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3b
DESCRIPTION OF THE FIGURES
Figure 1 shows the comparison of the amino acid sequences of
the rabies virus strain CVS-11 and E57 escape viruses. Virus-
infected cells were harvested 2 days post-infection and total
RNA was isolated. cDNA was generated and used for DNA
sequencing. Regions containing mutations are shown and the
mutations are indicated in bold. Figure 1A shows the
comparison of the nucleotide sequences. Numbers above amino
acids indicate amino acids numbers from rabies virus
glycoprotein including signal peptide. Figure 1B shows the
comparison of amino acid sequences. Schematic drawing of
rabies virus glycoprotein is shown on top. The black box
indicates the signal peptide, while the gray box indicates the
transmembrane domain. The sequences in Figure 1 are also
represented by SEQ ID Nos:130-141.
Figure 2 shows the comparison of the amino acid sequences of
the rabies virus strain CVS-11 and EJB escape viruses. Virus-
infected cells were harvested 2 days post-infection and total
RNA was isolated. cDNA was generated and used for DNA
sequencing. Regions containing mutations are shown and the
mutations are indicated in bold. Figure 2A shows the
comparison of the nucleotide sequences. Numbers above amino
acids indicate amino acid numbers from rabies virus
glycoprotein including the signal peptide. Figure 2B shows the
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comparison of amino acid sequences. Schematic drawing of
rabies virus glycoprotein is shown on top. The black box
indicates the signal peptide, while the gray box indicates the
transmembrane domain. The sequences in Figure 2 are also
represented by SEQ ID Nos:142-151.
Figure 3 shows the vector PDV-C06.
Figure 4 shows a competition ELISA of anti-rabies virus scFvs
and the biotinylated anti-rabies virus antibody called CR-57.
ELISA plates coated with purified rabies virus'G protein were
incubated with the respective scFvs before addition of CR-
57bio (0.5 gg/ml). Subsequently, CR-57bio binding was monitored
in absence and presence of scFvs.
Figure 5 shows a competition ELISA of anti-rabies virus scFvs
and the anti-rabies virus antibody called CR-57. ELISA plates
.Y coated with purified rabies virus G protein were incubated
with CR-57 (1 gg/ml) before addition of excess scFvs.
Subsequently, scFv binding was monitored in absence and
presence of CR-57.
Figure 6 shows a competition ELISA assay of anti-rabies virus
G protein IgGs and the anti-rabies virus antibody called CR-
57. G protein (ERA strain) was incubated with unlabeled IgGs
(shown on the X-axis). Biotinylated CR57 (CR57bio) was added
and allowed to bind to the G protein before visualization by
means of streptavidin-HRP. ELISA signals are shown as
percentage of CR57bio binding alone.
Figure 7 shows a competition FACS assay of anti-rabies virus G
protein IgGs and the anti-rabies virus antibody called CR-57.
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G protein (ERA strain) expressing PER.C6 cells were incubated
with unlabeled IgGs (shown on the X-axis). Biotinylated CR57
(CR57bio) was added and allowed to bind to the G protein
expressing cells before visualization by means of
5 streptavidin-PE. FACS signals are shown as percentage of
CR57bio binding alone.
Figure 8 shows the comparison of the amino acid sequences of
CVS-11 and E98.escape viruses. Virus-infected cells were
harvested 2 days post-infection and total RNA was isolated.
cDNA was generated and used for DNA sequencing. Region
containing a point mutation is shown and the mutation is
indicated in bold. Figure 8A shows the comparison of the
nucleotide sequences. The number above the nucleotide
indicates the mutated nucleotide (indicated in bold) from
rabies virus glycoprotein open reading frame without signal
peptide sequence. Figure 8B shows the comparison of amino acid
sequences. The number above the amino acid indicates the
mutated amino acid (indicated in bold) from rabies virus
glycoprotein without signal peptide sequence.
Figure 9 shows a phylogenetic tree of 123 rabies street
viruses (123 rabies virus G glycoprotein sequences,
Neighbor joining, Kimura-2-parameter method, 500 bootstraps).
Bold indicates viruses harboring the N>D mutation as observed
in E98 escape viruses.
Figure 10 shows neutralizing epitopes on rabies glycoprotein.
A schematic drawing of the rabies virus glycoprotein is shown
depicting the antigenic sites including the novel CR57
epitope. The signal peptide (19 amino acids) and transmembrane
domain are indicated by black boxes. Disulfide bridges are
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indicated. Amino acid numbering is from the mature protein
minus the signal peptide sequence.
DESCRIPTION OF THE INVENTION
Herebelow follow definitions of terms as used in the
invention.
DEFINITIONS
Binding molecule
As used herein the term "binding molecule" refers to an intact
immunoglobulin including monoclonal antibodies, such as
chimeric, humanized or human monoclonal antibodies, or to an
antigen-binding and/or variable domain comprising fragment of
an immunoglobulin that competes with the intact immunoglobulin
for specific binding to the binding partner of the
immunoglobulin, e.g. rabies virus or a fragment thereof such
as for instance the G protein. Regardless of structure, the
antigen-binding fragment binds with the same antigen that is
recognised by the intact immunoglobulin. An antigen-binding
fragment can comprise a peptide or polypeptide comprising an
amino acid sequence of at least 2 contiguous amino acid
residues, at least 5 contiguous amino acid residues, at least
10 contiguous amino acid residues, at least 15 contiguous
amino acid residues, at least 20 contiguous amino acid
residues, at least 25 contiguous amino acid residues, at least
contiguous amino acid residues, at least 35 contiguous
amino acid residues, at least 40 contiguous amino acid
residues, at least 50 contiguous amino acid residues, at least
30 60 contiguous amino residues, at least 70 contiguous amino
acid residues, at least contiguous 80 amino acid residues, at
least contiguous 90 amino acid residues, at least contiguous
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100 amino acid residues, at least contiguous 125 amino acid
residues, at least 150 contiguous amino acid residues, at
least contiguous 175 amino acid residues, at least 200
contiguous amino acid residues, or at least contiguous 250
amino acid residues of the amino acid sequence of the binding
molecule.
The term "binding molecule", as used herein includes all
immunoglobulin classes and subclasses known in the art.
Depending on the amino acid sequence of the constant domain of
their heavy chains, binding molecules can be divided into the
five major classes of intact antibodies: IgA, IgD, IgE, IgG,
and IgM, and several of these may be further divided into
subclasses (isotypes), e.g., IgAl, IgA2, IgG1, IgG2, IgG3 and
IgG4.
Antigen-binding fragments include, inter alia, Fab,
F(ab'), F(ab')2, Fv, dAb, Fd, complementarity determining
region (CDR) fragments, single-chain antibodies (scFv),
bivalent single-chain antibodies, single-chain phage
antibodies, diabodies, triabodies, tetrabodies, (poly)peptides
that contain at least a fragment of an immunoglobulin that is
sufficient to confer specific antigen binding to the
(poly)peptide, etc. The above fragments may be produced
synthetically or by enzymatic or chemical cleavage of intact
immunoglobulins or they may be genetically engineered by
recombinant DNA techniques. The methods of production are well
known in the art and are described, for example, in
Antibodies: A Laboratory Manual, Edited by: E. Harlow and D,
Lane (1988), Cold Spring Harbor Laboratory, Cold Spring
Harbor, New York, which is incorporated herein by reference. A
binding molecule or antigen-binding fragment thereof may have
one or more binding sites. If there is more than one binding
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site, the binding sites may be identical to one another or
they may be different.
The binding molecule can be a naked or unconjugated
binding molecule but can also be part of an immunoconjugate. A
naked or unconjugated binding molecule is intended to refer to
a binding molecule that is not conjugated, operatively linked
or otherwise physically or functionally associated with an
effector moiety or tag, such as inter alia a toxic substance,
a radioactive substance, a liposome, an enzyme. It will be
understood that naked or unconjugated binding molecules do not
exclude binding molecules that have been stabilized,
multimerized, humanized or in any other way manipulated, other
than by the attachment of an effector moiety or tag.
Accordingly, all post-translationally modified naked and
unconjugated binding molecules are included herewith,
including where the, modifications are made in the natural
binding molecule-producing cell environment, by a recombinant
binding molecule-producing cell, and are introduced-,by the
hand of man after initial binding molecule preparation. Of
course, the term naked or unconjugated binding molecule does
not exclude the ability of the binding molecule to form
functional associations with effector cells and/or molecules
after administration to the body, as some of such interactions
are necessary in order to exert a biological effect. The lack
of associated effector group or tag is therefore applied in
definition to the naked or unconjugated binding molecule in
vitro, not in vivo.
Complementarity determining regions (CDR)
The term "complementarity determining regions" as used herein
means sequences within the variable regions of binding
molecules, such as immunoglobulins, that usually contribute to
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a large extent to the antigen binding site which is
complementary in shape and charge distribution to the epitope
recognized on the antigen. The CDR regions can be specific for
linear epitopes, discontinuous epitopes, or conformational
epitopes of proteins or protein fragments, either as present
on the protein in its native conformation or, in some cases,
as present on the proteins as denatured, e.g., by
solubilization in SDS. Epitopes may also consist of
posttranslational modifications of proteins.
Functional variant
The term "functional variant", as used herein, refers to a
binding molecule that comprises a nucleotide and/or amino acid
sequence that is altered by one or more nucleotides and/or
15' amino acids compared to the nucleotide and/or amino acid
sequences of the parent binding molecule and that is still
capable of competing for binding to the binding partner, e.g.
rabies virus or a fragment thereof, with the parent binding
molecule. In other words, the modifications in the amino acid
and/or nucleotide sequence of the parent binding molecule do
not significantly affect or alter the binding characteristics
of the binding molecule encoded by the nucleotide sequence or
containing the amino acid sequence, i.e. the binding molecule
is still able to recognize and bind its target. The functional
variant may have conservative sequence modifications including
nucleotide and amino acid substitutions, additions and
deletions. These modifications can be introduced by standard
techniques known in the art, such as site-directed mutagenesis
and random PCR-mediated mutagenesis, and may comprise natural
as well as non-natural nucleotides and amino acids.
Conservative amino acid substitutions include the ones in
which the amino acid residue is replaced with an amino acid
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residue having similar structural or chemical properties.
Families of amino acid residues having similar side chains
have been defined in the art. These families include amino
acids with basic side chains (e.g., lysine, arginine,
5 histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., asparagine,
glutamine, serine, threonine, tyrosine, cysteine, tryptophan),
nonpolar side chains (e.g., glycine, alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine), beta-branched
10 side chains (e.g., threonine, valine, isoleucine) and aromatic
side chains (e.g., tyrosine, phenylalanine, tryptophan). It
will be clear to the skilled artisan that other
classifications of amino acid residue families than the one
used above can also be employed. Furthermore, a variant may
have non-conservative amino acid substitutions, e.g.,
replacement of an amino acid with an amino acid residue having
different structural or chemical properties. Similar minor
variations,may also include amino acid deletions or
insertions, or both. Guidance in determining which amino acid
residues may be substituted, inserted, or deleted without
abolishing immunological activity may be found using computer
programs well known in the art.
A mutation in a nucleotide sequence can be a single
alteration made at a locus (a point mutation), such as
25, transition or transversion mutations, or alternatively,
multiple nucleotides may be inserted, deleted or changed at a
single locus. In addition, one or more alterations may be made
at any number of loci within a nucleotide sequence. The
mutations may be performed by any suitable method known in the
art.
Host
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The term "host", as used herein, is intended to refer to an
organism or a cell into which a vector such as a cloning
vector or an expression vector has been introduced. The
organism or cell can be prokaryotic or eukaryotic. It should
be understood that this term is intended to refer not only to
the particular subject organism or cell, but to the progeny of
such an organism or cell as well. Because certain
modifications may occur in succeeding generations due to
either mutation or environmental influences, such progeny may
not, in fact, be identical to the parent organism or cell, but
are still included within the scope of the term "host" as used
herein.
Human
The term "human", when applied to binding molecules as defined
herein, refers to molecules that are either directly derived
from a human or based upon a human sequence. When a binding
molecule is derived from or based on a human sequence and
subsequently modified, it is still to be considered human as
used throughout the specification. In other words, the term
human, when applied to binding molecules is intended to
include binding molecules having variable and constant regions
derived from human germline immunoglobulin sequences based on
variable or constant regions either or not occurring in a
human or human lymphocyte or in modified form. Thus, the human
binding molecules may include amino acid residues not encoded
by human germline immunoglobulin sequences, comprise
substitutions and/or deletions (e.g., mutations introduced by
for instance random or site-specific mutagenesis in vitro or
by somatic mutation in vivo). "Based on" as used herein refers
to the situation that a nucleic acid sequence may be exactly
copied from a template, or with minor mutations, such as by
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error-prone PCR methods, or synthetically made matching the
template exactly or with minor modifications. Semisynthetic
molecules based on human sequences are also considered to be
human as used herein.
Monoclonal antibody
The term "monoclonal antibody" as used herein refers to a
preparation of antibody molecules of single molecular
composition, i.e. primary structure, i.e. having a single
amino acid sequence. A monoclonal antibody displays a single
binding specificity and affinity for a particular epitope.
Accordingly, the term "human monoclonal antibody" refers to an
antibody displaying a single binding specificity which have
variable and constant regions derived from or based on human
germline immunoglobulin sequences or derived from completely
synthetic sequences. The method of preparing the monoclonal
antibody is not relevant.
Nucleic acid molecule
The term "nucleic acid molecule" as used in the present
invention refers to a polymeric form of nucleotides and
includes both sense and antisense strands of RNA, cDNA,
genomic DNA, and synthetic forms and mixed polymers of the
above. A nucleotide refers to a ribonucleotide,
deoxynucleotide or a modified form of either type of
nucleotide.. The term also includes single- and double-stranded
forms of DNA. In addition, a polynucleotide may include either
or both naturally-occurring and modified nucleotides linked
together by naturally-occurring and/or non-naturally occurring
nucleotide linkages. The nucleic acid molecules may be
modified chemically or biochemically or may contain non-
natural or derivatized nucleotide bases, as will be readily
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appreciated by those of skill in the art. Such modifications
include, for example, labels, methylation, substitution of one
or more of the naturally occurring nucleotides with an analog,
internucleotide modifications such as uncharged linkages
(e.g., methyl phosphonates, phosphotriesters,
phosphoramidates, carbamates, etc.), charged linkages (e.g.,
phosphorothioates, phosphorodithioates, etc.), pendent
moieties (e.g., polypeptides), intercalators (e.g., acridine,
psoralen, etc.), chelators, alkylators, and modified linkages
(e.g., alpha anomeric nucleic acids, etc.). The above term is
also intended to include any topological conformation,
including single-stranded, double-stranded, partially
duplexed, triplex, hairpinned, circular and padlocked
conformations. Also included are synthetic molecules that
mimic polynucleotides in their ability to bind to a designated
sequence via hydrogen bonding and other chemical interactions.
Such molecules are known in the art and include, for example,
those in which peptide linkages substitute for phosphate
linkages in the backbone of the molecule. A reference to a
nucleic acid sequence encompasses its complement unless
otherwise specified. Thus, a reference to a nucleic acid
molecule having a particular sequence should be understood to
encompass its complementary strand, with its complementary
sequence. The complementary strand is also useful, e.g., for
antisense therapy, hybridization probes and PCR primers.
Pharmaceutically acceptable excipient
By "pharmaceutically acceptable excipient" is meant any inert
substance that is combined with an active molecule such as a
drug, agent, or binding molecule for preparing an agreeable or
convenient dosage form. The "pharmaceutically acceptable
excipient" is an excipient that is non-toxic, or at least of
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which the toxicity is acceptable for its intended use, to
recipients at the dosages and concentrations employed and is
compatible with other ingredients of the formulation
comprising the drug, agent or binding molecule.
Post exposure prophylaxis
"Post exposure prophylaxis" (PEP) is indicated for persons
possibly exposed to a rabid animal. Possible exposures include
bite exposure (i.e. any penetration of the skin by teeth)
including animal bites, and non-bite exposure. Non-bite
exposures include exposure to large amounts of aerosolized
rabies virus in laboratories or caves and surgical recipients
of corneas transplanted from patients who died of rabies. The
contamination of open wounds, abrasions, mucous membranes, or
theoretically, scratches, with saliva or other potentially
infectious material (such as neural tissue) from a rabid
animal also constitutes a non-bite exposure. Other contact by
itself, such as petting a rabid animal and contact with blood,
urine, or feces of a rabid animal, does not constitute an
exposure and is not an indication for prophylaxis. PEP should
begin as soon as possible after an exposure. If no exposure
has occurred post exposure prophylaxis is not necessary. In
all post exposure prophylaxis regimens, except for persons
previously immunized, active and passive immunizations should
be used concurrently.
Specifically Binding
The term "specifically binding", as used herein, in reference
to the interaction of a binding molecule, e.g. an antibody,
and its binding partner, e.g. an antigen, means that the
interaction is dependent upon the presence of a particular
structure, e.g. an antigenic determinant or epitope, on the
CA 02775886 2012-04-19
binding partner. In other words, the antibody preferentially
binds or recognizes the binding partner even when the binding
partner is present in a mixture of other molecules or
organisms. The binding may be mediated by covalent or non-
5 covalent interactions or a combination of both. In yet other
words, the term "specifically binding" means
immunospecifically binding to an antigen or a fragment thereof
and not immunospecifically binding to other antigens. A
binding molecule that immunospecifically binds to an antigen
10 may bind to other peptides or polypeptides with lower affinity
as determined by, e.g., radioimmunoassays (RIA), enzyme-linked
immunosorbent assays (ELISA), BIACORE, or other assays known
in the art. Binding molecules or fragments thereof that
immunospecifically bind to an antigen may be cross-reactive
15 with related antigens. Preferably, binding molecules or
fragments thereof that immunospecifically bind to an antigen
do not cross-react with other antigens.
Therapeutically effective amount
The term "therapeutically effective amount" refers to an
amount of the binding molecule as defined herein that is
effective for post-exposure prophylaxis of rabies.
Vector
The term "vector" denotes a nucleic acid molecule into which a
second nucleic acid molecule can be inserted for introduction
into a host where it will be replicated, and in some cases
expressed. In other words, a vector is capable of transporting
a nucleic acid molecule to which it has been linked. Cloning
as well as expression vectors are contemplated by the term
"vector", as used herein. Vectors include, but are not limited
to, plasmids, cosmids, bacterial artificial chromosomes (BAC)
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and yeast artificial chromosomes (YAC) and vectors derived
from bacteriophages or plant or animal (including human)
viruses. Vectors comprise an origin of replication recognized
by the proposed host and in case of expression vectors,
promoter and other regulatory regions recognized by the host.
A vector containing a second nucleic acid molecule is
introduced into a cell for example by transformation,
transfection, or by making use of bacterial or viral entry
mechanisms. Other ways of introducing nucleic acid into cells
are known, such as electroporation or particle bombardment
often used with plant cells, and the like. The method of
introducing nucleic acid into cells depends among other things
on the type of cells, and so forth. This is not critical to
the invention. Certain vectors are capable of autonomous
replication in a host into which they are introduced (e.g.,
vectors having a bacterial origin of replication can replicate
in bacteria). Other vectors can be integrated into the genome
of a host upon introduction into the host, and thereby are
replicated along with the host genome.
SUMMARY OF THE INVENTION
The invention provides binding molecules capable of
specifically binding to and neutralizing rabies virus.
Furthermore, the invention pertains to nucleic acid molecules
encoding at least the binding region of these binding
molecules. The invention further provides for the use of the
binding molecules of the invention in the post exposure
prophylaxis of a subject at risk of developing a condition
resulting from rabies virus.
DETAILED DESCRIPTION OF THE INVENTION
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In a first aspect the present invention encompasses
binding molecules capable of specifically binding to rabies
virus. Preferably, the binding molecules of the invention also
have rabies virus-neutralizing activity. Preferably, the
binding molecules of the invention are human binding
molecules. Alternatively, they may also be binding molecules
of other animals. Rabies virus is part of the Lyssavirus
genus. In total, the Lyssavirus genus includes eleven
genotypes: rabies virus (genotype 1), Lagos bat virus
(genotype 2), Mokola virus (genotype 3), Duvenhage virus
(genotype 4), European bat lyssavirus 1 (genotype 5), European
bat lyssavirus 2 (genotype 6), Australian bat lyssavirus
(genotype 7), Aravan virus (genotype 8), Khujand virus
(genotype 9), Irkut virus (genotype 10) and West Caucasian
virus (genotype 11). Besides binding to rabies virus, the
binding molecules of the invention may also be capable of
binding to other genotypes of the Lyssavirus genus.
Preferably, the binding molecules may also be capable of
neutralizing other genotypes of the Lyssavirus genus.
Furthermore, the binding molecules of the invention may even
be capable of binding to and/or neutralizing viruses, other
than Lyssaviruses, of the rhabdovirus family. This family
includes the genera cytorhabdovirus, ephemerovirus,
lyssavirus, nucleorhabdovirus, rhabdovirus and vesiculovirus.
The binding molecules may be capable of specifically
binding to rabies virus in its natural form or in its
inactivated/attenuated form. Inactivation of rabies virus may
be performed by treatment with inter alia beta-propiolactone
(BPL) (White and Chappel, 1982), heating at 56 C for more than
30 minutes, gamma irradiation, treatment with
acetylethylenimine or ethylenimine or treatment with ascorbic
acid and copper sulfate for 72 hours (Madhusudana et al.,
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2004). General viral inactivation methods well known to the
skilled artisan such as inter alia pasteurization (wet heat),
dry heat treatment, vapor heat treatment, treatment with low
pH, treatment with organic solvent/detergent, nanofiltration;
W light irradiation may also be used. Preferably, the
inactivation is performed by treatment with beta-propiolactone
(BPL). Methods to test if rabies virus is still infective or
partly or completely inactivated are well known to the person
skilled in the art and can among others be found in Laboratory
techniques in rabies, Edited by: F.-X Meslin, M.M. Kaplan and
H. Koprowski (1996), 4th edition, Chapter 36, World Health
Organization, Geneva.
The binding molecules may also be capable of specifically
binding to one or more fragments of the rabies virus such as
inter alia a preparation of one or more proteins and/or
(poly)peptides derived from rabies virus or a cell transfected
with a rabies virus protein and/or (poly)peptide. For methods
of treatment and/or prevention such as methods for post
exposure prophylaxis of rabies virus the binding molecules are
preferably capable of specifically binding to surface
accessible proteins of rabies virus such as the M (see Ameyama
et al. 2003) or G protein. For diagnostical purposes the human
binding molecules may also be capable of specifically binding
to proteins not present on the surface of rabies virus. The
amino acid sequence of surface accessible and internal
proteins of various known strains of rabies virus can be found
in the EMBL-database and/or other databases.
Preferably, the fragment at least comprises an antigenic
determinant recognized by the human binding molecules of the
invention. An "antigenic determinant" as used herein is a
moiety, such as a rabies virus (poly)peptide, (glyco)protein,
or analog or fragment thereof, that is capable of binding to a
CA 02775886 2012-04-19
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human binding molecule of the invention with sufficiently high
affinity to form a detectable antigen-binding molecule
complex.
The binding molecules according to the invention can be
intact immunoglobulin molecules such as polyclonal or
monoclonal antibodies, in particular human monoclonal
antibodies, or the binding molecules can be antigen-binding
fragments including, but not limited to, Fab, F(ab'), F(ab')2,
Fv, dAb, Fd, complementarity determining region (CDR)
fragments, single-chain antibodies (scFv), bivalent single-
chain antibodies, single-chain phage antibodies, diabodies,
triabodies, tetrabodies, and (poly)peptides that contain at
least a fragment of an immunoglobulin that is sufficient to
confer specific antigen binding to the rabies virus or
fragment thereof. The binding molecules of the invention can
be used in non-isolated or isolated form. Furthermore, the
binding molecules of the invention can be used alone or in a
mixture comprising at least one human binding molecule (or
variant or fragment thereof). In other words, the binding
molecules can be used in combination, e.g., as a
pharmaceutical composition comprising two or more binding
molecules, variants or fragments thereof. For example, binding
molecules having rabies virus-neutralizing activity can be
combined in a single therapy to achieve a desired
prophylactic, therapeutic or diagnostic effect.
RNA viruses such as rabies virus make use of their own
RNA polymerase during virus replication. These RNA polymerases
tend to be error-prone. This leads to the formation of so-
called quasi-species during a viral infection. Each quasi-
species has a unique RNA genorne, which could result in
differences in amino acid composition of viral proteins. If
such mutations occur in structural viral proteins, the virus
CA 02775886 2012-04-19
could potentially escape from the host's immune system due to
a change in T or B cell epitopes. The likelihood of this to
happen is higher when individuals are treated with a mixture
of two binding molecules, such as human monoclonal antibodies,
5 than with a polyclonal antibody mixture (HRIG). Therefore, a
prerequisite for a mixture of two human monoclonal antibodies
for treatment of rabies is that the two antibodies recognize
non-overlapping, non-competing epitopes on their target
antigen, i.e. rabies virus glycoprotein. The chance of the
10 occurrence of rabies escape viruses is thereby minimized. As a
consequence thereof, the binding molecules of the invention
preferably are capable of reacting with different, non-
overlapping, non-competing epitopes of the rabies virus, such
as epitopes on the rabies virus G protein. The mixture of
15 binding molecules may further comprise at least one other
therapeutic agent such as a medicament suitable for the post
exposure prophylaxis of rabies.
Typically, binding molecules according to the invention
can bind to their binding partners, i.e. rabies virus or
20 fragments thereof such as rabies virus proteins, with an
affinity constant (Kd-value) that is lower than 0.2*10-4 M,
1.0*10-5 M, 1.0*10-6 M, 1.0*10_7. M, preferably lower than 1.0*10-
8 M, more preferably lower than 1.0*10-9 M, more preferably
lower than 1.0*10-10 M, even more preferably lower than 1.0*10-
11 M, and in particular lower than 1.0*10-12 M. The affinity
constants can vary for antibody isotypes. For example,
affinity binding for an IgM isotype refers to a binding
affinity of at least about 1.0*10-7 M. Affinity constants can
for instance be measured using surface plasmon resonance, i.e.
an optical phenomenon that allows for the analysis of real-
time biospecific interactions by detection of alterations in
protein concentrations within a biosensor matrix, for example
CA 02775886 2012-04-19
21
using the BIACORE system (Pharmacia Biosensor AB, Uppsala,
Sweden).
The binding molecules according to the invention may bind
to rabies virus in purified/isolated or non-purified/non-
isolated form. The binding molecules may bind to rabies virus
in soluble form such as for instance in a sample or may bind
to rabies virus bound or attached to a carrier or substrate,
e.g., microtiter plates, membranes and beads, etc. Carriers or
substrates may be made of glass, plastic (e.g., polystyrene),
polysaccharides, nylon, nitrocellulose, or teflon, etc. The
surface of such supports may be solid or porous and of any
convenient shape. Alternatively, the binding molecules may
also bind to fragments of rabies virus such as proteins or
(poly)peptides of the rabies virus. In an embodiment the
binding molecules are capable of specifically binding to the
rabies virus G protein or fragment thereof. The rabies virus
proteins or (poly)peptides may either be in soluble form or
may bind to rabies virus bound or attached to a carrier or
substrate as described above. In another embodiment cells
tranfected with the G protein may be used as binding partner
for the binding molecules.
In a preferred embodiment of the invention, the binding
molecules of the invention neutralize rabies virus
infectivity. This may be achieved by preventing the attachment
of rabies virus to its receptors on host cells, such as inter
alia the murine p75 neurotrophin receptor, the neural cell
adhesion molecule (CD56) and the acetylcholine receptor, or
inhibition of the release of RNA into the cytoplasm of the
cell or prevention of RNA transcription or translation. In a
specific embodiment, the binding molecules of the invention
prevent rabies virus from infecting host cells by at least
99%, at least 95%, at least 90%, at least 85%, at least 80%,
*Trade-mark
CA 02775886 2012-04-19
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at least 75%, at least 70%, at least 60%, at least 50%, at
least 45%, at least 40%, at least 45%, at least 35%, at least
30%, at least 25%, at least 20%, or at least 10% relative to
infection of host cells by rabies virus in the absence of said
binding molecules. Neutralization can for instance be measured
as described in Laboratory techniques in rabies, Edited by:
F.-X Meslin, M.M. Kaplan and H. Koprowski (1996), 4th edition,
Chapters 15-17, World Health Organization, Geneva.
Furthermore, the human binding molecules of the invention may
be complement fixing binding molecules capable of assisting in
the lysis of enveloped rabies virus. The human binding
molecules of the invention might also act as opsonins and
augment phagocytosis of rabies virus either by promoting its
uptake via Fc or C3b receptors or by agglutinating rabies
virus to make it more easily phagocytosed.
In a preferred embodiment, the binding molecules
according to the invention comprise at least a CDR3 region
comprising the amino acid sequence selected from the group
consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ
ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID N0:12, SEQ ID
N0:13, SEQ ID N0:14, SEQ ID N0:15, SEQ ID N0:16, SEQ ID N0:17,
SEQ ID N0:18, SEQ ID N0:19, SEQ ID NO:20, SEQ ID N0:21, SEQ ID
N0:22, SEQ ID N0:23 and SEQ ID N0:24. In an embodiment the
CDR3 region is a heavy chain CDR3 region.
In yet another embodiment, the binding molecules
according to the invention comprise a variable heavy chain
comprising essentially an amino acid sequence selected from
the group consisting of SEQ ID N0:26, SEQ ID NO:27, SEQ ID
NO:28, SEQ ID N0:29, SEQ ID NO:30, SEQ ID N0:31, SEQ ID N0:32,
SEQ ID N0:33, SEQ ID N0:34, SEQ ID N0:35, SEQ ID N0:36, SEQ ID
N0:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID N0:40, SEQ ID NO:41,
CA 02775886 2012-04-19
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SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID
NO:46, SEQ ID NO:47, SEQ ID NO:48 and SEQ ID NO:49. In a
preferred embodiment the binding molecules according to the
invention comprise a variable heavy chain comprising
essentially an amino acid sequence comprising amino acids 1-
119 of SEQ ID NO:335.
In a further embodiment, the binding molecules according
to the invention comprise a variable heavy chain comprising
the amino acid sequence of SEQ ID NO:26 and a variable light
chain comprising the amino acid sequence of SEQ ID NO:50, a
variable heavy chain comprising the amino acid sequence of SEQ
ID NO:27 and a variable light chain comprising the amino acid
sequence of SEQ ID NO:51, a variable heavy chain comprising
the amino acid sequence of SEQ ID NO:28 and a variable light
chain comprising the amino acid sequence of SEQ ID NO:52, a
variable heavy chain comprising the amino acid sequence of SEQ
ID NO:29 and a variable light chain comprising the amino acid
sequence of SEQ ID NO:53, a variable heavy chain comprising
the amino acid sequence of SEQ ID NO:30 and a variable light
chain comprising the amino acid sequence of SEQ ID NO:54, a
variable heavy chain comprising the amino acid sequence of SEQ
ID NO:31 and a variable light chain comprising the amino acid
sequence of SEQ ID NO:55, a variable heavy chain comprising
the amino acid sequence of SEQ ID NO:32 and a variable light
chain comprising the amino acid sequence of SEQ ID NO:56, a
variable heavy chain comprising the amino acid sequence of SEQ
ID NO:33 and a variable light chain comprising the amino acid
sequence of SEQ ID NO:57, a variable heavy chain comprising
the amino acid sequence of SEQ ID NO:34 and a variable light
chain comprising the amino acid sequence of SEQ ID NO:58, a
variable heavy chain comprising the amino acid sequence of SEQ
ID NO:35 and a variable light chain comprising the amino acid
CA 02775886 2012-04-19
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sequence of SEQ ID NO:59, a variable heavy chain comprising
the amino acid sequence of SEQ ID NO:36 and a variable light
chain comprising the amino acid sequence of SEQ ID NO:60, a
variable heavy chain comprising the amino acid sequence of SEQ
ID NO:37 and a variable light chain comprising the amino acid
sequence of SEQ ID NO:61, a variable heavy chain comprising
the amino acid sequence of SEQ ID NO:38 and a variable light
chain comprising the amino acid sequence of SEQ ID NO:62, a
variable heavy chain comprising the amino acid sequence of SEQ
ID NO:39 and a variable light chain comprising the amino acid
sequence of SEQ ID NO:63, a variable heavy chain comprising
the amino acid sequence of SEQ ID NO:40 and a variable light
chain comprising the amino acid sequence of SEQ ID NO:64, a
variable heavy chain comprising the amino acid sequence of SEQ
ID NO:41 and a variable light chain comprising the amino acid
sequence of SEQ ID NO:65, a variable heavy chain comprising
the amino acid sequence of SEQ ID NO:42 and a variable light
chain comprising the amino acid sequence of SEQ ID NO:6.6, a
variable heavy chain comprising the amino acid sequence of SEQ
ID NO:43 and a variable light chain comprising the amino acid
sequence of SEQ ID NO:67, a variable heavy chain comprising
the amino acid sequence of SEQ ID NO:44 and a variable light
chain comprising the amino acid sequence of SEQ ID NO:68, a
variable heavy chain comprising the amino acid sequence of SEQ
ID NO:45 and a variable light chain comprising the amino acid
sequence of SEQ ID NO:69, a variable heavy chain comprising
the amino acid sequence of SEQ ID NO:46 and a variable light
chain comprising the amino acid sequence of SEQ ID NO:70, a
variable heavy chain comprising the amino acid sequence of SEQ
ID NO:47 and a variable light chain comprising the amino acid
sequence of SEQ ID NO:71, a variable heavy chain comprising
the amino acid sequence of SEQ ID NO:48 and a variable light
CA 02775886 2012-04-19
chain comprising the amino acid sequence of SEQ ID NO:72, a
variable heavy chain comprising the amino acid sequence of SEQ
ID NO:49 and a variable light chain comprising the amino acid
sequence of SEQ ID NO:73. In a preferred embodiment the human
5 binding molecules according to the invention comprise a
variable heavy chain comprising the amino acid sequence
comprising amino acids 1-119 of SEQ ID NO:335 and a variable
light chain comprising the amino acid sequence comprising
amino acids 1-107 of SEQ ID NO:337.
10 In a preferred embodiment the binding molecules having
rabies virus neutralizing activity of the invention are
administered in IgG format, preferably IgGl format.
Another aspect of the invention includes functional
variants of binding molecules as defined herein. Molecules are
15 considered to be functional variants of a binding molecule
according to the invention, if the variants are capable of
competing for specifically binding to rabies virus or a
fragment thereof with the parent binding molecules. In other
words, when the functional variants are still capable of
20 binding to rabies virus or a fragment thereof. The functional
variants should also still have rabies virus neutralizing
activity. Functional variants include, but are not limited to,
derivatives that are substantially similar in primary
structural sequence, but which contain e.g. in vitro or in
25 vivo modifications, chemical and/or biochemical, that are not
found in the parent binding molecule. Such modifications
include inter alia acetylation, acylation, ADP-ribosylation,
amidation, covalent attachment of flavin, covalent attachment
of a heme moiety, covalent attachment of a nucleotide or
nucleotide derivative, covalent attachment of a lipid or lipid
derivative, covalent attachment of phosphotidylinositol,
cross-linking, cyclization, disulfide bond formation,
CA 02775886 2012-04-19
26
demethylation, formation of covalent cross-links, formation of
cystine, formation of pyroglutamate, formylation, gamma-
carboxylation, glycosylation, GPI-anchor formation,
hydroxylation, iodination, methylation, myristoylation,
oxidation, pegylation, proteolytic processing,
phosphorylation, prenylation, racemization, selenoylation,
sulfation, transfer-RNA mediated addition of amino acids to
proteins such as arginylation, ubiquitination, and the like.
Alternatively, functional variants can be binding
molecules as defined in the present invention comprising an
amino acid sequence containing substitutions, insertions,
deletions or combinations thereof of one or more amino acids
compared to the amino acid sequences of the parent binding
molecules. Furthermore, functional variants can comprise
truncations of the amino acid sequence at either or both the
amino or carboxy termini. Functional variants according to the
invention may have the same or different, either higher or
lower, binding affinities compared to the parent binding
molecule but are still capable of binding to rabies virus or a
fragment thereof and are still capable of neutralizing rabies
virus. For instance, functional variants according to the
invention may have increased or decreased binding affinities
for rabies virus or a fragment thereof compared to the parent
binding molecules or may have a higher or lower rabies virus
neutralizing activity. Preferably, the amino acid sequences of
the variable regions, including, but not limited to, framework
regions, hypervariable regions, in particular the CDR3
regions, are modified. Generally, the light chain and the
heavy chain variable regions comprise three hypervariable
regions, comprising three CDRs, and more conserved regions,
the so-called framework regions (FRs). The hypervariable
regions comprise amino acid residues from CDRs and amino acid
CA 02775886 2012-04-19
27
residues from hypervariable loops. Functional variants
intended to fall within the scope of the present invention
have at least about 50% to about 99%, preferably at least
about 60% to about 99%, more preferably at least about 70% to
about 99%, even more preferably at least about 80% to about
99%, most preferably at least about 90% to about 99%, in
particular at least about 95% to about 99%, and in particular
at least about 97% to about 99% amino acid sequence homology
with the parent binding molecules as defined herein. Computer
algorithms such as inter alia Gap or Bestfit known to a person
skilled in the art can be used to optimally align amino acid
sequences to be compared and to define similar or identical
amino acid residues.
In another embodiment, functional variants may be
produced when the parent binding molecule comprises a
glycosylation site in its sequence that results in
glycosylation of the binding molecule upon expression in
eukaryotic cells and hence might abrogate the binding to the
antigen. The functional variant produced no longer contains
the glycosylation site, but will be capable of binding to
rabies virus and still have neutralizing activity.
Functional variants can be obtained by altering the
parent binding molecules or parts thereof by general molecular
biology methods known in the art including, but not limited
to, error-prone PCR, oligonucleotide-directed mutagenesis and
site-directed mutagenesis. Furthermore, the functional
variants may have complement fixing activity, be capable of
assisting in the lysis of enveloped rabies virus and/or act as
opsonins and augment phagocytosis of rabies virus either by
promoting its uptake via Fc or Cab receptors or by
agglutinating rabies virus to make it more easily
phagocytosed.
CA 02775886 2012-04-19
28
In yet a further aspect, the invention includes
immunoconjugates, i.e. molecules comprising at least one
binding molecule or functional variant thereof as defined
herein and further comprising at least one tag, such as inter
alia a detectable moiety/agent. Also contemplated in the
present invention are mixtures of immunoconjugates according
to the invention or mixtures of at least one immunoconjugate
according to the invention and another molecule, such as a
therapeutic agent or another binding molecule or
immunoconjugate. In a further embodiment, the immunoconjugates
of the invention may comprise one or more tags. These tags can
be the same or distinct from each other and can be
joined/conjugated non-covalently to the binding molecules. The
tag(s) can also be joined/conjugated directly to the binding
molecules through covalent bonding, including, but not limited
to, disulfide bonding, hydrogen bonding, electrostatic
bonding, recombinant fusion and conformational bonding.
Alternatively, the tag(s) can be joined/conjugated to the
binding molecules by means of one or more linking compounds.
Techniques for conjugating tags to binding molecules are well
known to the skilled artisan.
The tags of the immunoconjugates of the present invention
may be therapeutic agents, but preferably they are detectable
moieties/agents. Immunoconjugates comprising a detectable
agent can be used diagnostically to, for example, assess if a
subject has been infected with rabies virus or monitor the
development or progression of a rabies virus infection as part
of a clinical testing procedure to, e.g., determine the
efficacy of a given treatment regimen. However, they may also
be used for other detection and/or analytical and/or
diagnostic purposes. Detectable moieties/agents include, but
are not limited to, enzymes, prosthetic groups, fluorescent
CA 02775886 2012-04-19
29
materials, luminescent materials, bioluminescent materials,
radioactive materials, positron emitting metals, and
nonradioactive paramagnetic metal ions.
The tags used to label the binding molecules for
detection and/or analytical and/or diagnostic purposes depend
on the specific detection/analysis/diagnosis techniques and/or
methods used such as inter alia immunohistochemical staining
of (tissue) samples, flow cytometric detection, scanning laser
cytometric detection, fluorescent immunoassays, enzyme-linked
immunosorbent assays (ELISA's), radioimmunoassays (RIA's),
bioassays (e.g., neutralization assays), Western blotting
applications, etc. For immunohistochemical staining of tissue
samples preferred labels are enzymes that catalyze production
and local deposition of a detectable product. Enzymes
typically conjugated to binding molecules to permit their
immunohistochemical visualization are well-known and include,
but are not limited to, acetylcholinesterase, alkaline
phosphatase, beta-galactosidase, glucose oxidase, horseradish
peroxidase, and urease. Typical substrates for production and
deposition of visually detectable products are also well known
to the skilled person in the art. Next to that,
immunoconjugates of the invention can be labeled using
colloidal gold or they can be labeled with radioisotopes, such
as 33p, 32p, 355, 3H, and 125I. Binding molecules of the invention
can be attached to radionuclides directly or indirectly via a
chelating agent by methods well known in the art.
When the binding molecules of the present invention are
used for flow cytometric detections, scanning laser cytometric
detections, or fluorescent immunoassays, they can usefully be
labeled with fluorophores. A wide variety of fluorophores
useful for fluorescently labeling the binding molecules of the
present invention are known to the skilled artisan. When the
CA 02775886 2012-04-19
binding molecules of the present invention are used for
secondary detection using labeled avidin, streptavidin,
captavidin or neutravidin, the binding molecules may be
labeled with biotin to form suitable prosthetic group
5 complexes.
When the immunoconjugates of the invention are used for
in vivo diagnostic use, the binding molecules can also be made
detectable by conjugation to e.g. magnetic resonance imaging
(MRI) contrast agents, such as gadolinium
10 diethylenetriaminepentaacetic acid, to ultrasound contrast
agents or to X-ray contrast agents, or by radioisotopic
labeling.
Furthermore, the binding molecules, functional variants
thereof or immunoconjugates of the invention can also be
15 attached to solid supports, which are particularly useful for
in vitro immunoassays or purification of rabies virus or a
fragment thereof. Such solid supports might be porous or
nonporous, planar or nonplanar and include,. but are not
limited to, glass, cellulose, polyacrylamide, nylon,
20 polystyrene, polyvinyl chloride or polypropylene supports. The
human binding molecules can also for example usefully be
conjugated to filtration media, such as NHS-activated
Sepharose or CNBr-activated Sepharose for purposes of
immunoaffinity chromatography. They can also usefully be
25 attached to paramagnetic microspheres, typically by biotin-
streptavidin interaction. The microspheres can be used for
isolation of rabies virus or a fragment thereof from a sample
containing rabies virus or a fragment thereof. As another
example, the human binding molecules of the present invention
30 can usefully be attached to the surface of a microtiter plate
for ELISA.
CA 02775886 2012-04-19
31
The binding molecules of the present invention or
functional variants thereof can be fused to marker sequences,
such as a peptide to facilitate purification. Examples
include, but are not limited to, the hexa-histidine tag, the
hemagglutinin (HA) tag, the myc tag or the flag tag.
Alternatively, an antibody can be conjugated to a second
antibody to form an antibody heteroconjugate. In another
aspect the human binding molecules of the invention may be
conjugated/attached to one or more antigens. Preferably, these
antigens are antigens which are recognized by the immune
system of a subject to which the binding molecule-antigen
conjugate is administered. The antigens may be identical but
may also differ from each other. Conjugation methods for
attaching the antigens and binding molecules are well known in
the art and include, but are not limited to, the use of cross-
linking agents. The human binding molecules will bind to
rabies virus and the antigens attached to the human binding
molecules will initiate a-powerful T-cell attack on the
conjugate which will eventually lead to the destruction of the
rabies virus.
Next to producing immunoconjugates chemically by
conjugating, directly or indirectly via for instance a linker,
the immunoconjugates can be produced as fusion proteins
comprising the human binding molecules of the invention and a
suitable tag. Fusion proteins can be produced by methods known
in the art such as, e.g., recombinantly by constructing
nucleic acid molecules comprising nucleotide sequences
encoding the human binding molecules in frame with nucleotide
sequences encoding the suitable tag(s) and then expressing the
nucleic-acid molecules.
It is another aspect of the present invention to provide
a nucleic acid molecule encoding at least a binding molecule
CA 02775886 2012-04-19
32
or functional variant thereof according to the invention. Such
nucleic acid molecules can be used as intermediates for
cloning purposes, e.g. in the process of affinity maturation
described above. In a preferred embodiment, the nucleic acid
molecules are isolated or purified.
The skilled man will appreciate that functional variants
of these nucleic acid molecules are also intended to be a part
of the present invention. Functional variants are nucleic acid
sequences that can be directly translated, using the standard
genetic code, to provide an amino acid sequence identical to
that translated from the parent nucleic acid molecules.
Preferably, the nucleic acid molecules encode binding
molecules comprising a CDR3 region, preferably a heavy chain
CDR3 region, comprising an amino acid sequence selected from
the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,
SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12,
SEQ ID NO:13,_,SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ-ID
NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,
SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24.
Even more preferably, the nucleic acid molecules encode
human binding molecules comprising a variable heavy chain
comprising essentially an amino acid sequence selected from
the group consisting of SEQ ID NO:26, SEQ ID NO:27, SEQ ID
NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32,
SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID
NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41,
SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID
NO:46, SEQ ID NO:47, SEQ ID NO:48 and SEQ ID NO:49. In a
particularly preferred embodiment the nucleic acid molecules
encode binding molecules comprising a variable heavy chain
CA 02775886 2012-04-19
33
comprising essentially an amino acid sequence comprising amino
acids 1-119 of SEQ ID NO:335.
In yet another embodiment, the nucleic acid molecules
encode binding molecules comprising a variable heavy chain
comprising the amino acid sequence of SEQ ID NO:26 and a
variable light chain comprising the amino acid sequence of SEQ
ID NO:50, or they encode a variable heavy chain comprising the
amino acid sequence of SEQ ID NO:27 and a variable light chain
comprising the amino acid sequence of SEQ ID NO:51, or they
encode a variable heavy chain comprising the amino acid
sequence of SEQ ID NO:28 and a variable light chain comprising
the amino acid sequence of SEQ ID NO:52, or they encode a
variable heavy chain comprising the amino acid sequence of SEQ
ID NO:29 and a variable light chain comprising the amino acid
sequence of SEQ ID NO:53, or they encode a variable heavy
chain comprising the amino acid sequence of SEQ ID NO:30 and a
variable light chain comprising the amino'acid sequence of SEQ
ID NO:54,..,.or they encode a variable heavy chain comprising the
amino acid sequence of SEQ ID NO:31 and a variable light chain
comprising the amino acid sequence of SEQ ID NO:55, or they
encode a variable heavy chain comprising the amino acid
sequence of SEQ ID NO:32 and a variable light chain comprising
the amino acid sequence of SEQ ID NO:56, or they encode a
variable heavy chain comprising the amino acid sequence of SEQ
ID NO:33 and a variable light chain comprising the amino acid
sequence of SEQ ID NO:57, or they encode a variable heavy
chain comprising the amino acid sequence of SEQ ID NO:34 and a
variable light chain comprising the amino acid sequence of SEQ
ID NO:58, or they encode a variable heavy chain comprising the
amino acid sequence of SEQ ID NO:35 and a variable light chain
comprising the amino acid sequence of SEQ ID NO:59, or they
encode a variable heavy chain comprising the amino acid
CA 02775886 2012-04-19
34
sequence of SEQ ID NO:36 and a variable light chain comprising
the amino acid sequence of SEQ ID NO:60, or they encode a
variable heavy chain comprising the amino acid sequence of SEQ
ID NO:37 and a variable light chain comprising the amino acid
sequence of SEQ ID NO:61, or they encode a variable heavy
chain comprising the amino acid sequence of SEQ ID NO:38 and a
variable light chain comprising the amino acid sequence of SEQ
ID NO:62, or they encode a variable heavy chain comprising the
amino acid sequence of SEQ ID NO:39 and a variable light chain
comprising the amino acid sequence of SEQ ID NO:63, or they
encode a variable heavy chain comprising the amino acid
sequence of SEQ ID NO:40 and a variable light chain comprising
the amino acid sequence of SEQ ID NO:64, or they encode a
variable heavy chain comprising the amino acid sequence of SEQ
ID NO:41 and a variable light chain comprising the amino acid
sequence of SEQ ID NO:65, or they encode a variable heavy
chain comprising the amino acid sequence of SEQ ID NO:42 and a
variable light chain comprising the amino acid sequence of SEQ
ID NO:66, or they encode a variable heavy chain comprising the
amino acid sequence of SEQ ID NO:43 and a variable light chain
comprising the amino acid sequence of SEQ ID NO:67, or they
encode a variable heavy chain comprising the amino acid
sequence of SEQ ID NO:44 and a variable light chain comprising
the amino acid sequence of SEQ ID NO:68, or they encode a
variable heavy chain comprising the amino acid sequence of SEQ
ID NO:45 and a variable light chain comprising the amino acid
sequence of SEQ ID NO:69, or they encode a variable heavy
chain comprising the amino acid sequence of SEQ ID NO:46 and a
variable light chain comprising the amino acid sequence of SEQ
ID NO:70, or they encode a variable heavy chain comprising the
amino acid sequence of SEQ ID NO:47 and a variable light chain
comprising the amino acid sequence of SEQ ID NO:71, or they
CA 02775886 2012-04-19
encode a variable heavy chain comprising the amino acid
sequence of SEQ ID NO:48 and a variable light chain comprising
the amino acid sequence of SEQ ID NO:72, or they encode a
variable heavy chain comprising the amino acid sequence of SEQ
5 ID NO:49 and a variable light chain comprising the amino acid
sequence of SEQ ID NO:73. In a preferred embodiment the
nucleic acid molecules encode human binding molecules
comprising a variable heavy chain comprising the amino acid
sequence comprising amino acids 1-119 of SEQ ID N0:335 and a
10 variable light chain comprising the amino acid sequence
comprising amino acids 1-107 of SEQ ID N0:337.
In a specific embodiment of the invention the nucleic
acid molecules encoding the variable heavy chain of the
binding molecules of the invention comprise essentially a
15 nucleotide sequence selected from the group consisting of SEQ
ID N0:74, SEQ ID NO:75, SEQ ID N0:76, SEQ ID N0:77, SEQ ID
N0:78, SEQ ID N0:79, SEQ ID N0:80, SEQ ID N0:81, SEQ ID N0:82,
SEQ ID N0:83, SEQ,ID N0:84, SEQ ID N0:85, SEQ ID N0:86, SEQ ID.,
N0:87, SEQ ID N0:88, SEQ ID N0:89, SEQ ID N0:90, SEQ ID N0:91,
20 SEQ ID N0:92, SEQ ID N0:93, SEQ ID N0:94, SEQ ID N0:95, SEQ ID
N0:96 and SEQ ID N0:97. Preferably, the nucleic acid molecules
encoding the variable heavy chain of the binding molecules of
the invention comprise essentially a nucleotide sequence
comprising nucleotides 1-357 of SEQ ID N0:334.
25 In yet another specific embodiment of the present
invention, the nucleic acid molecules encoding the variable
light chain of the binding molecules of the invention comprise
essentially a nucleotide sequence selected of the group
consisting of SEQ ID N0:98, SEQ ID N0:99, SEQ ID N0:100, SEQ
30 ID N0:101, SEQ ID N0:102, SEQ ID N0:103, SEQ ID N0:104, SEQ ID
N0:105, SEQ ID NO:106,.SEQ ID N0:107, SEQ ID N0:108, SEQ ID
N0:109, SEQ ID N0:110, SEQ ID N0:111, SEQ ID N0:112, SEQ ID
CA 02775886 2012-04-19
36
NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID
NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120 and SEQ ID
NO:121. Preferably, the nucleic acid molecules encoding the
variable light chain of the human binding molecules of the
invention comprise essentially a nucleotide sequence
comprising nucleotides 1-321 of SEQ ID NO:336.
It is another aspect of the invention to provide vectors,
i.e. nucleic acid constructs, comprising one or more nucleic
acid molecules according to the present invention. Vectors can
be derived from plasmids such as inter alia F, R1, RP1, Col,
pBR322, TOL, Ti, etc; cosmids; phages such as lambda,
lambdoid, M13, Mu, P1, P22, Qp, T-even, T-odd, T2, T4, T7, etc;
plant viruses such as inter alia alfalfa mosaic virus,
bromovirus, capillovirus, carlavirus, carmovirus, caulivirus,
clostervirus, comovirus, cryptovirus, cucumovirus,
dianthovirus, fabavirus, fijivirus, furovirus, geminivirus,
hordeivirus, ilarvirus, luteovirus, machlovirus, marafivirus,
.-necrovirus, nepovirus, phytorepvirus, plant rhabdovirus,
potexvirus, potyvirus, sobemovirus, tenuivirus, tobamovirus,
tobravirus, tomato spotted wilt virus, tombusvirus, tymovirus,
etc; or animal viruses such as inter alia adenovirus,
arenaviridae, baculoviridae, birnaviridae, buryaviridae,
calciviridae, cardioviruses, coronaviridae, corticoviridae,
cystoviridae, Epstein-Barr virus, enteroviruses, filoviridae,
flaviviridae, Foot-and-Mouth disease virus, hepadnaviridae,
hepatitis viruses, herpesviridae, immunodeficiency viruses,
influenza virus, inoviridae, iridoviridae, orthomyxoviridae,
papovaviruses, paramyxoviridae, parvoviridae, picornaviridae,
poliovirus, polydnaviridae, poxviridae, reoviridae,
retroviruses, rhabdoviridae, rhinoviruses, Semliki Forest
virus, tetraviridae, togaviridae, toroviridae, vaccinia virus,
vescular stomatitis virus, etc. Vectors can be used for
CA 02775886 2012-04-19
37
cloning and/or for expression of the human binding molecules
of the invention and might even be used for gene therapy
purposes. Vectors comprising one or more nucleic acid
molecules according to the invention operably linked to one or
more expression-regulating nucleic acid molecules are also
covered by the present invention. The choice of the vector is
dependent on the recombinant procedures followed and the host
used. Introduction of vectors in host cells can be effected by
inter alia calcium phosphate transfection, virus infection,
DEAE-dextran mediated transfection, lipofectamin transfection
or electroporation. Vectors may be autonomously replicating or
may replicate together with the chromosome into which they
have been integrated. Preferably, the vectors contain one or
more selection markers. The choice of the markers may depend
on the host cells of choice, although this is not critical to
the invention as is well known to persons skilled in the art.
They include, but are not limited to, kanamycin, neomycin,
puromycin, hygromycin, z.eocin, thymidine kinase gene from
Herpes simplex virus (HSV-TK), dihydrofolate reductase gene
from mouse (dhfr). Vectors comprising one or more nucleic acid
molecules encoding the human binding molecules as described
above operably linked to one or more nucleic acid molecules
encoding proteins or peptides that can be used to isolate the
binding molecules are also covered by the invention. These
proteins or peptides include, but are not limited to,
glutathione-S-transferase, maltose binding protein, metal-
binding polyhistidine, green fluorescent protein, luciferase
and beta-galactosidase.
Hosts containing one or more copies of the vectors
mentioned above are an additional subject of the present
invention. Preferably, the hosts are host cells. Host cells
include, but are not limited to, cells of mammalian, plant,
CA 02775886 2012-04-19
38
insect, fungal or bacterial origin. Bacterial cells include,
but are not limited to, cells from Gram positive bacteria such
as several species of the genera Bacillus, Streptomyces and
Staphylococcus or cells of Gram negative bacteria such as
several species of the genera Escherichia, such as E. coli,
and Pseudomonas. In the group of fungal cells preferably yeast
cells are used. Expression in yeast can be achieved by using
yeast strains such as inter alia Pichia pastoris,
Saccharomyces cerevisiae and Hansenula polymorpha.
Furthermore, insect cells such as cells from Drosophila and
Sf9 can be used as host cells. Besides that, the host cells
can be plant cells. Transformed (transgenic) plants or plant
cells are produced by known methods, for example,
Agrobacterium-mediated gene transfer, transformation of leaf
discs, protoplast transformation by polyethylene glycol-
induced DNA transfer, electroporation, sonication,
microinjection or bolistic gene transfer. Additionally, a
suitable expression system can be a.baculovirus system.
Expression systems using mammalian cells such as Chinese
Hamster Ovary (CHO) cells, COS cells, BHK cells or Bowes
melanoma cells are preferred in the present invention.
Mammalian cells provide expressed proteins with
posttranslational modifications that are most similar to
natural molecules of mammalian origin. Since the present
invention deals with molecules that may have to be
administered to humans, a completely human expression system
would be particularly preferred. Therefore, even more
preferably, the host cells are human cells. Examples of human
cells are inter alia HeLa, 911, AT1080, A549, 293 and HEK293T
cells. Preferred mammalian cells are human retina cells such
as 911 cells or the cell line deposited at the European
Collection of Cell Cultures (ECACC), CAMR, Salisbury,
CA 02775886 2012-04-19
39
Wiltshire SP4 OJG, Great Britain on 29 February 1996 under
number 96022940 and marketed under the trademark PER.C6
(PER.C6 is a registered trademark of Crucell Holland B.V.).
For the purposes of this application "PER.C6" refers to cells
deposited under number 96022940 or ancestors, passages up-
stream or downstream as well as descendants from ancestors of
deposited cells, as well as derivatives of any of the
foregoing.
In preferred embodiments, the human producer cells
comprise at least a functional part of a nucleic acid sequence
encoding an adenovirus El region in expressible format. In
even more preferred embodiments, said host cells are derived
from a human retina and immortalised with nucleic acids
comprising adenoviral El sequences, such as the cell line
deposited at the European Collection of Cell Cultures (ECACC),
CANR, Salisbury, Wiltshire SP4 OJG, Great Britain on 29
February 1996 under number 96022940 and marketed under the
trademark PER.C6 . Production of recombinant proteins in host
cells can be performed according to methods well known in the
art. The use of the cells marketed under the trademark PER.C6
as a production platform for proteins of interest has been
described in WO 00/63403 the disclosure of which is
incorporated herein by reference in its entirety.
A method of producing a binding molecule or a functional
variant according to the invention is an additional part of
the invention. The method comprises the steps of a) culturing
a host according to the invention under conditions conducive
to the expression of the binding molecule or functional
variant thereof, and b) optionally, recovering the expressed
binding molecule or functional variant thereof. The expressed
binding molecules or functional variants thereof can be
recovered from the cell free extract, but preferably they are
CA 02775886 2012-04-19
recovered from the culture medium. Methods to recover
proteins, such as binding molecules, from cell free extracts
or culture medium are well known to the man skilled in the
art. Binding molecules or functional variants thereof as
5 obtainable by the above described method are also a part of
the present invention.
Alternatively, next to the expression in hosts, such as
host cells, the binding molecules or functional variants
thereof of the invention can be produced synthetically by
10 conventional peptide synthesizers or in cell-free translation
systems using RNA nucleic acid derived from DNA molecules
according to the invention. Binding molecule or functional
variants thereof as obtainable by the above described
synthetic production methods or cell-free translation systems
15 are also a part of the present invention.
In another embodiment binding molecules or functional
variants thereof according to the present invention may be
generated by transgenic non-human mammals, such as for.
instance transgenic mice or rabbits, that express human
20 immunoglobulin genes. Preferably, the transgenic non-human
mammals have a genome comprising a human heavy chain transgene
and a human light chain transgene encoding all or a portion of
the human binding molecules as described above. The transgenic
non-human mammals can be immunized with a purified or enriched
25 preparation of rabies virus or a fragment thereof. Protocols
for immunizing non-human mammals are well established in the
art. See Using Antibodies: A Laboratory Manual, Edited by: E.
Harlow, D. Lane (1998), Cold Spring Harbor Laboratory, Cold
Spring Harbor, New York and Current Protocols in Immunology,
30 Edited by: J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M.
Shevach, W. Strober (2001), John Wiley & Sons Inc., New York,
the disclosures of which are incorporated herein by reference.
CA 02775886 2012-04-19
41
In a further aspect, the invention provides a method of
identifying binding molecules such as human monoclonal
antibodies or fragments thereof according to the invention or
nucleic acid molecules according to the invention capable of
specifically binding to rabies virus and comprises the steps
of a) contacting a collection of binding molecules on the
surface of replicable genetic packages with the rabies virus
or a fragment thereof under conditions conducive to binding,
b) selecting at least once for replicable genetic packages
binding to the rabies virus or the fragment thereof, and c)
separating and recovering the replicable genetic packages
binding to the rabies virus or the fragment thereof.
The selection step may be performed in the presence of
rabies virus. The rabies virus may be isolated or non-
isolated, e.g. present in serum and/or blood of an infected
individual. In another embodiment the rabies virus is
inactivated. Alternatively, the selection step may be
performed in the presence of a fragment.of rabies virus such
as an extracellular part of the rabies virus, one or more
(poly)peptides derived from rabies virus such as the G
protein, fusion proteins comprising these proteins or
(poly)peptides, and the like. In another embodiment cells
transfected with rabies virus G protein are used for selection
procedures.
In yet a further aspect, the invention provides a method
of obtaining a binding molecule or a nucleic acid molecule
according to the invention, wherein the method comprises the
steps of a) performing the above described method of
identifying binding molecules, such as human monoclonal
antibodies or fragments thereof according to the invention, or
nucleic acid molecules according to the invention, and b)
isolating from the recovered replicable genetic packages the
CA 02775886 2012-04-19
42
binding molecule and/or the nucleic acid encoding the binding
molecule. Once a new monoclonal antibody has been established
or identified with the above mentioned method of identifying
binding molecules or nucleic acid molecules encoding the
binding molecules, the DNA encoding the scFv or Fab can be
isolated from the bacteria or replicable genetic packages and
combined with standard molecular biological techniques to make
constructs encoding bivalent scFvs or complete human
immunoglobulins of a desired specificity (e.g. IgG, IgA or
IgM). These constructs can be transfected into suitable cell
lines and complete human monoclonal antibodies can be produced
(see Huls et al., 1999; Boel et al., 2000).
A replicable genetic package as used herein can be
prokaryotic or eukaryotic and includes cells, spores,
bacteria, viruses, (bacterio)phage and polysomes. A preferred
replicable genetic package is a phage. The human binding
molecules, such as for instance single chain Fv's, are
displayed on the replicable genetic package, i.e. they are
attached to a group or molecule located at an exterior surface
of the replicable genetic package. The replicable genetic
package is a screenable unit comprising a human binding
molecule to be screened linked to a nucleic acid molecule
encoding the binding molecule. The nucleic acid molecule
should be replicable either in vivo (e.g., as a vector) or in
vitro (e.g., by PCR, transcription and translation). In vivo
replication can be autonomous (as for a cell), with the
assistance of host factors (as for a virus) or with the
assistance of both host and helper virus (as for a phagemid).
Replicable genetic packages displaying a collection of human
binding molecules are formed by introducing nucleic acid
molecules encoding exogenous binding molecules to be displayed
into the genomes of the replicable genetic packages to form
CA 02775886 2012-04-19
43
fusion proteins with endogenous proteins that are normally
expressed from the outer surface of the replicable genetic
packages. Expression of the fusion proteins, transport to the
outer surface and assembly results in display of exogenous
binding molecules from the outer surface of the replicable
genetic packages. In a further aspect the invention pertains
to a human binding molecule capable of binding rabies virus or
a fragment thereof and being obtainable by the identification
method as described above.
In yet a further aspect the invention relates to a method
of identifying a binding molecule potentially having
neutralizing activity against rabies virus, wherein the method
comprises the steps of (a) contacting a collection of binding
molecules on the surface of replicable genetic packages with
the rabies virus under conditions conducive to binding, (b)
separating and recovering binding molecules that bind to the
rabies virus from binding molecules that do not bind, (c)
isolating at least... one recovered binding molecule, (d)
verifying if the binding molecule isolated has neutralizing
activity against the rabies virus, characterized in that the
rabies virus in step a is inactivated. The inactivated rabies
virus may be purified before being inactivated. Purification
may be performed by means of well known purification methods
suitable for viruses such as for instance centrifugation
through a glycerol cushion. The inactivated rabies virus in
step a may be immobilized to a suitable material before use.
Alternatively, the rabies virus in step a may still be active.
In another alternative embodiment a fragment of a rabies
virus, such as a polypeptide of a rabies virus such as the G
protein, is used in step a. In yet another embodiment cells
transfected with rabies virus G protein are used for selecting
binding molecule potentially having neutralizing activity
CA 02775886 2012-04-19
44
against rabies virus. As indicated herein, when cells
expressing rabies virus G protein were included in the
selection method the number of selected neutralizing
antibodies was higher compared to selection methods wherein
only purified rabies virus G protein and/or inactivated rabies
virus was used.
In a further embodiment the method of identifying a
binding molecule potentially having neutralizing activity
against rabies virus as described above further comprises the
step of separating and recovering, and optionally isolating,
human binding molecules containing a variable heavy 3-30
germline gene. A person skilled in the art can identify the
specific germline gene by methods known in the art such as for
instance nucleotide sequencing. The step of separating and
recovering binding molecules containing a variable heavy 3-30
germline gene can be performed before or after step c. As
indicated below-the majority of rabies virus neutralizing
human monoclonal antibodies found in the present invention
comprises this specific Vu germline gene.
Phage display methods for identifying and obtaining
(neutralizing) binding molecules, e.g. antibodies, are by now
well-established methods known by the person skilled in the
art. They are e.g. described in US Patent Number 5,696,108;
Burton and Barbas, 1994; de Kruif et al., 1995b; and Phage
Display: A Laboratory Manual. Edited by: CF Barbas, DR Burton,
JK Scott and GJ Silverman (2001), Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York. All these
references are herewith incorporated herein in their entirety.
For the construction of phage display libraries,
collections of human monoclonal antibody heavy and light chain
variable region genes are expressed on the surface of
bacteriophage, preferably filamentous bacteriophage,
CA 02775886 2012-04-19
particles, in for example single-chain Fv (scFv) or in Fab
format (see de Kruif et al., 1995b). Large libraries of
antibody fragment-expressing phages typically contain more
than 1.0*109 antibody specificities and may be assembled from
5 the immunoglobulin V regions expressed in the B lymphocytes of
immunized- or non-immunized individuals. In a specific
embodiment of the invention the phage library of human binding
molecules, preferably scFv phage library, is prepared from RNA
isolated from cells obtained from a subject that has been
10 vaccinated against rabies or exposed to a rabies virus. RNA
can be isolated from inter alia bone marrow or peripheral
blood, preferably peripheral blood lymphocytes. The subject
can be an animal vaccinated or exposed to rabies virus, but is
preferably a human subject which has been vaccinated or has
15 been exposed to rabies virus. Preferably the human subject has
been vaccinated. A collection of human binding molecules on
the surface of replicable genetic packages, such as a scFv
phage library, as described above is another aspect of the
present invention.
20 Alternatively, phage display libraries may be constructed
from immunoglobulin variable regions that have been partially
assembled in vitro to introduce additional antibody diversity
in the library (semi-synthetic libraries). For example, in
vitro assembled variable regions contain stretches of
25 synthetically produced, randomized or partially randomized DNA
in those regions of the molecules that are important for
antibody specificity, e.g. CDR regions. Rabies virus specific
phage antibodies can be selected from the libraries by
immobilising target antigens such as antigens from rabies
30 virus on a solid phase and subsequently exposing the target
antigens to a phage library to allow binding of phages
expressing antibody fragments specific for the solid phase-
CA 02775886 2012-04-19
46
bound antigen(s). Non-bound phages are removed by washing and
bound phages eluted from the solid phase for infection of
Escherichia coli (E.coli) bacteria and subsequent propagation.
Multiple rounds of selection and propagation are usually
required to sufficiently enrich for phages binding
specifically to the target antigen(s). If desired, before
exposing the phage library to target antigens the phage
library can first be subtracted by exposing the phage library
to non-target antigens bound to a solid phase. Phages may also
be selected for binding to complex antigens such as complex
mixtures of rabies virus proteins or (poly)peptides, host
cells expressing one or more rabies virus proteins or
(poly)peptides of rabies virus, or (inactivated) rabies virus
itself. Antigen specific phage antibodies can be selected from
the library by incubating a solid phase with bound thereon a
preparation of inactivated rabies virus with the phage
antibody library to let for example the scFv or Fab part of
the phage.bind to the proteins/polypeptides of the rabies
virus preparation. After incubation and several washes to
remove unbound and loosely attached phages, the phages that
have bound with their scFv or Fab part to the preparation are
eluted and used to infect Escherichia coli to allow
amplification of the new specificity. Generally, one or more
selection rounds are required to separate the phages of
interest from the large excess of non-binding phages.
Alternatively, known proteins or (poly)peptides of the rabies
virus can be expressed in host cells and these cells can be
used for selection of phage antibodies specific for the
proteins or (poly)peptides. A phage display method using these
host cells can be extended and improved by subtracting non-
relevant binders during screening by addition of an excess of
host cells comprising no target molecules or non-target
CA 02775886 2012-04-19
47
molecules that are similar, but not identical, to the target,
and thereby strongly enhance the chance of finding relevant
binding molecules (This process is referred to as the
MAbstract process. MAbstract is a registered trademark of
Crucell Holland B.V., see also US Patent Number 6,265,150
which is incorporated herein by reference).
In yet a further aspect, the invention provides
compositions comprising at least one binding molecule, at
least one functional variant or fragment thereof, at least one
immunoconjugate according to the invention or a combination
thereof. The compositions may further comprise inter alia
stabilising molecules, such as albumin or polyethylene glycol,
or salts. Preferably, the salts used are salts that retain the
desired biological activity of the human binding molecules and
do not impart any undesired toxicological effects. If
necessary, the human binding molecules of the invention may be
coated in or on a material to protect them from the action of
acids or other natural or non-natural conditions that may
inactivate the binding molecules.
In yet a further aspect, the invention provides
compositions comprising at least one nucleic acid molecule as
defined in the present invention. The compositions may
comprise aqueous solutions such as aqueous solutions
containing salts (e.g., NaCl or salts as described above),
detergents (e.g., SDS) and/or other suitable components.
Furthermore, the present invention pertains to
pharmaceutical compositions comprising at least one n binding
molecule according to the invention, at least one functional
variant or fragment thereof, at least one immunoconjugate
according to the invention, at least one composition according
to the invention, or combinations thereof. The pharmaceutical
CA 02775886 2012-04-19
48
composition of the invention further comprises at least one
pharmaceutically acceptable excipient.
In a preferred embodiment the pharmaceutical composition
according to the invention comprises at least one additional
binding molecule, i.e. the pharmaceutical composition can be a
cocktail/mixture of binding molecules. The pharmaceutical
composition may comprise at least two binding molecules
according to the invention or at least one binding molecule
according to the invention and at least one further anti-
rabies virus binding molecule. Said further binding molecule
preferably comprises a CDR3 region comprising the amino acid
sequence of SEQ ID NO:25. The binding molecule comprising the
CDR3 region comprising the amino acid sequence of SEQ ID NO:25
may be a chimeric or humanized monoclonal antibody or
functional fragment thereof, but preferably it is a human
monoclonal antibody or functional fragment thereof. In an
embodiment, the binding molecule comprises a heavy chain
variable region comprising the amino acid sequence SEQ ID
NO:273. In another embodiment, the binding molecule comprises
a light chain variable region comprising the amino acid
sequence SEQ ID NO:275. In yet another embodiment the binding
molecule comprises a heavy and light chain comprising the
amino acid sequences of SEQ ID NO:123 and SEQ ID NO:125,
respectively. The binding molecules in the pharmaceutical
composition should be capable of reacting with different, non-
competing epitopes of the rabies virus. The epitopes may be
present on the G protein of rabies virus and may be different,
non-overlapping epitopes. The binding molecules should be of
high affinity and should have a broad specificity. Preferably,
they neutralize as many fixed and street strains of rabies
virus as possible. Even more preferably, they also exhibit
neutralizing activity towards other genotypes of the
CA 02775886 2012-04-19
49
Lyssavirus genus or even with other viruses of the rhabdovirus
family, while exhibiting no cross-reactivity with other
viruses or normal cellular proteins. Preferably, the binding
molecule is capable of neutralizing escape variants of the
other binding molecule in the cocktail.
Another aspect of the present invention pertains to a
pharmaceutical composition comprising at least two rabies
virus neutralizing binding molecules, preferably (human)
binding molecules according to the invention, characterized in
that the binding molecules are capable of reacting with
different, non-competing epitopes of the rabies virus. In an
embodiment the pharmaceutical composition comprises a first
rabies virus neutralizing binding molecule which is capable of
reacting with an epitope located in antigenic site I of the
rabies virus G protein and a second rabies virus neutralizing
binding molecule which is capable of reacting with an epitope
located in antigenic site III of the rabies virus G protein.
...The antigenic structure of the rabies glycoprotein was
initially defined by Lafon et al. (1983). The antigenic sites
were identified using a panel of mouse mAbs and their
respective mAb resistant virus variants. Since then, the
antigenic sites have been mapped by identification of the
amino acid mutations in the glycoprotein of mAb resistant
variants (see Seif et al_, 1985; Prehaud et al., 1988; and
Benmansour et al., 1991). The majority of rabies neutralizing
mAbs are directed against antigenic site II (see Benmansour et
al., 1991), which is a discontinuous conformational epitope
comprising of amino acid 34-42 and amino acid 198-200 (see
Prehaud et al., 1988). Antigenic site III is a continuous
conformational epitope at amino acid 330-338 and harbors two
charged residues, K330 and R333, that affect viral
pathogenicity (see Seif et al., 1985; Coulon et al., 1998; and
CA 02775886 2012-04-19
Dietzschold et al., 1983). The conformational antigenic site I
was defined by only one mAb, 509-6, and located at amino acid
231 (see Benmansour et al., 1991; and Lafon et al., 1983).
Antigenic site IV is known to harbor overlapping linear
5 epitopes (see Tordo, 1996; Bunschoten et al., 1989; Luo et
al., 1997; and Ni et al., 1995). Benmansour at al. (1991) also
described the presence of minor site a located at position
342-343, which is distinct from antigenic site III despite its
close proximity. Alignment of the CR-57 epitope with the
10 currently known linear and conformational neutralizing
epitopes on rabies glycoprotein (Figure 10) revealed that the
CR-57 epitope is located in the same region as the
conformational antigenic site I, defined by the single mAb
509-6. Based on nucleotide and amino acid sequences of the
15 glycoprotein of the escape viruses of CR04-098, the epitope
recognized by this antibody appears to be located in the same
region as the continuous conformational antigenic site III.
In a preferred embodiment the pharmaceutical composition
comprises a first rabies virus neutralizing binding molecule
20 comprising at least a CDR3 region, preferably heavy chain CDR3
region, comprising the amino acid sequence of SEQ ID N0:25 and
a second rabies virus neutralizing binding molecule comprising
at least a CDR3 region, preferably heavy chain CDR3 region,
comprising the amino acid sequence selected from the group
25 consisting of SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:14, SEQ ID
NO:15, SEQ ID N0:16 and SEQ ID NO:22. More preferably, the
second rabies virus neutralizing binding molecule comprises at
least a CDR3 region, preferably heavy chain CDR3 region,
comprising the amino acid sequence of SEQ ID NO:14.
30 Preferably, the first rabies virus neutralizing binding
molecule comprises a heavy and light chain comprising the
amino acid sequences of SEQ ID NO:123 and SEQ ID NO:125,
CA 02775886 2012-04-19
51
respectively, and the second rabies.virus neutralizing binding
molecule comprises a heavy and light chain comprising the
amino acid sequences of SEQ ID NO:335 and SEQ ID NO:337,
respectively. Preferably, the heavy and light chain of the
first rabies virus neutralizing binding molecule are encoded
by SEQ ID N0:122 and SEQ ID N0:124, respectively, and the
heavy and light chain of the second rabies virus neutralizing
binding molecule are encoded by SEQ ID N0:334 and SEQ ID
N0:336, respectively.
A pharmaceutical composition comprising two binding
molecules, wherein the pI of the binding molecules is
divergent may have a problem when choosing a suitable buffer
which optimally stabilizes both binding molecules. When
adjusting the pH of the buffer of the composition such that it
increases the stability of one binding molecule, this might
decrease the stability of the other binding molecule. Decrease
of stability or even instability of a binding molecule may
lead to its precipitation or aggregation or to its spontaneous-
degradation resulting in loss of the functionality of the
binding molecule. Therefore, in another aspect the invention
provides a pharmaceutical composition comprising at least two
binding molecules, preferably human binding molecules,
characterized in that the binding molecules have isoelectric
points (pI) that differ less than about 1.5, 1.4, 1.3, 1.2,
1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, preferably less
than (and including) 0.25 pI units from one another. The pI
can be measured experimentally, e.g. by means of isoelectric
focusing, or be calculated based on the amino acid sequence of
the binding molecules. in an embodiment the binding molecules
are binding molecules according to the present invention and
the pharmaceutical composition is a pharmaceutical composition
according to the invention. Preferably, the binding molecules
CA 02775886 2012-04-19
52
are monoclonal antibodies, e.g. human monoclonal antibodies
such as IgG1 antibodies. Preferably, the binding molecules are
capable of binding to and/or neutralizing an infectious agent,
e.g. a virus, a bacterium, a yeast, a fungus or a parasite. In
an embodiment the binding molecules are capable of binding to
and/or neutralizing a lyssavirus, e.g. rabies virus. In a
specific embodiment both binding molecules have a calculated
pI that is in the range between 8.0-9.5, preferably 8.1-9.2,
more preferably 8.2-8.5. Preferably, the binding molecules
have the heavy chain CDR3 region of SEQ ID NO:14 and SEQ ID
NO:25, respectively.
In another embodiment the invention provides a cocktail
of two or more human or other animal binding molecules,
including but not limited to antibodies, wherein at least one
binding molecule is derived from an antibody phage or other
replicable package display technique and at least one binding
molecule is obtainable by a hybridoma technique. When
divergent techniques being used, the selection of binding
molecules having a compatible pI is also very useful in order
to obtain a composition wherein each binding molecule is
sufficiently stable for storage, handling and subsequent use.
In another embodiment the binding molecules present in
the pharmaceutical composition of the invention augment each
others neutralizing activity, i.e. they act synergistically
when combined. In other words, the pharmaceutical compositions
may exhibit synergistic rabies virus, and even lyssavirus,
neutralizing activity. As used herein, the term "synergistic"
means that the combined effect of the binding molecules when
used in combination is greater than their additive effects
when used individually. The ranges and ratios of the components
of the pharmaceutical compositions of the invention should be
determined based on their individual potencies and tested in
CA 02775886 2012-04-19
53
in vitro neutralization assays or animal models such as
hamsters.
Furthermore, the pharmaceutical composition according to
the invention may comprise at least one other therapeutic,
prophylactic and/or diagnostic agent. Said further therapeutic
and/or prophylactic agents may be anti-viral agents such as
ribavirin or interferon-alpha.
The binding molecules or pharmaceutical compositions of
the invention can be tested in suitable animal model systems
prior to use in humans. Such animal model systems include, but
are not limited to, mice, rats, hamsters, monkeys, etc.
Typically, pharmaceutical compositions must be sterile
and stable under the conditions of manufacture and storage. The
human binding molecules, variant or fragments thereof,
immunoconjugates, nucleic acid molecules or compositions of
the present invention can be in powder form for reconstitution
in the appropriate pharmaceutically acceptable excipient
before or at the time of delivery. In the case of sterile
powders for the preparation of sterile injectable solutions,
the preferred methods of preparation are vacuum drying and
freeze-drying (lyophilization) that yield a powder of the
active ingredient plus any additional desired ingredient from
a previously sterile-filtered solution thereof.
Alternatively, the binding molecules, variant or
fragments thereof, immunoconjugates, nucleic acid molecules or
compositions of the present invention can be in solution and
the appropriate pharmaceutically acceptable excipient can be
added and/or mixed before or at the time of delivery to
provide a unit dosage injectable form. Preferably, the
pharmaceutically acceptable excipient used in the present
invention is suitable to high drug concentration, can maintain
proper fluidity and, if necessary, can delay absorption.
CA 02775886 2012-04-19
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The choice of the optimal route of administration of the
pharmaceutical compositions will be influenced by several
factors including the physico-chemical properties of the
active molecules within the compositions, the urgency of the
clinical situation and the relationship of the plasma
concentrations of the active molecules to the desired
therapeutic effect. For instance, if necessary, the human
binding molecules of the invention can be prepared with
carriers that will protect them against rapid release, such as
a controlled release formulation, including implants,
transdermal patches, and microencapsulated delivery systems.
Biodegradable, biocompatible polymers can inter alia be used,
such as ethylene vinyl acetate, poly-anhydrides, poly-glycolic
acid, collagen, poly-orthoesters, and poly-lactic acid.
Furthermore, it may be necessary to coat the human binding
molecules with, or co-administer the binding molecules with, a
material or compound that prevents the inactivation of the
human binding molecules. For example, the human binding
molecules may be administered to a subject in an appropriate
carrier, for example, liposomes, or a diluent.
The routes of administration can. generally be divided
into two main categories, oral and parenteral administration.
The preferred administration of the human binding molecules
and pharmaceutical compositions of the invention is into and
around the wound and intramuscularly in the gluteal region.
Formulations of the human binding molecules and pharmaceutical
compositions are dependent on the routes of administration.
In a further aspect, the binding molecules, functional
variants, immunoconjugates, compositions, or pharmaceutical
compositions of the invention can be used as a medicament. So,
a method of treatment and/or prevention of a lyssavirus
infection using the human binding molecules, functional
CA 02775886 2012-04-19
variants, immunoconjugates, compositions, or pharmaceutical
compositions of the invention is another part of the present
invention. The lyssavirus can be a virus from any of the known
genotypes, but is preferably rabies virus. The above-mentioned
5 molecules or compositions can be used in the postexposure
prophylaxis of rabies.
The molecules or compositions mentioned above may be
employed in conjunction with other molecules useful in
diagnosis, prophylaxis and/or treatment of rabies virus. They
10 can be used in vitro, ex vivo or in vivo. For instance, the
human binding molecules, functional variants, immunoconjugates
or pharmaceutical compositions of the invention can be co-
administered with a vaccine against rabies. Alternatively, the
vaccine may also be administered before or after
15 administration of the molecules or compositions of the
invention. Administration of the molecules or compositions of
the invention with a vaccine is suitable for post exposure
prophylaxis. Rabies-vaccines inclu4e, but are not limited to,
purified chick embryo cell (PCEC) vaccine (RabAvert), human
20 diploid cell vaccine (HDCV; Imovax vaccine) or rabies vaccine
adsorbed (RVA).
The molecules are typically formulated in the
compositions and pharmaceutical compositions of the invention
in a therapeutically or diagnostically effective amount.
25 Dosage regimens can be adjusted to provide the optimum desired
response (e.g., a therapeutic response). A suitable dosage
range may for instance be 0.1-100 IU/kg body weight,
preferably 1.0-50 IU/kg body weight and more preferably 10-30
IU/kg body weight, such as 20 IU/kg body weight.
30 Preferably, a single bolus of the binding molecules or
pharmaceutical compositions of the invention are administered.
The molecules and pharmaceutical compositions according to the
CA 02775886 2012-04-19
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present invention are preferably sterile. Methods to render
these molecules and compositions sterile are well known in the
art. The dosing regimen of post exposure prophylaxis is
administration of five doses of rabies vaccine intramuscularly
in the deltoid muscle on days 0, 3, 7, 14 and 28 days after
exposure in individuals not previously immunized against
rabies virus. The human binding molecules or pharmaceutical
compositions according to the invention should be administered
into and around the wounds on day 0 or otherwise as soon as
possible after exposure, with the remaining volume given
intramuscularly at a site distant from the vaccine. Non-
vaccinated individuals are advised to be administered anti-
rabies virus human binding molecules, but it is clear to the
skilled artisan that vaccinated individuals in need of such
treatment may also be administered anti-rabies virus human
binding molecules.
In another aspect, the invention concerns the use of
binding molecules or functional variants thereof,.,
immunoconjugates according to the invention, nucleic acid
molecules according to the invention, compositions or
pharmaceutical compositions according to the invention in the
preparation of a medicament for the diagnosis, prophylaxis,
treatment, or combination thereof, of a condition resulting
from an infection by a lyssavirus. The lyssavirus can be a
virus from any of the known genotypes but is preferably rabies
virus. Preferably the molecules mentioned above are used in
the preparation of a medicament for the post exposure
prophylaxis of rabies.
Next to that, kits comprising at least one binding
molecule according to the invention, at least one functional
variant thereof according to the invention, at least one
immunoconjugate according to the invention, at least one
CA 02775886 2012-04-19
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nucleic acid molecule according to the invention, at least one
composition according to the invention, at least one
pharmaceutical composition according to the invention, at
least one vector according to the invention, at least one host
according to the invention or a combination thereof are also a
part of the present invention. Optionally, the above described
components of the kits of the invention are packed in suitable
containers and labeled for diagnosis, prophylaxis and/or
treatment of the indicated conditions. The above-mentioned
components may be stored in unit or multi-dose containers, for
example, sealed ampoules, vials, bottles, syringes, and test
tubes, as an aqueous, preferably sterile, solution or as a
lyophilized, preferably sterile, formulation for
reconstitution. The containers may be formed from a variety of
materials such as glass or plastic and may have a sterile
access port (for example the container may be an intravenous
solution bag or a vial having a stopper pierceable by a
hypodermic injection needle). The kit may further comprise
more containers comprising a pharmaceutically acceptable
buffer, such as phosphate-buffered saline, Ringer's solution
and dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including
other buffers, diluents, filters, needles, syringes, culture
medium for one or more of the suitable hosts. Associated with
the kits can be instructions customarily included in
commercial packages of therapeutic, prophylactic or diagnostic
products, that contain information about for example the
indications, usage, dosage, manufacture, administration,
contraindications and/or warnings concerning the use of such
therapeutic, prophylactic or diagnostic products.
Currently, HRIG products are used for post exposure
prophylaxis of rabies. An adult dose of HRIG of 1500 IU (75 kg
CA 02775886 2012-04-19
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individual, 20 IU/kg) is only available in a volume of 10 ml.
More concentrated HRIG products are not possible as the
currently obtainable 10 ml dose contains 1-1.5 gram of total
IgG. In view thereof the current HRIG products have two
drawbacks. Firstly, it is often not anatomically feasible to
administer the recommended full dose in and around the bite
wounds and secondly the administration of the current volume
dose of HRIG is associated with significant pain. The present
invention gives a solution to these drawbacks as it provides a
pharmaceutical composition comprising a full adult dose in a
volume of approximately 2 ml or less, if desirable. Such a
pharmaceutical composition may comprise for example two
binding molecules capable of neutralizing rabies virus,
preferably CR57 and CR04-098. The pharmaceutical composition
further comprises a pharmaceutically acceptable excipient and
has a volume of around 2 ml. More is also possible, but less
desirable in view of the pain associated with injecting larger
volumes. Less than 2 ml. is also possible. The pharmaceutical
composition comprises the full adult dose (in IU) necessary
for successful post exposure prophylaxis. In an embodiment the
pharmaceutical composition is stored in 10 ml vial such as for
instance a 10 ml ready-to-use vial (type I glass) with a
stopper. By providing a 10 ml vial the option is given to
dilute the pharmaceutical composition towards a higher volume
in case an individual presents a large wound surface area. The
invention also provides a kit comprising at least a container
(such as a vial) comprising the pharmaceutical composition.
The kit may further comprise a second container which holds a
diluent suitable for diluting the pharmaceutical composition
towards a higher volume. Suitable diluents include, but are
not limited to, the pharmaceutically acceptable excipient of
the pharmaceutical composition and a saline solution.
CA 02775886 2012-04-19
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Furthermore, the kit may comprise instructions for diluting
the pharmaceutical composition and/or instructions for
administering the pharmaceutical composition, whether diluted
or not.
The invention further pertains to a method of detecting a
rabies virus in a sample, wherein the method comprises the
steps of a) contacting a sample with a diagnostically
effective amount of a binding molecule, a functional variant
or an immunoconjugate according to the invention, and b)
determining whether the binding molecule, functional variant,
or immunoconjugate specifically binds to a molecule of the
sample. The sample may be a biological sample including, but
not limited to blood, serum, tissue or other biological
material from (potentially) infected subjects. The
(potentially) infected subjects may be human subjects, but
also animals that are suspected as carriers of rabies virus
might be tested for the presence of rabies virus using the
human binding molecules, functional variants or
immunoconjugates of the invention. The sample may first be
manipulated to make it more suitable for the method of
detection. Manipulation means inter alia treating the sample
suspected to contain and/or containing rabies virus in such a
way that the rabies virus will disintegrate into antigenic
components such as proteins, (poly)peptides or other antigenic
fragments. Preferably, the binding molecules, functional
variants or immunoconjugates of the invention are contacted
with the sample under conditions which allow the formation of
an immunological complex between the human binding molecules
and rabies virus or antigenic components thereof that may be
present in the sample. The formation of an immunological
complex, if any, indicating the presence of rabies virus in
the sample, is then detected and measured by suitable means.
CA 02775886 2012-04-19
Such methods include, inter alia, homogeneous and
heterogeneous binding immunoassays, such as radioimmunoassays
(RIA), ELISA, immunofluorescence, immunohistochemistry, FACS,
BIACORE and Western blot analyses.
5 Furthermore, the binding molecules of the invention can
be used to identify epitopes of rabies virus proteins such as
the G protein. The epitopes can be linear, but also structural
and/or conformational. In one embodiment, binding of binding
molecules of the invention to a series of overlapping
10 peptides, such as 15-mer peptides, of a protein from rabies
virus such as the rabies virus G protein can be analyzed by
means of PEPSCAN analysis (see inter alia WO 84/03564, WO
93/09872, Slootstra et al. 1996). The binding of human binding
molecules to each peptide can be tested in a PEPSCAN-based
15 enzyme-linked immuno assay (ELISA). In another embodiment, a
random peptide library comprising peptides from rabies virus'
proteins can be screened for peptides capable of binding to
the human binding molecules of the invention. In the above
assays the use of rabies virus neutralizing human binding
20 molecules may identify one or more neutralizing epitopes. The
peptides/epitopes found can be used as vaccines and for the
diagnosis of rabies.
In a further aspect, the invention provides a method of
screening a binding molecule or a functional variant of a
25 binding molecule for specific binding to a different,
preferably non-overlapping epitope of rabies virus as the
epitope bound by a binding molecule or functional variant of
the invention, wherein the method comprises the steps of a)
contacting a binding molecule or a functional variant to be
30 screened, a binding molecule or functional variant of the
invention and rabies virus or a fragment thereof (such as for
instance the rabies virus G protein), b) measure if the
CA 02775886 2012-04-19
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binding molecule or functional variant to be screened is
capable of competing for specifically binding to the rabies
virus or fragment thereof with the binding molecule or
functional variant of the invention. If no competition is
measured the binding molecules or functional variants to be
screened bind to a different epitope. In a specific embodiment
of the above screening method, human binding molecules or
functional variants thereof may be screened to identify human
binding molecules or functional variants capable of binding a
different epitope than the epitope recognized by the binding
molecule comprising the CDR3 region comprising the amino acid
sequence of SEQ ID NO:25. Preferably, the epitopes are non-
overlapping or non-competing. It is clear to the skilled
person that the above screening method can also be used to
identify binding molecules or functional variants thereof
capable of binding to the same epitope. In a further step it
may be determined if the screened binding molecules that are
not capable of competing for specifically binding to the
rabies virus or fragment thereof have neutralizing activity.
It may also be determined if the screened binding molecules
that are capable of competing for specifically binding to the
rabies virus or fragment thereof have neutralizing activity.
Neutralizing anti-rabies virus binding molecules or functional
variants thereof found in the screening method are another
part of the present invention. In the screening method
"specifically binding to the same epitope" also contemplates
specific binding to substantially or essentially the same
epitope as the epitope bound by the human binding molecules of
the invention. The capacity to block, or compete with, the
binding of the human binding molecules of the invention to
rabies virus typically indicates that a binding molecule to be
screened binds to an epitope or binding site on the rabies
CA 02775886 2012-04-19
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virus that structurally overlaps with the binding site on the
rabies virus that is immunospecifically recognized by the
binding molecules of the invention. Alternatively, this can
indicate that a binding molecule to be screened binds to an
epitope or binding site which is sufficiently proximal to the
binding site immunospecifically recognized by the binding
molecules of the invention to sterically or otherwise inhibit
binding of the binding molecules of the invention to rabies
virus or a fragment thereof.
In general, competitive inhibition is measured by means
of an assay, wherein an antigen composition, i.e. a
composition comprising rabies virus or fragments (such as G
proteins) thereof, is admixed with reference binding molecules
and binding molecules to be screened. In an embodiment the
reference binding molecule may be one of the human binding
molecules of the invention and the binding molecule to be
screened may be another human binding molecule of the
invention. In another embodiment the reference binding
molecule may be the binding molecule comprising the CDR3
region comprising the amino acid sequence of SEQ ID NO:25 and
the binding molecule to be screened may be one of the human
binding molecules of the invention. In yet another embodiment
the reference binding molecule may be one of-the human binding
molecule of the invention and the binding molecule to be
screened may be the binding molecule comprising the CDR3
region comprising the amino acid sequence of SEQ ID NO:25.
Usually, the binding molecules to be screened are present in
excess. Protocols based upon ELISAs are suitable for use in
such simple competition studies. In certain embodiments, one
may pre-mix the reference binding molecules with varying
amounts of the binding molecules to be screened (e.g., 1:10,
1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90 or 1:100) for a
CA 02775886 2012-04-19
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period of time prior to applying to the antigen composition.
In other embodiments, the reference binding molecules and
varying amounts of binding molecules to be screened can simply
be admixed during exposure to the antigen composition. In any
event, by using species or isotype secondary antibodies one
will be able to detect only the bound reference binding
molecules, the binding of which will be reduced by the
presence of a binding molecule to be screened that recognizes
substantially the same epitope. In conducting a binding
molecule competition study between a reference binding
molecule and any binding molecule to be screened (irrespective
of species or isotype), one may first label the reference
binding molecule with a detectable label, such as, e.g.,
biotin, an enzymatic, a radioactive or other label to enable
subsequent identification. In these cases, one would pre-mix
or incubate the labeled reference binding molecules with the
binding molecules to be screened at various ratios (e.g.,
1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90 or 1:100)
and (optionally after a suitable period of time) then assay
the reactivity of the labeled reference binding molecules and
compare this with a control value in which no potentially
competing binding molecule was included in the incubation. The
assay may again be any one of a range of immunological assays
based upon antibody hybridization, and the reference binding
molecules would be detected by means of detecting their label,
e.g., using streptavidin in the case of biotinylated reference
binding molecules or by using a chromogenic substrate in
connection with an enzymatic label (such as 3,3'5,5'-
tetramethylbenzidine (TMB) substrate with peroxidase enzyme)
or by simply detecting a radioactive label. A binding molecule
to be screened that binds to the same epitope as the reference
binding molecule will be able to effectively compete for
CA 02775886 2012-04-19
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binding and thus will significantly reduce reference binding
molecule binding, as evidenced by a reduction in bound label.
Binding molecules binding different non-competing epitopes
will show no reduction. The reactivity of the (labeled)
reference binding molecule in the absence of a completely
irrelevant binding molecule would be the control high value.
The control low value would be obtained by incubating the
labeled reference binding molecule with unlabelled reference
binding molecules of exactly the same type, when competition
would occur and reduce binding of the labeled reference
binding molecule. In a test assay, a significant reduction in
labeled reference binding molecule reactivity in the presence
of a binding molecule to be screened is indicative of a
binding molecule that recognizes the same epitope, i.e., one
that "cross-reacts" with the labeled reference binding
molecule. If no reduction is shown, the binding molecule may
bind a different non-competing epitope.
Binding molecules identified,,by these competition assays
("competitive binding molecules") include, but are not limited
to, antibodies, antibody fragments and other binding agents
that bind to an epitope or binding site bound by the reference
binding molecule as well as antibodies, antibody fragments and
other binding agents that bind to an epitope or binding site
sufficiently proximal to an epitope bound by the reference
binding molecule for competitive binding between the binding
molecules to be screened and the reference binding molecule to
occur. Preferably, competitive binding molecules of the
invention will, when present in excess, inhibit specific
binding of a reference binding molecule to a selected target
species by at least 10%, preferably by at least 25%, more
preferably by at least 50%, and most preferably by at least
75%-90% or even greater. The identification of one or more
CA 02775886 2012-04-19
competitive binding molecules that bind to about,
substantially, essentially or at the same epitope as the
binding molecules of the invention is a straightforward
technical matter. As the identification of competitive binding
5 molecules is determined in comparison to a reference binding
molecule, it will be understood that actually determining the
epitope to which the reference binding molecule and the
competitive binding molecule bind is not in any way required
in order to identify a competitive binding molecule that binds
10 to the same or substantially the same epitope as the reference
binding molecule. Alternatively, binding molecules binding to
different non-competing epitopes identified by these
competition assays may also include, but are not limited to,
antibodies, antibody fragments and other binding agents.
15 In another aspect the invention provides a method of
identifying a binding molecule potentially having neutralizing
activity against an infectious agent causing disease in a
living being,,or a nucleic acid molecule encoding a binding,
molecule potentially having neutralizing activity against an
20 infectious agent causing disease in a living being, wherein
the method comprises the steps of a) contacting a collection
of binding molecules on the surface of replicable genetic
packages with at least a cell expressing a protein of the
infectious agent causing disease in a living being on its
25 surface under conditions conducive to binding, b) separating
and recovering binding molecules that bind to the cell
expressing a protein of the infectious agent causing disease
in a living being on its surface from binding molecules that
do not bind said cell, c) isolating at least one recovered
30 binding molecule, d) verifying if the binding molecule
isolated has neutralizing activity against the infectious
agent causing disease in a living being. The cell expressing a
CA 02775886 2012-04-19
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protein of the infectious agent causing disease in a living
being on its surface can be a cell transfected with the
protein. A person skilled in the art is aware that antigens of
the infectious agent other than proteins can also be
successfully used in the method. In a specific embodiment the
cell is a PER.C6 cell. However, other (El-immortalized) cell
lines could also be used to express the proteins such as BHK,
CHO, NSO, HEK293, or 911 cells. In an embodiment the binding
molecule is human. The infectious agent can be a virus, a
bacterium, a yeast, a fungus or a parasite. In an embodiment
the protein is a protein normally expressed on the surface of
the infectious agent or comprises at least a part of a protein
that is surface accessible. In a specific embodiment the
collection of binding molecules on the surface of replicable
genetic packages are subtracted/counterselected with the cells
used for expressing of the protein of the infectious agent,
i.e. the cells are identical to the cells used in step a with
the proviso that they do not express the protein of the
infectious agent on their surface. The cells used for
subtraction/counterselection can be untransfected cells.
Alternatively, the cells can be transfected with a protein or
(extracellular) part thereof that is similar and/or highly
homologous in sequence or structure with the respective
protein of the infectious agent and/or that is derived from an
infectious agent of the same family or even genus.
Another aspect of the invention pertains to a binding
molecule as defined herein having rabies virus neutralizing
activity, characterized in that the human binding molecule
comprises at least a heavy chain CDR3 region comprising the
amino acid sequence comprising SEQ ID NO:25 and further
characterized in that the human binding molecule has a rabies
virus neutralizing activity of at least 2500 IU/mg protein.
CA 02775886 2012-04-19
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More preferably, said human binding molecule has a rabies
virus neutralizing activity of at least 2800 IU/mg protein, at
least 3000 IU/mg protein, at least 3200 IU/mg protein, at
least 3400 IU/mg protein, at least 3600 IU/mg protein, at
least 3800 IU/mg protein, at least 4000 IU/mg protein, at
least 4200 IU/mg protein, at least 4400 IU/mg protein, at
least 4600 IU/mg protein, at least 4800 IU/mg protein, at
least 5000 IU/mg protein, at least 5200 IU/mg protein, at
least 5400 IU/mg protein. The neutralizing activity of the
binding molecule was measured by an in vitro neutralization
assay (modified RFFIT (rapid fluorescent focus inhibition
test)). The assay is described in detail in the example
section infra.
In an embodiment the binding molecule comprises a
variable heavy chain comprising the amino acid sequence
comprising SEQ ID NO:273. In another embodiment the binding
molecule comprises a heavy chain comprising the amino acid
sequence comprising SEQ ID NO:123. The variable light chain of
the binding molecule may comprise the amino acid sequence
comprising SEQ ID NO:275. The light chain of the binding
molecule may comprise the amino acid sequence comprising SEQ
ID NO:125.
A nucleic acid molecule encoding the binding molecules as
described above is also a part of the present invention.
Preferably, the nucleic acid molecule comprises the nucleotide
sequence comprising SEQ ID NO:122. In addition the nucleic
acid molecule may also comprise the nucleotide sequence
comprising SEQ ID NO:124. A vector comprising the nucleic acid
molecules and a host cell comprising such a vector are also
provided herein. Preferably, the host cell is a mammalian cell
such as a human cell. Examples of cells suitable for
production of human binding molecules are inter alia HeLa,
CA 02775886 2012-04-19
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911, AT1080, A549, 293 and HEK293T cells. Preferred mammalian
cells are human retina cells such as 911 cells or the cell
line deposited at the European Collection of Cell Cultures
(ECACC), CAMR, Salisbury, Wiltshire SP4 OJG, Great Britain on
29 February 1996 under number 96022940 and marketed under the
trademark PER.C6 (PER.C6 is a registered trademark of Crucell
Holland B.V.). For the purposes of this application "PER.C6"
refers to cells deposited under number 96022940 or ancestors,
passages up-stream or downstream as well as descendants from
ancestors of deposited cells, as well as derivatives of any of
the foregoing.
EXAMPLES
To illustrate the invention, the following examples are
provided. The examples are not intended to limit the scope of
the invention in any way.
Example 1
Epitope recognition of human anti-rabies antibodies CR-57 and
CR-JB
To address whether the human monoclonal antibodies called
CR-57 and CR-JB recognize non-overlapping, non-competing
epitopes, escape viruses of the human monoclonal antibodies
called CR-57 and CR-JB were generated. CR-57 and CR-JB were
generated essentially as described (see Jones et al., 2003),
via introduction of the variable heavy and light chain coding
regions of the corresponding antibody genes into a single
human IgGl expression vector named pcDNA3002(Neo). The
resulting vectors pgS057C11 and pgSOJBC11 were used for
transient expression in cells from the cell line deposited at
the European Collection of Cell Cultures (ECACC), CAMR,
CA 02775886 2012-04-19
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Salisbury, Wiltshire SP4 OJG, Great Britain on 29 February
1996 under number 96022940 and marketed under the trademark
PER.C6 . The nucleotide and amino acid sequences of the heavy
and light chains of these antibodies are shown in SEQ ID
NO:122 - 129, respectively. Serial dilutions (0.5 ml) of
rabies virus strain CVS-11 (dilutions ranging from 10-1 - 10-8)
were incubated with a constant amount (-4 IU/ml) of antibody
CR-57 or CR-JB (0.5 ml) for 1 hour at 37 C/5% CO2 before
addition to wells containing mouse neuroblastoma cells (MNA
cells) or BSR cells (Baby Hamster Kidney-like cell line).
After 3 days of selection in the presence of either human
monoclonal antibody CR-57 or CR-JB, medium (1 ml) containing
potential escape viruses was harvested and stored at 4 C until
further use. Subsequently, the cells were acetone-fixed for 20
minutes at 4 C, and stained overnight at 37 C/5% C02 with an
anti-rabies N-FITC antibody conjugate (Centocor). The number
of foci per well were scored by immunofluorescence and medium
of wells containing one to six foci were chosen for virus
amplification. All E57 escape viruses were generated from 1
single focus with the exception of E57B1 (3 foci). EJB escape
viruses were isolated from 1 focus (EJB3F), 3 foci (EJB2B, 4
foci (EJB2C), 5 foci (EJB2E, 2F), or 6 foci (EJB2D),
respectively. Each escape virus was first amplified on a small
scale on BSR or MNA cells depending on their growth
characteristics. These small virus batches were then used to
further amplify the virus on a large scale on MNA or BSR
cells. Amplified virus was then titrated on MNA cells to
determine the titer of each escape virus batch as well as the
optimal dilution of the escape virus (giving 80-100 %
infection after 24 hours) for use in a virus neutralization
assay.
CA 02775886 2012-04-19
Modified RFFIT (rapid fluorescent focus inhibition test)
assays were performed to examine cross-protection of E57 (the
escape viruses of CR-57) and EJB (the escape viruses of CR-JB)
with CR-JB and CR-57, respectively. Therefore, CR-57 or CR-JB
5 was diluted by serial threefold dilutions starting with a 1:5
dilution. Rabies virus (strain CVS-11) was added to each
dilution at a concentration that gives 80-100% infection.
Virus/IgG mix was incubated for 1 hour at 37 C/5% CO2 before
addition to MNA cells. 24 hours post-infection (at 34 C/5% CO2)
10 the cells were acetone-fixed for 20 minutes at 4 C, and stained
for minimally 3 hours with an anti-rabies virus N-FITC
antibody conjugate (Centocor). The wells were then analyzed
for rabies virus infection under a fluorescence microscope to
determine the 50% endpoint dilution. This is the dilution at
15 which the virus infection is blocked by 50% in this assay. To
calculate the potency, an international standard (Rabies
Immune Globulin Lot R3, Reference material from the laboratory
of Standards and Testing DMPQ/CBER/FDA) was included in each
modified RFFIT. The 50% endpoint dilution of this standard
20 corresponds with a potency of 2 IU/ml. The neutralizing
potency of the single human monoclonal antibodies CR-57 and
CR-JB as well as the combination of these antibodies were
tested.
EJB viruses were no longer neutralized by CR-JB or CR-57
25 (see Table 1), suggesting both antibodies bound to and induced
amino acid changes in similar regions of the rabies virus
glycoprotein. E57 viruses were no longer neutralized by CR-57,
whereas 4 out of 6 E57 viruses were still neutralized by CR-
JB, although with a lower potency (see Table 1). A mixture of
30 the antibodies CR-57 and CR-JB (in a 1:1 IU/mg ratio) gave
CA 02775886 2012-04-19
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similar results as observed with the single antibodies (data
not shown).
To identify possible mutations in the rabies virus
glycoprotein the nucleotide sequence of the glycoprotein open
reading frame (ORF) of each of the EJB and E57 escape viruses
was determined. Viral RNA of each of the escape viruses and
CVS-11 was isolated from virus-infected MNA cells and
converted into cDNA by standard RT-PCR. Subsequently, cDNA was
used for nucleotide sequencing of the rabies virus
glycoprotein ORFs in order to identify mutations.
Both E57 and EJB escape viruses showed mutations in the
same region of the glycoprotein (see Figure 1 and 2,
respectively; see for all the sequences described in Figures 1
and 2 SEQ ID NO:130 - 151). This indicates that both
antibodies recognize overlapping epitopes. From the above can
be concluded that the combination of CR-57 and CR-JB in a
cocktail does not prevent the escape of neutralization-
resistant variants and is therefore not an ideal
immunoglobulin preparation for rabies post exposure
prophylaxis.
Example 2
Construction of a ScFv phage display library using peripheral
blood lymphocytes of rabies vaccinated donors.
From four rabies vaccinated human subjects 50 ml blood
was drawn from a vein one week after the last boost.
Peripheral blood lymphocytes (PBL) were isolated from these
blood samples using Ficoll cell density fractionation. The
blood serum was saved and frozen at -20 C. The presence of
anti-rabies antibodies in the sera was tested positive using a
FACS staining on rabies virus glycoprotein transfected 293T
cells. Total RNA was prepared from the PBL using organic phase
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separation (TRIZOLTM) and subsequent ethanol precipitation. The
obtained RNA was dissolved in DEPC-treated ultrapure water and
the concentration was determined by OD 260 nm measurement.
Thereafter, the RNA was diluted to a concentration of 100
ng/pl. Next, 1 pg of RNA was converted into cDNA as follows:
To 10 ul total RNA, 13 pl DEPC-treated ultrapure water and 1
u1 random hexamers (500 ng/pl) were added and the obtained
mixture was heated at 65 C for 5 minutes and quickly cooled on
wet-ice. Then, 8 p1 5X First-Strand buffer, 2 pl dNTP (10 mM
each), 2 ul DTT (0.1 M), 2 pl Rnase-inhibitor (40 U/ul) and 2
ul Supers criptTMIII MMLV reverse transcriptase (200 U/pl) were
added to the mixture, incubated at room temperature for 5
minutes and incubated for 1 hour at 50 C. The reaction was
terminated by heat inactivation, i.e. by incubating the
mixture for 15 minutes at 75 C.
The obtained cDNA products were diluted to a final volume
of 200 ul with DEPC-treated ultrapure water. The OD 260 nm of
a 50 times diluted solution (in 10 mM Tris buffer) of the
dilution of the obtained cDNA products gave a value of 0.1.
For each donor 5 to 10 pl of the diluted cDNA products
were used as template for PCR amplification of the
immunoglobulin gamma heavy chain family and kappa or lambda
light chain sequences using specific oligonucleotide primers
(see Tables 2-7). PCR reaction mixtures contained, besides the
diluted cDNA products, 25 pmol sense primer and 25 pmol anti-
sense primer in a final volume of 50 pl of 20 mM Tris-HC1 (pH
8.4), 50 mM KC1, 2.5 mM MgC12, 250 pM dNTPs and 1.25 units Taq
polymerase. In a heated-lid thermal cycler having a
temperature of 96 C, the mixtures obtained were quickly melted
for 2 minutes, followed by 30 cycles of: 30 seconds at 96 C,
30 seconds at 60 C and 60 seconds at 72 C.
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In a first round amplification, each of seventeen light
chain variable region sense primers (eleven for the lambda
light chain (see Table 2) and six for the kappa light chain
(see Table 3) were combined with an anti-sense primer
recognizing the C-kappa called HuCk 5'-ACACTCTCCCCTGTTGAAGCT
CTT-3' (see SEQ ID NO:152) or C-lambda constant region HuCX2
5'-TGAACATTCTGTAGGGGCCACTG-3' (see SEQ ID NO:153) and HuCX7
5'-AGAGCATTCTGCAGGGGCCACTG-3' (see SEQ ID NO:154) (the HuCA2
and HuCX7 anti-sense primers were mixed to equimolarity before
use), yielding 4 times 17 products of about 600 basepairs.
These products were purified on a 2% agarose gel and isolated
from the gel using Qiagen gel-extraction columns. 1/10 of each
of the isolated products was used in an identical PCR reaction
as described above using the same seventeen sense primers,
whereby each lambda light chain sense primer was combined with
one of the three Jlambda-region specific anti-sense primers
and each kappa light chain sense primer was combined with one
of the five Jkappa-region specific anti-sense primers. The
primers used in the second amplification were extended with
restriction sites (see Table 4) to enable directed cloning in
the phage display vector PDV-C06 (see Figure 3 and SEQ ID
NO:155). This resulted in 4 times 63 products of approximately
350 basepairs that were pooled to a total of 10 fractions.
This number of fractions was chosen to maintain the natural
distribution of the different light chain families within the
library and not to over or under represent certain families.
The number of alleles within a family was used to determine
the percentage of representation within a library (see Table
5). In the next step, 2.5 pg of pooled fraction and 100 pg
PDV-C06 vector were digested with Sall and NotI and purified
from gel. Thereafter, a ligation was performed overnight at
16 C as follows. To 500 ng PDV-C06 vector 70 ng pooled
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fraction was added in a total volume of 50 pl ligation mix
containing 50 mM Tris-HC1 (pH 7.5), 10 mM MgC12, 10 mM DTT, 1
mM ATP, 25 Vg/ml BSA and 2.5 pl T4 DNA Ligase (400 U/pl). This
procedure was followed for each pooled fraction. The ligation
mixes were purified by phenol/chloroform, followed by a
chloroform extraction and ethanol precipitation, methods well
known to the skilled artisan. The DNA obtained was dissolved
in 50 pl ultrapure water and per ligation mix two times 2.5 pl
aliquots were electroporated into 40 pl of TG1 competent E.
coli bacteria according to the manufacturer's protocol
(Stratagene). Transformants were grown overnight at 37 C in a
total of 30 dishes (three dishes per pooled fraction;
dimension of dish: 240 mm x 240 mm) containing 2TY agar
supplemented with 50 jig/ml ampicillin and 4.5% glucose. A
(sub)library of variable light chain regions was obtained by
scraping the transformants from the agar plates. This
(sub)library was directly used for plasmid DNA preparation
using a QiagenTm QIAFilter MAXI prep kit.
For each donor the heavy chain immunoglobulin sequences
were amplified from the same cDNA preparations in a similar
two round PCR procedure and identical reaction parameters as
described above for the light chain regions with the proviso
that the primers depicted in Tables 6 and 7 were used. The
first amplification was performed using a set of nine sense
directed primers (see Table 6; covering all families of heavy
chain variable regions) each combined with an IgG specific
constant region anti-sense primer called HuCIgG 5'-GTC CAC CTT
GGT GTT GCT GGG CTT-3' (SEQ ID NO:156) yielding four times
nine products of about 650 basepairs. These products were
purified on a 2% agarose gel and isolated from the gel using
Qiagen gel-extraction columns. 1/10 of each of the isolated
products was used in an identical PCR reaction as described
CA 02775886 2012-04-19
above using the same nine sense primers, whereby each heavy
chain sense primer was combined with one of the four JH-region
specific anti-sense primers. The primers used in the second
round were extended with restriction sites (see Table 7) to
5 enable directed cloning in the light chain (sub)library
vector. This resulted per donor in 36 products of
approximately 350 basepairs. These products were pooled for
each donor per used (VH) sense primer into nine fractions. The
products obtained were purified using Qiagen PCR Purification
10 columns. Next, the fractions were digested with Sfil and XhoI
and ligated in the light chain (sub)library vector, which was
cut with the same restriction enzymes, using the same ligation
procedure and volumes as described above for the light chain
(sub)library. Alternatively, the fractions were digested with
15 Ncol and XhoI and ligated in the light chain vector, which was
cut with the same restriction enzymes, using the same ligation
procedure and volumes as described above for the light chain
(sub)library. Ligation purification and subsequent
transformation of the resulting definitive library was also
20 performed as described above for the light chain (sub)library
and at this point the ligation mixes of each donor were
combined per VH pool. The transformants were grown in 27
dishes (three dishes per pooled fraction; dimension of dish:
240 mm x 240 mm) containing 2TY agar supplemented with 50
25 jig/ml ampicillin and 4.5% glucose. All bacteria were harvested
in 2TY culture medium containing 50 ug/ml ampicillin and 4.5%
glucose, mixed with glycerol to 15% (v/v) and frozen in 1.5 ml
aliquots at -80 C. Rescue and selection of each library were
performed as described below.
Example 3
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Selection of phages carrying single chain Fv fragments
specifically recognizing rabies virus glycoprotein
Antibody fragments were selected using antibody
phage display libraries, general phage display technology
and MAbstract technology, essentially as described in
US Patent Number 6,265,150 and in WO 98/15833. The
antibody phage libraries used were two different
semi-synthetic scFv phage libraries (JK1994 and WT2000)
and the immune scFv phage libraries (RAB-03-GO1 and
RAB-04-GO1) prepared as described in Example 2 above.
The first semi-synthetic scFv phage library (JK1994)
has been described in de Kruif et al. (1995b), the
second one (WT2000) was build essentially as described in de
Kruif et al. (1995b). Briefly, the library has a semi-
synthetic format whereby variation was incorporated in the
heavy and light chain V genes using degenerated
oligonucleotides that incorporate variation within CDR
regions. Only VH3 heavy chain genes were used, in combination
with kappa- and lambda light chain genes. CDR1 and CDR3 of the
heavy chain and CDR3 of the light chain were recreated
synthetically in a PCR-based approach similar as described in
de Kruif et al. (1995b). The thus created V region genes were
cloned sequentially in scFv format in a phagemid vector and
amplified to generate a phage library as described before.
Furthermore, the methods and helper phages as described in WO
02/103012 were used in the present invention. For identifying phage
antibodies recognizing rabies virus glycoprotein phage selection
experiments were performed using whole rabies virus (rabies virus
Pitman-Moore strain) inactivated by treatment with beta-propiolactone,
purified rabies virus glycoprotein (rabies
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virus ERA strain), and/or transfected cells expressing rabies
virus G protein (rabies virus ERA strain).
The G protein was purified from the rabies virus ERA
strain as follows. To a virus solution, 1/10 volume of 10%
octyl-beta-glucopyranoside was added and mixed gently. Upon a
30 minutes incubation at 4 C the virus sample was centrifuged
(36,000 rpm, 4 C) in a SW51 rotor. The supernatant was
collected and dialyzed overnight at 4 C against 0.1 M
Tris/EDTA. Subsequently, the glycoprotein was collected from
the dialysis chamber, aliquotted, and stored at -80 C until
further use. The protein concentration was determined by OD
280 nm and the integrity of the G protein was analyzed by SDS-
PAGE.
Whole inactivated rabies virus or rabies virus G protein
were diluted in phosphate buffered saline (PBS), 2-3 ml was
added to MaxiSorp Nunc-Immuno Tubes (Nunc) and incubated
overnight at 4 C on a rotating wheel. An aliquot of a phage
library (500 pl, approximately 1013 cfu, amplified using CT
helper phage (see WO 02/103012)) was blocked in blocking
buffer (2% Protifar in PBS) for 1-2 hours at room temperature.
The blocked phage library was added to the immunotube (either
preincubated with or without CR-57 scFv to block the epitope
recognized by CR-57), incubated for 2 hours at room
temperature, and washed with wash buffer (0.1% Tween-20
(Serva) in PBS) to remove unbound phages. Bound phages were
then eluted from the antigen by incubation for 10 minutes at
room temperature with 1 ml of 50 mM Glycine-HC1 pH 2.2.
Subsequently, the eluted phages were mixed with 0.5 ml of 1 M
Tris-HC1 pH 7.5 to neutralize the pH. This mixture was used to
infect 5 ml of a XL1-Blue E. coli culture that had been grown
at 37 C to an OD 600 nm of approximately 0.3. The phages were
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allowed to infect the XL1-Blue bacteria for 30 minutes at
37 C. Then, the mixture was centrifuged for 10 minutes, at
3200*g at room temperature and the bacterial pellet was
resuspended in 0.5 ml 2-trypton yeast extract (2TY) medium.
The obtained bacterial suspension was divided over two 2TY
agar plates supplemented with tetracyclin, ampicillin and
glucose. After incubation overnight of the plates at 37 C, the
colonies were scraped from the plates and used to prepare an
enriched phage library, essentially as described by De Kruif
et al. (1995a) and WO 02/103012. Briefly, scraped bacteria
were used to inoculate 2TY medium containing ampicillin,
tetracycline and glucose and grown at a temperature of 37 C to
an OD 600 nm of -0.3. CT helper phages were added and allowed
to infect the bacteria after which the medium was changed to
2TY containing ampicillin, tetracycline and kanamycin.
Incubation was continued overnight at 30 C. The next day, the
bacteria were removed from the 2TY medium by centrifugation
after which the phages in the medium were precipitated using
polyethylene glycol (PEG) 6000/NaCl. Finally, the phages were
dissolved in 2 ml of PBS with 1% bovine serum albumin (BSA),
filter-sterilized and used for the next round of selection.
Phage selections were also performed with rabies virus
glycoprotein transfected cells. The cells used were cells from
the cell line deposited at the European Collection of Cell
Cultures (ECACC), CAMR, Salisbury, Wiltshire SP4 OJG, Great
Britain on 29 February 1996 under number 96022940 and marketed
under the trademark PER.C6 . They are hereinafter referred to
as PER.C6 cells. Here, the blocked phage library (2 ml) was
first added to 1*107 subtractor cells (in DMEM/10% FBS) and
incubated for 1 hour at 4 C on a rotating wheel. The subtractor
cells were PER.C6 cells that expressed the Vesicular
Stomatitis Virus (VSV) glycoprotein ecto domain on their
CA 02775886 2012-04-19
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surface fused to the rabies virus transmembrane and
cytoplasmic domain. With this subtraction step phages
recognizing either VSV glycoprotein or antigens specific for
PER.C6 cells were removed from the phage library. The
phage/cell mixture was centrifuged (5 minutes at 4 C at 500xg)
to remove cell-bound phages, and the supernatant was added to
a new tube containing 3 ml of 1*107 subtractor cells. The
subtraction step was repeated twice with the respective
supernatant. Subsequently, the subtracted phages were
incubated for 1.5 hours at 4 C on a rotating wheel with the
rabies virus glycoprotein expressing transfected cells (PER.C6
cells (3*106 cells)). Before that, the transfected cells were
preincubated either with or without CR-57 scFv to block the
epitope recognized by CR-57. After incubation the cells were
washed five times with 1 ml of DMEM/10%FBS (for each wash, the
cells were resuspended and transferred to new tube), phages
were eluted and processed as described above.
Typically, two rounds of selections were performed before
isolation of individual phage antibodies. After the second
round of selection, individual E. coli colonies were used to
prepare monoclonal phage antibodies. Essentially, individual
colonies were grown to log-phase in 96 well plate format and
infected with VCSM13 helper phages after which phage antibody
production was allowed to proceed overnight. The produced
phage antibodies were PEG/NaCl-precipitated and filter-
sterilized and tested in ELISA for binding to both whole
inactivated rabies virus and purified rabies virus G protein.
From the selection a large panel of phage antibodies was
obtained that demonstrated binding to both whole inactivated
rabies virus and rabies virus G protein (see example below).
Two selection strategies were followed with the above-
described immune libraries. In the first strategy 736 phage
CA 02775886 2012-04-19
antibodies were selected after two selection rounds using in
the first and second selection round inactivated virus or
purified G protein. In the second strategy 736 phage
antibodies were selected after two selection rounds using in
5 the first selection round cell surface expressed recombinant G
protein and in the second selection round inactivated virus or
purified G protein. The number of unique phage antibodies
obtained by the first strategy was 97, while the second
strategy yielded 70 unique ones. The 97 unique phage
10 antibodies found by means of the first strategy gave rise to
18 neutralizing antibodies and the 70 unique clones identified
by means of the second strategy yielded 33 neutralizing
antibodies. This clearly demonstrates that selections that
included rabies virus glycoprotein tranfected cells, i.e. cell
15 surface expressed recombinant G protein, as antigen appeared
to yield more neutralizing antibodies compared to selections
using only purified G protein and/or inactivated virus.
Example 4
20 Validation of the rabies virus glycoprotein specific single-
chain phage antibodies.
Selected single-chain phage antibodies that were obtained
in the screens described above, were validated in ELISA for
specificity, i.e. binding to rabies virus G protein, purified
25 as described supra. Additionally, the single-chain phage
antibodies were also tested for binding to 5% FBS. For this
purpose, the rabies virus G protein or 5% FBS preparation was
coated to MaxisorpTM ELISA plates. After coating, the plates
were blocked in PBS/1% Protifar for 1 hour at room
30 temperature. The selected single-chain phage antibodies were
incubated for 15 minutes in an equal volume of PBS/1% Protifar
to obtain blocked phage antibodies. The plates were emptied,
CA 02775886 2012-04-19
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and the blocked phage antibodies were added to the wells.
Incubation was allowed to proceed for one hour, the plates
were washed in PBS containing 0.1% Tween-20 and bound phage
antibodies were detected (using OD 492 nm measurement) using
an anti-M13 antibody conjugated to peroxidase. As a control,
the procedure was performed simultaneously using no single-
chain phage antibody, a negative control single chain phage
antibody directed against CD8 (SC02-007) or a positive control
single chain phage antibody directed against rabies virus
glycoprotein (scFv S057). As shown in Table 8, the selected
phage antibodies called SC04-001, SC04-004, SC04-008, SC04-
010, SC04-018, SC04-021, SC04-026, SC04-031, SC04-038, SC04-
040, SC04-060, SC04-073, SC04-097, SC04-098, SC04-103, SC04-
104, SC04-108, SC04-120, SC04-125, SC04-126, SC04-140, SC04-
144, SC04-146, and SC04-164 displayed significant binding to
the immobilized purified rabies virus G protein, while no
binding to FBS was observed. Identical results were obtained
in ELISA using the whole inactivated rabies virus prepared as
described supra (data not shown).
Example 5
Characterization of the rabies virus specific scFvs
From the 'selected specific single chain phage antibody
(scFv) clones plasmid DNA was obtained and nucleotide
sequences were determined according to standard techniques.
The nucleotide sequences of the scFvs (including restriction
sites for cloning) called SC04-001, SC04-004, SC04-008, SC04-
010, SC04-018, SC04-021, SC04-026, SC04-031, SC04-038, SC04-
040, SC04-060, SC04-073, SC04-097, SC04-098, SC04-103, SC04-
104, SC04-108, SC04-120, SC04-125, SC04-126, SC04-140, SC04-
144, SC04-146, and SC04-164 are shown in SEQ ID NO:157, SEQ ID
NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID N0:165, SEQ ID
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NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID
NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID
NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID
NO:191, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID
NO:199, SEQ ID NO:201 and SEQ ID NO:203, respectively. The
amino acid sequences of the scFvs called SC04-001, SC04-004,
SC04-008, SC04-010, SC04-018, SC04-021, SC04-026, SC04-031,
SC04-038, SC04-040, SC04-060, SC04-073, SC04-097, SC04-098,
SC04-103, SC04-104, SC04-108, SC04-120, SC04-125, SC04-126,
SC04-140, SC04-144, SC04-146, and SC04-164 are shown in SEQ ID
NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID
NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID
NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ ID
NO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID
NO:190, SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID
NO:198, SEQ ID NO:200, SEQ ID NO:202 and SEQ ID NO:204,
respectively.
The VH and VL gene identity (see Tomlinson IN, Williams
SC, Ignatovitch 0, Corbett SJ, Winter G. V-BASE Sequence
Directory. Cambridge United Kingdom: MRC Centre for Protein
Engineering (1997)) and heavy chain CDR3 compositions of the
scFvs specifically binding the rabies virus G protein are
depicted in Table 9.
Example 6
In vitro neutralization of rabies virus by rabies virus
specific scFvs (modified RFFIT)
In order to determine whether the selected scFvs were
capable of blocking rabies virus infection, in vitro
neutralization assays (modified RFFIT) were performed. The
scFv preparations were diluted by serial threefold dilutions
starting with a 1:5 dilution. Rabies virus (strain CVS-11) was
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added to each dilution at a concentration that gives 80-100 %
infection. Virus/scFv mix was incubated for 1 hour at 37 C/5%
CO2 before addition to MNA cells. 24 hours post-infection (at
34 C/5% C02) the cells were acetone-fixed for 20 minutes at
4 C, and stained for minimally 3 hours with an anti-rabies N-
FITC antibody conjugate (Centocor). The cells were then
analyzed for rabies virus infection under a fluorescence
microscope to determine the 50% endpoint dilution. This is the
dilution at which the virus infection is blocked by 50% in
this assay (see Example 1). Several scFvs were identified that
showed neutralizing activity against rabies virus (see Table
10).
Additionally, it was investigated by means of the in
vitro neutralization assay (modified RFFIT) as described
above, if the selected scFvs were capable of neutralizing the
E57 escape viruses as prepared in Example 1 (E57A2, E57A3,
E57B1, E57B2, E57B3 and E57C3). Several scFvs were identified
that showed neutralizing activity against the E57 escape
viruses (see Tables 11A and 11B).
Example 7
Rabies virus G protein competition ELISA with scFvs
To identify antibodies that bind to non-overlapping, non-
competing epitopes, a rabies glycoprotein competition ELISA
was performed. Nunc-ImmunoTM Maxisorp F96 plates (Nunc) were
coated overnight at 4 C with a 1:1000 dilution of purified
rabies virus glycoprotein (1 mg/ml; rabies virus ERA strain)
in PBS (50 l). Uncoated protein was washed away before the
wells were blocked with 100 l PBS/1% Protifar for 1 hour at
room temperature. Subsequently, the blocking solution was
discarded and 50 l of the non-purified anti-rabies virus scFvs
CA 02775886 2012-04-19
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in PBS/1% Protifar (2x diluted) was added. Wells were washed
five times with 100 l of PBS/0.05% Tween-20. Then, 50 l
biotinylated anti-rabies virus competitor IgG, CR-57bio, was
added to each well, incubated for 5 minutes at room
temperature, and the wells were washed five times with 100 gi
of PBS/0.05% Tween-20. To detect the binding of CR-57bio, 50 l
of a 1:2000 dilution of streptavidin-HRP antibody (Becton
Dickinson) was added to the wells and incubated for 1 hour at
room temperature. Wells were washed again as above and the
ELISA was further developed by addition of 100 l of OPD
reagens (Sigma). The reaction was stopped by adding 50 gl 1 M
H2SO4 before measuring the OD at 492 nm.
The signal obtained with CR-57bio alone could be reduced
to background levels when co-incubated with scFv S057, i.e.
the scFv form of CR-57 (for nucleotide and amino acid sequence
of S057 see SEQ ID NO:205 and 206, respectively) or scFv SOJB,
i.e. the scFv form of CR-JB (for nucleotide and amino acid
sequence of SOJB see SEQ ID NO:312 and 313, respectively).
This indicates that the scFvs S057 and SOJB compete with the
interaction of CR-57bio to rabies virus glycoprotein by
binding to the same epitope or to an overlapping epitope as
CR-57bio, respectively. In contrast, an irrelevant scFv called
SC02-007, i.e_ a scFv binding to CD8, did not compete for
binding. The anti-rabies virus scFvs called SC04-004, SC04-
010, SC04-024, SC04-060, SC04-073, SC04-097, SC04-098, SC04-
103, SC04-104, SC04-120, SC04-125, SC04-127, SC04-140, SC04-
144 and SC04-146 did also not compete with CR-57bio,
indicating that these scFvs bind to a different epitope than
the epitope recognized by CR-57 (see Figure 4).
Similar results were obtained with the following
experiment. First, the rabies virus antibody CR-57 was added
CA 02775886 2012-04-19
to wells coated with rabies virus G protein. Next, the
competing scFvs were added. In this set-up the anti-rabies
virus scFvs were detected with anti-VSV-HRP by virtue of the
presence of a VSV-tag in the scFv amino acid sequences (see
5 Figure 5).
Example 8
Construction of fully human immunoglobulin molecules (human
monoclonal anti-rabies virus antibodies) from the selected
10 anti-rabies virus single chain Fv's
Heavy and light chain variable regions of the scFvs
called SC04-001, SC04-008, SC04-018, SC04-040 and SC04-126
were PCR-amplified using oligonucleotides to append
restriction sites and/or sequences for expression in the IgG
15 expression vectors pSyn-C03-HCyl (see SEQ ID No:277) and pSyn-
C04-CX (see SEQ ID No:278), respectively. The Vx and VL genes
were amplified using the oligonucleotides as shown in Table 12
and 13, respectively, and the PCR products were cloned into
the vectors pSyn-C03-HCyl and pSyn-C04-CX, respectively.
20 Heavy and light chain variable regions of the scFvs
called SC04-004, SC04-010, SC04-021, SC04-026, SC04-031, SC04-
038, SC04-060, SC04-073, SC04-097, SC04-098, SC04-103, SC04-
104, SC04-108, SC04-120, SC04-125, SC04-140, SC04-144, SC04-
146 and SC04-164 were also PCR-amplified using
25 oligonucleotides to append restriction sites and/or sequences
for expression in the IgG expression vectors pSyn-C03-HCyl and
pSyn-C05-CK (see SEQ ID No:279), respectively. The VH and VL
genes were amplified using the oligonucleotides as given in
Table 12 and 13, respectively, and the PCR products were
30 cloned into the vectors pSyn-C03-HCyl and pSyn-C05-Ck,
respectively. The oligonucleotides are designed such that they
correct any deviations from the germline sequence that have
CA 02775886 2012-04-19
86
been introduced during library construction, due to the
limited set of oligonucleotides that have been used to amplify
the large repertoire of antibody genes. Nucleotide sequences
for all constructs were verified according to standard
techniques known to the skilled artisan.
The resulting expression constructs pgGl04-001C03,
pgGl04-008C03, pgG104-018C03, pgG104-040C03 and pgGl04-126C03
encoding the anti-rabies virus human IgG1 heavy chains in
combination with the relevant pSyn-C04-VX construct encoding
the corresponding light chain were transiently expressed in
293T cells and supernatants containing IgG1 antibodies were
obtained. The expression constructs pgG104-004C03, pgGl04-
010C03, pgGl04-021C03, pgG104-026C03, pgGl04-031C03, pgG104-
038C03, pgGl04-060C03, pgG104-073C03, pgGl04-097C03, pgG104-
098C03, pgG104-103C03, pgG104-104C03, pgGl04-108C03, pgG104-
120C03, pgGl04-125C03, pgGl04-140C03, pgG104-144C03, pgGl04-
146C03 and pgG104-164C03 encoding the anti-rabies virus human
IgGi.heavy chains in combination with the relevantpSyn-C05-Vx
construct encoding the corresponding light chain were
transiently expressed in 293T cells and supernatants
containing IgG1 antibodies were obtained.
The nucleotide and amino acid sequences of the heavy and
light chains of the antibodies called CR04-001, CR04-004,
CR04-008, CR04-010, CR04-018, CR04-021, CR04-026, CR04-031,
CR04-038, CR04-040, CR04-060, CR04-073, CR04-097, CR04-098,
CR04-103, CR04-104, CR04-108, CR04-120, CR04-125, CR04-126,
CR04-140, CR04-144, CR04-146 and CR04-164 were determined
according to standard techniques. Subsequently, the
recombinant human monoclonal antibodies were purified over a
protein-A column followed by a buffer exchange on a desalting
column using standard purification methods used generally for
CA 02775886 2012-04-19
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immunoglobulins (see for instance WO 00/63403).
Additionally, for CR04-098, a single human IgG1
expression vector named pgGl04-098010 was generated as
described above for vectors pgS057CII and pgSOJBCII encoding
CR-57 and CR-JB, respectively (see Example 1). The nucleotide
and amino acid sequences of the heavy and light chains of
antibody CR04-098 encoded by vector pgG104-098C10 are shown in
SEQ ID NO:334 - 337, respectively. Vectors pgS057C11 (see
Example 1) and pgGl04-098C10 were used for stable expression
of CR-57 and CR04-098, respectively, in cells from the cell
line deposited at the European Collection of Cell Cultures
(ECACC), CAMR, Salisbury, Wiltshire SP4 OJG, Great Britain on
29 February 1996 under number 96022940 and marketed under the
trademark PER.C6 . The stably produced CR-57 and CR04-098 have
a calculated isoelectric point of 8.22 and 8.46, respectively.
The experimentally observed isoelectric points are between
8.1-8.3 for CR-57 and 9.0-9.2 for CR04-098...The recombinant
human monoclonal antibodies were purified as described above.
Unless otherwise stated, for CR04-001, CR04-004, CR04-008,
CR04-010, CR04-018, CR04-021, CR04-026, CR04-031, CR04-038,
CR04-040, CR04-060, CR04-073, CR04-097, CR04-098, CR04-103,
CR04-104, CR04-108, CR04-120, CR04-125, CR04-126, CR04-140,
CR04-144, CR04-146 and CR04-164 use was made of recombinant
human monoclonal antibodies transiently expressed by the two
vector system as described above and for CR57 use was made of
recombinant human monoclonal antibody transiently expressed by
the one vector system as described in Example 1.
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Example 9
Rabies virus G protein competition ELISA with IgGs
To address whether the human monoclonal anti-rabies virus
G protein IgGs bind to non-overlapping, non-competing
epitopes, competition experiments are performed. Wells with
coated rabies virus G protein are incubated with increasing
concentrations (0-50 gg/ml) of unlabeled anti-rabies virus G
protein IgG for 1 hour at room temperature. Then, 50 pl of a
different biotinylated anti-rabies virus IgG (1 g/ml) is added
to each well, incubated for 5 minutes at room temperature, and
immediately washed five times with 100 gl of PBS/0.05% Tween-
20. Subsequently, wells are incubated for 1 hour at room
temperature with 50 gl of a 1:2000 dilution of streptavidin-HRP
(Becton Dickinson), washed and developed as described above. A
decrease in signal with increasing concentration of unlabeled
IgG indicates that the two antibodies are competing with each
other and recognize the same epitope or overlapping epitopes.
Alternatively, wells coated with rabies virus G protein
(ERA strain) were incubated with 50 g/ml of unlabeled anti-
rabies virus G protein IgG for 1 hour at room temperature.
Then, 50 pl of biotinylated CR57 (0.5-5 gg/ml; at subsaturated
levels) was added to each well. The further steps were
performed as described supra. The signals obtained were
compared to the signal obtained with only biotinylated CR57
(see Figure 6; no competitor). From Figure 6 can be deduced
that the signal could not be reduced with the antibody called
CR02-428 which served as a negative control. In contrast,
competition with unlabeled CR57 (positive control) or CR-JB
reduced the signal to background levels. From Figure 6 can
further be deduced that none of the anti-rabies virus G
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protein IgGs competed significantly with CR-57, which is in
agreement with the scFv competition data as described in
Example 7.
In addition, competition experiments were performed on
rabies virus G protein (ERA strain) transfected PER.C6 cells by
means of flow cytometry. Transfected cells were incubated with
20 gl of unlabeled anti-rabies virus G protein IgG (50 gg/ml)
for 20 minutes at 4 C. After washing of the cells with PBS
containing 1% BSA, 20 gl of biotinylated CR57 (0.5-5 gg/ml; at
subsaturated levels) were added to each well, incubated for 5
minutes at 4 C, and immediately washed twice with 100 1 of PBS
containing 1% BSA. Subsequently, wells were incubated for 15
minutes at 4 C with 20 2l of a 1:200 dilution of streptavidin-
PE (Caltag), washed and developed as described above. The
signal obtained with biotinylated CR57 could not be reduced
significantly with the negative control antibody CR02-428 (see
Figure 7). In contrast, competition with unlabeled CR57
(positive control) or CR-JB reduced the signal to background
levels. None of the anti-rabies virus G protein IgGs competed
significantly with CR-57, with the exception of CR04-126 which
reduced the signal to approximately .30% (see Figure 7). The
latter did not compete in ELISA (see Figure 6). This may be
caused by the difference in the way the glycoprotein is
presented to the antibody in FACS experiments compared to
ELISA experiments. The binding of CR04-126 could be. more
dependent on the conformation of the glycoprotein, resulting
in the competitive effect observed with CR04-126 in the FACS-
based competition assay and not in the ELISA-based competition
assay. Additionally, CR04-008 and CR04-010 reduced the signal
to approximately 50% (see Figure 7) in the FACS-based
competition assay indicating that they might compete with
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CR57. For CR04-010 this was however not confirmed by the scFv
competition data or the ELISA-based competition assay. For the
other IgGs, the FACS data were in agreement with the
respective ELISA data of both the scFvs and the IgGs.
5
Example 10
Additive/synergistic effects of anti-rabies TgGs in in vitro
neutralization of rabies virus (modified RFFIT)
In order to determine whether the anti-rabies virus G
10 protein IgGs have additive or synergistic effects in
neutralization of rabies virus, different combinations of the
IgGs are tested. First, the potency (in IU/mg) of each
individual antibody is determined in a modified RFFIT (see
Example 1). Then, antibody combinations are prepared based on
15 equal amounts of IU/mg and tested in the modified RFFIT. The
potencies of each antibody combination can be determined and
compared with the expected potencies. If the potency of the
antibody combination is equal to the sum of the, potencies of
each individual antibody present in the combination, the
20 antibodies have an additive effect. If the potency of the
antibody combination is higher, the antibodies have a
synergistic effect in neutralization of rabies virus.
Alternatively, additive or synergistic effects can be
determined by the following experiment. First, the potency of
25 the antibodies to be tested, e.g. CR-57 and CR04-098, is
determined in a standard RFFIT (see Laboratory techniques in
rabies, Edited by: F.-X Meslin, M.M. Kaplan and H. Koprowski
(1996), 4th edition, Chapter 15, World Health Organization,
Geneva). Then, the antibodies are mixed in a 1:1 ratio based
30 on IU/ml. This antibody mixture, along with the individual
antibodies at the same concentration, are tested in six
independent RFFIT experiments to determine the 50%
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neutralizing endpoint. Subsequently, the combination index
(CI) is determined for the antibody mixture using the formula
CI = (Cl/Cxl) + (C2/Cx2) + (C1C2/Cx1Cx2) as described by Chou
et al. (1984). Cl and C2 are the amount (in pg) of monoclonal
antibody 1 and monoclonal antibody 2 that lead to 50%
neutralization when used in combination and Cxl and Cx2 are
the amount (in g) of monoclonal antibody 1 and monoclonal
antibody 2 that lead to 50% neutralization when used alone. CI
= 1, indicates an additive effect, CI < 1 indicates a
synergistic effect and CI > 1 indicates an antagonistic effect
of the monoclonal antibodies.
Example 11
Identification of epitopes recognized by recombinant human
anti-rabies virus antibodies by PEPSCAN-ELISA
15-mer linear and looped/cyclic peptides were synthesized
from the extracellular domain of the G protein of the rabies
virus strain ERA (see SEQ ID NO:207 for the complete amino
acid sequence of the glycoprotein G of the rabies virus strain
ERA, the extracellular domain consists of amino acids 20-458;
the protein-id of the glycoprotein of rabies virus strain ERA
in the EMBL-database is J02293) and screened using credit-card
format mini-PEPSCAN cards (455 peptide formats/card) as
described previously (Slootstra et al., 1996; WO 93/09872).
All peptides were acetylated at the amino terminus. In all
looped peptides position-2 and position-14 were replaced by a
cysteine (acetyl-XCXXXXXXXXXXXCX-minicard). If other cysteines
besides the cysteines at position-2 and position-14 were
present in a prepared peptide, the other cysteines were
replaced by an alanine. The looped peptides were synthesized
using standard Fmoc-chemistry and deprotected using trifluoric
acid with scavengers. Subsequently, the deprotected peptides
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were reacted on the cards with an 0.5 mM solution of 1,3-
bis(bromomethyl)benzene in ammonium bicarbonate (20 mM, pH
7.9/acetonitril (1:1 (v/v)). The cards were gently shaken in
the solution for 30-60 minutes, while completely covered in
the solution. Finally, the cards were washed extensively with
excess of H2O and sonicated in disrupt buffer containing 1%
SDS/0.1% beta-mercaptoethanol in PBS (pH 7.2) at 70 C for 30
minutes, followed by sonication in H2O for another 45 minutes.
The human monoclonal antibodies were prepared as described
above. Binding of these antibodies to each linear and looped
peptide was tested in a PEPSCAN-based enzyme-linked immuno
assay (ELISA). The 455-well creditcard-format polypropylene
cards, containing the covalently linked peptides, were
incubated with the antibodies (10 Vg/ml; diluted in blocking
solution, which contained 5% horse-serum (v/v) and 5%
ovalbumin (w/v)) (4 C, overnight). After washing, the peptides
were incubated with anti-human antibody peroxidase (dilution
1/1000) (1 hour, 25 C), and subsequently, after washing the
peroxidase substrate 2,2'-azino-di-3-ethylbenzthiazoline
sulfonate (ABTS) and 2 pl/ml 3% H202 was added. Controls (for
linear and looped) were incubated with anti-human antibody
peroxidase only. After 1 hour the color development was
measured. The color development of the ELISA was quantified
with a CCD-camera and an image processing system. The set-up
consisted of a CCD-camera and a 55 mm lens (Sony CCD Video
Camera XC-77RR, Nikon micro-nikkor 55 mm f/2.8 lens), a camera
adaptor (Sony Camera adaptor DC-77RR) and the Image Processing
Software package Optimas, version 6.5 (Media Cybernetics,
Silver Spring, MD 20910, U.S.A.). Optimas ran on a pentium II
computer system.
The human anti-rabies virus G protein monoclonal
antibodies were tested for binding to the 15-mer linear and
*Trade-mark
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looped/cyclic peptides synthesized as described supra. A
peptide is considered to relevantly bind to an antibody when
OD values are equal to or higher than two times the average
OD-value of all peptides (per antibody). See Table 14 for
results of the binding of the human monoclonal antibodies
called CR57, CRJB and CR04-010 to the linear peptides of the
extracellular domain of glycoprotein G of rabies virus strain
ERA. Regions' showing significant binding to the respective
antibodies are highlighted in grey (see Table 14).
Antibody CR57 bound to the linear peptides having an
amino acid sequence selected from the group consisting of
SLKGACKLKLCGVLG (SEQ ID NO:314), LKGACKLKLCGVLGL (SEQ ID
NO:315), KGACKLKLCGVLGLR (SEQ ID NO:316), GACKLKLCGVLGLRL (SEQ
ID NO:317), ACKLKLCGVLGLRLM (SEQ ID NO:318), CKLKLCGVLGLRLMD
(SEQ ID NO:319), KLKLCGVLGLRLMDG (SEQ ID NO:320),
LKLCGVLGLRLMDGT (SEQ ID NO:321) and KLCGVLGLRLMDGTW (SEQ ID
NO:322) (see Table 14). The peptides having the amino acid
sequences GACKLKLCGVLGLRL (SEQ ID NO:317), ACKLKLC-PVLGLRLM
(SEQ ID NO:318) have an OD-value that is lower than twice the
average value. Nevertheless these peptides were claimed,
because they are in the near proximity of a region of
antigenic peptides recognised by antibody CR57. Binding was
most prominent to the peptide with the amino acid sequence
KLCGVLGLRLMDGTW (SEQ ID NO:322).
Antibody CR04-010 bound to the linear peptides having an
amino acid sequence selected from the group consisting of
GFGKAYTIFNKTLME (SEQ ID NO:323), FGKAYTIFNKTLMEA (SEQ ID
NO:324), GKAYTIFNKTLMEAD (SEQ ID NO:325), KAYTIFNKTLMEADA (SEQ
ID NO:326), AYTIFNKTLMEADAH (SEQ ID NO:327), YTIFNKTLMEADAHY
(SEQ ID NO:328), TIFNKTLMEADAHYK (SEQ ID NO:329),
IFNKTLMEADAHYKS (SEQ ID NO:330) and FNKTLMEADAHYKSV (SEQ ID
NO:331). The peptides having the amino acid sequences
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AYTIFNKTLMEADAH (SEQ ID NO:327), YTIFNKTLMEADAHY (SEQ ID
NO:328) have an OD-value that is lower than twice the average
value. Nevertheless these peptides were claimed, because they
are in the near proximity of a region of antigenic peptides
recognized by antibody CR04-010. Binding was most prominent to
the peptides with the amino acid sequence TIFNKTLMEADAHYK (SEQ
ID NO:329), IFNKTLMEADAHYKS (SEQ ID NO:330) and
FNKTLMEADAHYKSV (SEQ ID NO:331).
CRJB and the antibodies called CR04-040, CR04-098 and
CR04-103 (data not shown) did not recognize a region of linear
antigenic peptides.
Any of the above peptides or parts thereof represents
good candidates of a neutralizing epitope of rabies virus and
could form the basis for a vaccine or for raising neutralizing
antibodies to treat and/or prevent a rabies virus infection.
SLKGACKLKLCGVLGLRLMDGTW (SEQ ID NO:332) and
GFGKAYTIFNKTLMEADAHYKSV (SEQ ID NO:333) are particularly
interesting regions of the glycoprotein based on their high
reactivity in PEPSCAN.
From the above PEPSCAN data can further be deduced that
the human monoclonal antibodies called CR57 and CR04-010 bind
to different regions of the rabies virus G protein indicating
that they recognize non-competing epitopes.
Example 12
Determination of neutralizing potency of anti-rabies G protein
IgGs using an in vitro neutralization assay (modified RFFIT).
The neutralizing potency of each of the produced human
monoclonal antibodies was determined in a modified RFFIT as
described in Example 1. Sixteen IgGs neutralized rabies strain
CVS-11 with a potency higher than 1000 IU/mg, whereas only two
IgGs had a potency lower than 2 IU/mg (see Table 15). Eight of
CA 02775886 2012-04-19
the sixteen antibodies outperformed transiently produced CR-57
with regard to potency, suggesting a higher efficiency in post
exposure prophylaxis of rabies virus than CR-57. The potency
of transiently produced CR-57 was approximately 3800 IU/mg
5 protein (see Tables 1 and 15), whereas stably produced CR-57
displayed a potency of 5400 IU/mg protein (data not shown).
Interestingly, the majority of the neutralizing human
monoclonal antibodies identified contain a variable heavy 3-30
germline gene (see Table 9).
10 Based on the affinity of the antibodies for rabies virus
(data not shown) and 100% endpoint dilution of the antibodies
in a modified RFFIT assay (data not shown), a panel of six
unique IgGs, i.e. CR04-010, CR04-040, CR04-098, CR04-103,
CR04-104, and CR04-144, were chosen for further development.
15 Within this panel, antibody CR04-098 was particularly
interesting as it displayed the highest potency, i.e.
approximately 7300 IU/mg protein (see Table 15). A similar
potency was also.,found for stably produced CR04-098 (data not
shown).
Example 13
In vitro neutralization of E57 escape viruses by anti-rabies
virus IgGs
To further characterize the novel human monoclonal anti-
rabies antibodies the neutralizing activity of the IgGs
against E57 escape viruses was tested in a modified RFFIT as
described above. The majority of the anti-rabies virus IgGs
had good neutralizing activity against all six E57 escape
viruses (see Table 16). In contrast, CR04-008, CR04-018 and
CR04-126 did not neutralize 6/6, 2/6 and 3/6 E57 escape
viruses, respectively. No neutralization means that no 50%
endpoint was reached at an antibody dilution of 1:100. CR04-
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021, CR04-108, CR04-120, CR04-125, and CR04-164 showed a
significant decrease in neutralizing activity against a number
of escape viruses. This suggests that the epitope of these
antibodies has been affected either directly or indirectly in
the E57 escape virus glycoprotein. On the basis of the above
several anti-rabies virus IgGs may be compatible with CR-57 in
an anti-rabies cocktail for post exposure prophylaxis
treatment. In particular, the panel of six unique IgGs as
identified above, i.e. antibodies CR04-010, CR04-040, CR04-
098, CR04-103, CR04-104, and CR04-144, displayed good
neutralizing potency towards the E57 escape viruses suggesting
that epitope(s) recognized by these antibodies was/were not
affected by the amino acid mutations induced by CR-57.
Antibody CR04-098 appeared most promising since it had a
potency higher than 3000 IU/mg for each of the escape viruses.
Example 14
Epitope recognition of anti-rabies antibodies CR-57,and CR04-
098
To confirm that the human monoclonal antibodies called
CR-57 and CR04-098 recognize non-overlapping, non-competing
epitopes, escape viruses of the human monoclonal antibody
called CR04-098 were generated essentially as described for
escape-viruses of CR57 (see Example 1). In short, the number
of foci per well was scored by immunofluorescence and medium
of wells containing preferably one focus were chosen for virus
amplification. All E98 escape viruses were generated from 1
single focus with the exception of E98-2 (2 foci) and E98-4 (4
foci). A virus was defined as an escape variant if the
neutralization index was <2.5 logs. The neutralization index
was determined by subtracting the number of infectious virus
particles/ml produced in BSR cell cultures infected with virus
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plus monoclonal antibody (- 4 IU/ml) from the number of
infectious virus particles/ml produced in BSR or MNA cell
cultures infected with virus alone ([log focus forming
units/ml virus in absence of monoclonal antibody minus log
ffu/ml virus in presence of monoclonal antibody]). An index
lower than 2.5 logs was considered as evidence of escape.
To further investigate that CR04-098 binds to a different
non-overlapping, non-competing epitope compared to CR-57, CR-
57 was tested against E98 escape viruses in a modified RFFIT
assay as described above. As shown in Table 17, CR-57 had good
neutralizing activity against all five E98 escape viruses.
Additionally, antibodies CR04-010 and CR04-144 were tested for
neutralizing activity against the E98 escape viruses. Both
antibodies did not neutralize the E98 escape viruses (data not
shown) suggesting that the epitope recognized by both
antibodies is either directly or indirectly affected by the
amino acid mutation induced by antibody CR04-098. The
antibodies CR04-018 and CR04-12.6 were tested for neutralizing
activity against only one of the E98 escape viruses, i.e. E98-
4. CR04-018 was capable of neutralizing the escape virus,
while CR04-126 only had a weak neutralizing potency towards
the escape virus. This suggests that the epitope recognized by
CR04-018 is not affected by the mutation induced by antibody
CR04-098. Additionally, the antibodies CR04-010, CR04-038,
CR04-040, CR04-073, CR04-103, CR04-104, CR04-108, CR04-120,
CR04-125, CR04-164 did not neutralize E98-4 suggesting that,
they recognize the same epitope as CR04-098 (data not shown).
To identify possible mutations in the rabies glycoprotein
of each of the E98 escape viruses, the nucleotide sequence of
the glycoprotein open reading frame (ORF) was determined as
described before for the E57 and EJB escape viruses. All E98
escape viruses showed the mutation N to D at amino acid
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position 336 of the rabies glycoprotein (see Figure 8). This
region of the glycoprotein has been defined as antigenic site
III comprising of amino acids 330-338 (numbering without
signal peptide). In contrast, CR-57 recognized an epitope
located at amino acids 226-231 (numbering without signal
peptide), which overlaps with antigenic site I. In addition to
the N336D mutation the E98 escape virus called E98-5 showed
the mutation H to Q at amino acid position 354 (codon change
CAT to CAG) of the rabies glycoprotein (data not shown).
Moreover, pepscan analysis of binding of CR57 to peptides
harbouring a mutated CR57 epitope (as observed in E57 escape
viruses) did show that interaction of CR57 was abolished (data
not shown). Strikingly, CR04-098 was still capable of binding
to the mutated glycoprotein (comprising the N336D mutation)
expressed on PER.C6 cells, as measured by flow cytometry
(data not shown), even though viruses containing this mutation
were no longer neutralized.
Furthermo,re, epitope mapping studies and affinity ranking
studies were performed using surface plasmon resonance
analysis using a BIAcore3000TM analytical system. Purified
rabies glycoprotein (ERA strain) was immobilized as a ligand
on a research grade CM5 4-flow channel (Fc) sensor chip
(Biacore AB, Sweden) using amine coupling. Ranking was
performed at 2.5 C with HBS-EP (Biacore AB, Sweden) as running
buffer. 50 pl of each antibody was injected at a constant flow
rate of 20 pl/min. Then, running buffer was applied for 750
seconds followed by regeneration of the CM5 chip with 5 pl 2M
NaOH, 5 pl 45 mM HCl and 5 pl 2 mM NaOH. The resonance signals
expressed as resonance units (RU) were plotted as a function
of time and the increase and decrease in RU as a measure of
association and dissociation, respectively, were determined
and used for ranking of the antibodies. The actual KD values
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for CR57 and CR04-098 as determined by surface plasmon
resonance analysis were 2.4 nM and 4.5 riM, respectively. The
epitope mapping studies further confirmed that CR57 and CR04-
098 bind to different epitopes on rabies glycoprotein.
Injection of CR57 resulted in a response of 58 RU (data not
shown). After injection of CR04-098 an additional increase in
response level (24 RU) was obtained, suggesting that binding
sites for CR04-098 were not occupied (data not shown). Similar
results were observed when the reverse order was applied
showing that each antibody reached similar RU levels
regardless of the order of injection (data not shown). These
results further demonstrate that CR57 and CR04-098 can bind
simultaneously and recognize different epitopes on the rabies
virus glycoprotein.
Overall, the above data further confirm that the
antibodies CR-57 and CR04-098 recognize distinct non-
overlapping epitopes, i.e. epitopes in antigenic site I and
III, respectively. The data are in good agreement with the
ELISA/FACS competition data indicating that CR-57 and CR04-098
do not compete for binding to ERA G and the good neutralizing
activity of antibody CR04-098 against all E57 escape viruses.
On the basis of these results and the fact that'in vitro
exposure of rabies virus to the combination of CR57 and CR04-
098 (selection in the presence of 4 IU/ml of either antibody)
yielded no escape viruses (data not shown), it was concluded
that the antibodies CR-57 and CR04-098 recognize non-
overlapping, non-competing epitopes and can advantageously be
used in an anti-rabies virus antibody cocktail for post-
exposure prophylaxis treatment.
Example 15
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Assessment of conservation of the epitope recognized by CR57
and CR04-098
The minimal binding region of CR-57 (amino acids KLCGVL
within SEQ ID NO:332, the region of the glycoprotein of rabies
virus recognized by CR57 as determined by means of PEPSCAN and
alanine scanning technology) was aligned with nucleotide
sequences of 229 genotype 1 rabies virus isolates to assess
the conservation of the epitope (see Table 18). The sample set
contained human isolates, bat isolates and isolates from
canines or from domestic animals most likely bitten by rabid
canines. Frequency analysis of the amino acids at each
position within the minimal binding region revealed that the
critical residues constituting the epitope were highly
conserved. The lysine at position one was conserved in 99.6%
of the isolates, while in only 1/229 isolates a conservative
K>R mutation was observed. Positions two and three (L and C)
were completely conserved. It is believed that the central
cysteine residue is structurally involved in the glycoprotein
folding and is conserved among all lyssaviruses (see Badrane
and Tordo, 2001). The glycine at position four was conserved
in 98.7% of the isolates, while in 3/229 isolates mutations
towards charged amino acids (G>R in 1/229;,G>E in 2/229) were
observed. The fifth position was also conserved with the
exception of one isolate where a conservative V>I mutation was
observed. At the sixth position, which is not a critical
residue as determined by an alanine-replacement scan,
significant heterogeneity was observed in the street isolates:
L in 70.7%, P in 26.7% and S in 2.6% of the strains,
respectively. Taken together, approximately 99 percent of the
rabies viruses that can be encountered are predicted to be
recognized by the CR-57 antibody.
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123 of these 229 virus isolates were analyzed for the
presence of mutations in both the CR-57 and CR04-098 epitope.
None of these 123 street viruses did contain mutations in both
epitopes. The N>D mutation as observed in the E98 escape
viruses was present in only five virus isolates. These viruses
were geographically distinct and isolated from animals in
Africa (see Figure 9 for phylogenetic tree; the five virus
isolates, i.e. AF325483, AF325482, AF325481, AF325480 and
AF325485, are indicated in bold). The phylogenetic analysis of
glycoprotein sequences revealed that rabies viruses with
mutated CR57 epitopes are only distantly related to rabies
viruses bearing a mutated CR04-098 epitope. Therefore, the
likelihood of encountering a rabies virus resistant to
neutralization by a cocktail of CR-57 and CR04-098 is
virtually absent.
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Table 1: Neutralizing potency of CR-57 and CR-JB against wild-
type and escape viruses.
Potency Potency Potency Potency
CR-57 CR-JB CR-57 CR-JB
Virus (IU/mg) (IU/mg) Virus (IU/mg) (III/mg)
CVS-11 3797 605 CVS-11 3797 605
E57A2 0 <0.2 EJB2B 0.004 0.6
E57A3 0 419 EJB2C <0.004 2
E57B1 0 93 EJB2D <0.004 3
E57B2 0 <0.3 EJB2E <0.2 <0.3
E57B3 0 419 EJB2F <0.06 3
E57C3 0 31 EJB3F <0.04 0.06
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Table 2: Human lambda chain variable region primers (sense).
Primer name Primer nucleotide SEQ ID NO
sequence
HuVA1A 5'-CAGTCTGTGCTGACT SEQ ID NO:208
CAGCCACC-3'
HuVA1B 5'-CAGTCTGTGYTGACG SEQ ID NO:209
CAGCCGCC-3'
HuVA1C 5'-CAGTCTGTCGTGACG SEQ ID NO:210
CAGCCGCC-3'
HuVX2 5'-CARTCTGCCCTGACT SEQ ID NO:211
CAGCCT-3'
HuVX3A 5'-TCCTATGWGCTGACT SEQ ID NO:212
CAGCCACC-3'
HuVA3B 5'-TCTTCTGAGCTGACT SEQ ID NO:213
CAGGACCC-3'
HuVA4 5'-CACGTTATACTGACT SEQ ID NO:214
CAACCGCC-3'
HuVA5 5'-CAGGCTGTGCTGACT SEQ ID NO:215
CAGCCGTC--3'
HuVX6 5'-AATTTTATGCTGACT =SEQ ID NO:216
CAGCCCCA-3'
HuVX7/8 5'-CAGRCTGTGGTGACY SEQ ID NO:217
CAGGAGCC-3'
HuVX9 5'-CWGCCTGTGCTGACT SEQ ID NO:218
CAGCCMCC-3'
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Table 3: Human kappa chain variable region primers (sense).
Primer name Primer nucleotide SEQ ID NO
sequence
HuVx1B 5'-GACATCCAGWTGACCC SEQ ID NO:219
AGTCTCC-3'
HuVx2 5'-GATGTTGTGATGACT SEQ ID NO:220
CAGTCTCC-3'
HuVx3 5'-GAAATTGTGWTGACR SEQ ID NO:221
CAGTCTCC-3'
HuVx4 5'-GATATTGTGATGACC SEQ ID NO:222
CACACTCC-3'
HuVx5 5'-GAAACGACACTCACG SEQ ID NO:223
CAGTCTCC-3'
HuVx6 5'-GAAATTGTGCTGACTC SEQ ID NO:224
AGTCTCC-3'
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Table 4: Human kappa chain variable region primers extended
with Sall restriction sites (sense), human kappa chain J-
region primers extended with Notl restriction sites (anti-
sense), human lambda chain variable region primers extended
with Sall restriction sites (sense) and human lambda chain J-
region primers extended with NotI restriction sites (anti-
sense).
Primer name Primer nucleotide SEQ ID NO
sequence
HuVx1B-SalI 5'-TGAGCACACAGGTCG SEQ ID NO:225
ACGGACATCCAGWTGACC
CAGTCTCC-3'
HuVx2-SalI 5'-TGAGCACACAGGTCG SEQ ID NO:226
ACGGATGTTGTGATGACT
CAGTCTCC-3'
HuVic3B-SalI 5'-TGAGCACACAGGTCG SEQ ID NO:227
ACGGAAATTGTGWTGACR
CAGTCTCC-3'
HuVx4B-SalI 5'-TGAGCACACAGGTCG SEQ ID NO:228
ACGGATATTGTGATGACC
CACACTCC-3'
HuVx5-SalI 5'-TGAGCACACAGGTCGACG SEQ ID NO:229
GAAACGACACTCACGCAGTCT
CC-3'
HuVK6-Sa1I 5'-TGAGCACACAGGTCG SEQ ID NO:230
ACGGAAATTGTGCTGACT
CAGTCTCC-3'
HuJxl-Notl 5'-GAGTCATTCTCGACTTGC SEQ ID NO:231
GGCCGCACGTTTGATTTCCAC
CTTGGTCCC-3'
HuJx2-NotI 5'-GAGTCATTCTCGACT SEQ ID NO:232
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TGCGGCCGCACGTTTGAT
CTCCAGCTTGGTCCC-3'
HuJx3-NotI 5'-GAGTCATTCTCGACTTGC SEQ ID NO:233
GGCCGCACGTTTGATATCCAC
TTTGGTCCC-3'
HuJx4-NotI 5'-GAGTCATTCTCGACT SEQ ID NO:234
TGCGGCCGCACGTTTGAT
CTCCACCTTGGTCCC-3'
HuJx5-NotI 5'-GAGTCATTCTCGACTTGC SEQ ID NO:235
GGCCGCACGTTTAATCTCCAG
TCGTGTCCC-3'
HuVA1A-SalI 5'-TGAGCACACAGGTCGACG SEQ ID NO:236
CAGTCTGTGCTGACTCAGCCA
CC-3'
HuVX1B-SalI 5'-TGAGCACACAGGTCGACG SEQ ID NO:237
CAGTCTGTGYTGACGCAGCCG
CC-3'
HuVA1C-SalI 5'-TGAGCACACAGGTCGACG SEQ ID NO:238
CAGTCTGTCGTGACGCAGCCG
CC-3'
HuVA2-Sall 5'-TGAGCACACAGGTCGACG SEQ ID NO:239
CARTCTGCCCTGACTCAGCCT-
3'
HuVA3A-Sall 5'-TGAGCACACAGGTCGACG SEQ ID NO:240
TCCTATGWGCTGACTCAGCCA
CC-3'
HuVX3B-SalI 5'-TGAGCACACAGGTCGACG SEQ ID NO:241
TCTTCTGAGCTGACTCAGGAC
CC-3'
HuVA4-SalI 5'-TGAGCACACAGGTCGACG SEQ ID NO:242
CACGTTATACTGACTCAACCG
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CC-3'
HuVA5-Sall 5'-TGAGCACACAGGTCGACG SEQ ID NO:243
CAGGCTGTGCTGACTCAGCCG
TC-3'
HuVA6-SalI 5'-TGAGCACACAGGTCGACG SEQ ID NO:244
AATTTTATGCTGACTCAGCCC
CA-3'
HuVA7/8-Sall 5'-TGAGCACACAGGTCGACG SEQ ID NO:245
CAGRCTGTGGTGACYCAGGAG
CC-3'
HuVA9-Sall 5'-TGAGCACACAGGTCGACG SEQ ID NO:246
CWGCCTGTGCTGACTCAGCCM
CC-3'
HuJA1-NotI 5'-GAGTCATTCTCGACTTGC SEQ ID NO:247
GGCCGCACCTAGGACGGTGAC
CTTGGTCCC-3'
HuJA2/3-NotI 5'-GAGTCATTCTCGACTTGC SEQ ID NO:248
GGCCGCACCTAGGACGGTCAG
CTTGGTCCC-3'
HuJX4/5-NotI 5'-GAGTCATTCTCGACTTGC SEQ ID NO:249
GGCCGCACYTAAAACGGTGAG
CTGGGTCCC-3'
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Table 5: Distribution of the different light chain products
over the 10 fractions.
Light chain Number of Fraction alleles/fraction
products alleles number
Vk1B/Jkl-5 19 1 and 2 9.5
Vk2/Jkl-5 9 3 9
Vk3B/Jkl-5 7 4 7
Vk4B/Jkl-5 1
Vk5/Jkl-5 1 5 5
Vk6/Jkl-5 3
VA1A/J11-3
VA1B/Jll-3 5 6 5
VA1C/Jl1-3
VX2/Jll-3 5 7 5
VX3A/Jll-3 9 8 9
VA3B/J11-3
VA4/J11-3 3
VA5/Jll-3 1V 9 5
VA6/Jll-3 1
VA7/8/J11-3 3 10 6
Vx9/Jll-3 3
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Table 6: Human IgG heavy chain variable region primers
(sense).
Primer name Primer nucleotide SEQ ID NO
sequence
HuVH1B/7A 5'-CAGRTGCAGCTGGTG SEQ ID NO:250
CARTCTGG-3'
HuVH1C 5'-SAGGTCCAGCTGGTR SEQ ID NO:251
CAGTCTGG-3'
HuVH2B 5'-SAGGTGCAGCTGGTG SEQ ID NO:252
GAGTCTGG-3'
HuVH3B 5'-SAGGTGCAGCTGGTG SEQ ID NO:253
GAGTCTGG-3'
HuVH3C 5'-GAGGTGCAGCTGGTG SEQ ID NO:254
GAGWCYGG-3'
HuVH4B 5'-CAGGTGCAGCTACAG SEQ ID NO:255
CAGTGGGG-3'
HuVH4C 5'-CAGSTGCAGCTGCAG SEQ ID NO:256
GAGTCSGG-3'
HuVH5B 5'-GARGTGCAGCTGGTG SEQ ID NO:257
CAGTCTGG-3'
HuVH6A 5'-CAGGTACAGCTGCAG SEQ ID NO:258
CAGTCAGG-3'
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Table 7: Human IgG heavy chain variable region primers
extended with SfiI/NcoI restriction sites (sense) and human
IgG heavy chain J-region primers extended with XhoI/BstEII
restriction sites (anti-sense).
Primer name Primer nucleotide SEQ ID NO
sequence
HuVH1B/7A-Sfil 5'-GTCCTCGCAACTGCG SEQ ID NO:259
GCCCAGCCGGCCATGGCC
CAGRTGCAGCTGGTGCAR
TCTGG-3'
HuVH1C-Sfil 5'-GTCCTCGCAACTGCG SEQ ID NO:260
GCCCAGCCGGCCATGGCC
SAGGTCCAGCTGGTRCAG
TCTGG-3'
HuVH2B-SfiI 5'-GTCCTCGCAACTGCG SEQ ID NO:261
GCCCAGCCGGCCATGGCC
CAGRTCACCTTGAAGGAG
TCTGG-3'
HuVH3B-SfiI 5'-GTCCTCGCAACTGCGGCC SEQ ID NO:262
CAGCCGGCCATGGCCSAGGTG
CAGCTGGTGGAGTCTGG-3'
HuVH3C-Sfil 5'-GTCCTCGCAACTGCG SEQ ID NO:263
GCCCAGCCGGCCATGGCC
GAGGTGCAGCTGGTGGAG
WCYGG-3'
HuVH4B-SfiI 5'-GTCCTCGCAACTGCG SEQ ID NO:264
GCCCAGCCGGCCATGGCC
CAGGTGCAGCTACAGCAG
TGGGG-3'
HuVH4C-SfiI 5'-GTCCTCGCAACTGCGGCC SEQ ID NO:265
CAGCCGGCCATGGCCCAGSTG
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CAGCTGCAGGAGTCSGG-3'
HuVH5B-SfiI 5'-GTCCTCGCAACTGCG SEQ ID NO:266
GCCCAGCCGGCCATGGCC
GARGTGCAGCTGGTGCAG
TCTGG-3'
HuVH6A-Sfil 5'-GTCCTCGCAACTGCG SEQ ID NO:267
GCCCAGCCGGCCATGGCC
CAGGTACAGCTGCAGCAG
TCAGG-3'
HuJH1/2-XhoI 5'-GAGTCATTCTCGACTCGA SEQ ID NO:268
GACGGTGACCAGGGTGCC-3'
HuJH3-XhoI 5'-GAGTCATTCTCGACT SEQ ID NO:269
CGAGACGGTGACCATTGT
CCC-3'
HuJH4/5-XhoI 5'-GAGTCATTCTCGACT SEQ ID NO:270
CGAGACGGTGACCAGGGT
TCC-3'
HuJH6-XhoI 5'-GAGTCATTCTCGACTCGA SEQ ID NO:271
GACGGTGACCGTGGTCCC-3'
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Table 8: Binding of single-chain (scFv) phage antibodies to
rabies virus G protein (ERA strain) and to FBS as measured by
ELISA.
Name phage antibody Rabies virus G FBS
protein (OD492nm) (OD492nm)
SC04-001 0.828 0.053
SC04-004 0.550 0.054
SC04-008 0.582 0.058
SC04-010 0.915 0.043
SC04-018 0.247 0.052
SC04-021 0.278 0.052
SC04-026 0.212 0.054
SC04-031 0.721 0.065
SC04-038 0.653 0.061
SC04-040 0.740 0.053
SC04-060 0.923 0.056
SC04-073 0.657 0.054
SC4-097 0.835 0.056
SC04-098 0.798 0.060
SC04-103 0.606 0.059
SC04-104 0.566 0.063
SC04-108 0.363 0.052
SC04-120 0.571 0.052
SC04-125 0.735 0.049
SC04-126 0.232 0.051
SC04-140 0.865 0.057
SC04-144 0.775 0.054
SC04-146 0.484 0.057
SC04-164 0.547 0.057
control (S057) 0.650 0.055
control (02-007) 0.063 0.052
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Table 9: Data of the single-chain Fv's capable of binding
rabies virus G protein.
Name SEQ ID NO SEQ ID NO HCDR3 Vg-locus VL-locus
scFV of nucl. of amino (SEQ ID NO:)
sequence acid
(libr.) sequence
sc04-001 157 158 GLYGELFDY 3-20 V13 (31
(JK1994) (SEQ ID NO:1) (DP32) - V2-13)
159 160 VkI
sc04-004 DYLYPTTDFDY 3-23 (012/02 -
(WT2000) (SEQ ID N0:2) .(DP47) DPK9)
sc04-008 161 162
(RAB-03- MGFTGTYFDY 2-70 V13 (3h
GO1) (SEQ ID NO:3) (DP28) - V2-14)
sc04-010 163 164
(RAB-03- DGLDLTGTIQPFGY 3-30 VkI (L11
G01) (SEQ ID NO:4) (DP49) - DPK3)
sc04-018 165 166
(RAB-03- VSVTTGAFNI 4-04 V11 (lc
GOI) (SEQ ID N0:5) (DP70) - V1-16)
sc04-021 167 168
(RAB-03- GSVLGDAFDI 3-30
GOI) (SEQ ID N0:6) (DP49) VkI (L8)
sc04-026 169 170 VkII
(RAB-03- TSNWNYLDRFDP 5-51 (A19/03 -
G01) (SEQ ID NO:7) (DP73) DPK15)
sc04-031 171 172
(RAB-03- GSVLGDAFDI 3-30 VkI (L5
G01) (SEQ ID N0:8) (DP49) - DPKS)
scO4-038 173 174
(RAB-03- GSVLGDAFDI 3-30 VkI (L5
GOI) (SEQ ID NO:9) (DP49) - DPK5)
sc04-040 175 176
(RAB-03- GSKVGDFDY 3-30 V13 (3h
GOl) (SEQ ID N0:10) (DP49) - V2-14)
sc04-060 177 178 EKEKYSDRSGYSYY VkI
(RAB-04- YYYMDV 4-59 (012/02 -
G01) (SEQ ID NO:11) (DP71) DPK9)
sc04-073 179 180
(RAB-04- DGLDLTGTIQPFGY 3-30
G01) (SEQ ID N0:12) (DP49) VkI (L12)
sc04-097 181 182
(RAB-04- TASNLGRGGMDV 3-23
GO1) (SEQ ID N0:13) (DP47) VkI (L8)
sc04-098 183 184
(RAB-04- VAVAGTHFDY 3-30
G01) (SEQ ID NO:14) (DP49) VkI (A30)
sc04-103 185 186
(RAB-04- VAVAGESFDS 3-30 VkI (L5
G01) (SEQ ID NO:15) (DP49) - DPKS)
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sc04-104 187 188
(RAB-04- IVVVTALDAFDI 3-30
GO1) (SEQ ID NO:16) (DP49) VkI (L12)
sc04-108 189 190
(RAB-04- FMIVADDAFDI 3-30
GO1) (SEQ ID NO:17) (DP49) VkI (L1)
sc04-120 191 192
(RAB-04- GGKTGEFDY 3-30
GO1) (SEQ ID NO:18) (DP49) VkI (L8)
sc04-125 193 194
(RAB-04- IATAGTGFDY 3-30
GO1) (SEQ ID NO:19) (DP49) VkI (L8)
sc04-126 195 196
(RAB-04- MGFTGTYFDY 2-70 V13 (3h
GO1) (SEQ ID NO:20) (DP28) - V2-14)
sc04-140 197 198
(RAB-04- VTNPGDAFDI 3-30 VkI
G01) (SEQ ID NO:21) (DP49) (L4/18a)
sc04-144 199 200
(RAB-04- GGKTGEFDY 3-30
GO1) (SEQ ID NO:22) (DP49) VkI (L8)
sc04-146 201 202
(RAB-04- GGKTGEFDY 3-30 VkIII (L2
G01) (SEQ ID NO:23) (DP49) - DPK21)
sc04-164 203 204
(RAB-04- GSVLGDAFDI 3-30 VkI (L19
G01) (SEQ ID NO:24) (DP49) - DPK6)
205 206 ENLDNSGTYYYFSG
WFDP 1-69 V12 (2e
S057 (SEQ ID NO:25) (DP10) - V1-3)
312 313 RQHISSFPWFDS
(SEQ ID V13 (3h
SOJB NO:276) 2-05 - V2-14)
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Table 10: Data of assay for rabies virus neutralizing activity
of scFvs.
Name scFv 50% endpoint 50% endpoint Potency
dilution dilution WHO (IU/ml)
standard (2
IU/ml )
SC04-001 270 405 1.3
SC04-004 3645 405 18
SC04-008 >10935 405 >54
SC04-010 810 405 4
SC04-018 15 405 0.1
SC04-021 270 405 1.3
SC04-026 45 270 0.3
SC04-031 90 270 0.7
SC04-038 270 270 2
SC04-040 45 270 0.3
SC04-060 30 270 0.2
SC04-073 405 270 3
SC04-097 30 270 0.2
SC04-098 1215 270 9
SC04-103 45 270 0.3
SC04-104 135 270 1
SC04-108 135 270 1
SC04-120 810 270 6
SC04-125 405 270 3
SC04-126 10 2,70 0.1
SC04-140 135 270 1
SC04-144 810 270 6
SC04-146 405 270 3
SC04-164 45 270 0.3
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Table 11A: Data of assay for measuring neutralizing activity
of scFvs for E57 escape viruses E57A2, E57A3 and E57B1.
Name
E57A2 E57A3 E57B1
scFv
1* 2* 3* 1* 2* 3* 1* 2* 3*
SC04-001 10 90 0.2 10 90 0.2 30 45 1.3
SC04-004 810 90 18.0 1215 90 27.0 810 45 36.0
SC04-008 10 90 0.2 15 90 0.3 270 45 12.0
SC04-010 270 90 6.0 270 90 6.0 270 45 12.0
SC04-018 5 90 0.1 15 90 0.3 15 45 0.7
SC04-021 10 90 0.2 30 90 0.7 10 90 0.2
SC04-026 <5 90 0.0 <5 45 0.0 <5 90 0.0
SC04-031 10 90 0.2 30 90 0.7 10 90 0.2
SC04-038 90 90 2.0 90 90 2.0 45 90 1.0
SC04-040 15 90 0.3 5 90 0.1 5 90 0.1
SC04-060 5 90 0.1 5 90 0.1 <5 90 0.0
SC04-073 135 90 3.0 90 30 6.0 30 30 2.0
SC04-097 <5 90 0.0 <5 90 0.0 <5 90 0.0
SC04-098 810 90 18.0 270 30 18.0 270 30 18.0
SC04-103 <5 90 0.0 10 90 0.2 5 90 0.1
SC04-104 90 90 2.0 30 30 2.0*:' 30 30 2.0
SC04-108 15 90 0.3 <5 90 0.0 <5 90 0.0
SC04-120 45 90 1.0 30 30 2.0 10 30 0.7
SC04-125 135 90 3.0 135 30 9.0 90 30 6.0
SC04-126 <5 90 0.0 <5 45 0.0 <5 90 0.0
SC04-140 30 45 1.3 90 30 6.0 45 90 1.0
SC04-144 270 45 12.0 270 30 18.0 135 90 3.0
SC04-146 90 45 4.0 90 30 6.0 90 90 2.0
SC04-164 15 45 0.7 30 30 2.0 15 90 0.3
1* is 50% endpoint dilution
2* is 50% endpoint dilution WHO standard (2 IU/ml)
3* is Potency (III/ml)
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Table 11B: Data of assay for measuring neutralizing activity
of scFvs for E57 escape viruses E57B2, E57B3 and E57C3.
Name E57B2 E57B3 E57C3
SCFV
1 2* 3* 1* 2* 3* 1* 2* 3*
sC04-001 30 45 1.3 90 270 0.7 5 90 0.1
SC04-004 5 45 0.2 2430 270 18.0 270 90 6.0
SC04-008 5 45 0.2 45 270 0.3 10 90 0.2
SC04-010 45 45 2.0 405 270 3.0 270 90 6.0
SC04-018 15 45 0.7 15 270 0.1 30 90 0.7
SC04-021 10 90 0.2 30 270 0.2 10 90 0.2
SC04-026 <5 45 0.0 <5 45 0.0 <5 30 0.0
SC04-031 10 90 0.2 30 270 0.2 30 90 0.7
SC04-038 30 90 0.7 90 270 0.7 90 90 2.0
SC04-040 5 90 0.1 15 135 0.2 10 90 0.2
SC04-060 <5 90 0.0 10 135 0.1 5 90 0.1
SC04-073 30 90 0.7 90 270 0.7 90 90 2.0
SC04-097 <5 90 0.0 <5 135 0.0 <5 90 0.0
SC04-098 90 90 2.0 810 270 6.0 270 90 6.0
SC04-103 <5 90 0.0 10 135 0.1 10 90 0.2
SC04-104 45 90 1.0 45 270 0.3 90 90 2.0
SC04-108 10 90 0.2 <5 135 0.0 15 90 0.3
SC04-120 15 90 0.3 45 270 0.3 30 90 0.7
SC04-125 90 90 2.0 270 270 2.0 270 90 6.0
SC04-126 <5 45 0.0 <5 45 0.0 <5 30 0.0
SC04-140 30 90 0.7 90 90 2.0 270 90 6.0
SC04-144 90 90 2.0 270 90 6.0 405 90 9.0
SC04-146 30 90 0.7 90 90 2.0 90 90 2.0
SC04-164 15 90 0.3 15 90 0.3 30 90 0.7
1* is 50% endpoint dilution
2* is 50% endpoint dilution WHO standard (2 IU/ml)
3* is Potency (III/ml)
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Table 12: Oligonucleotides used for PCR amplification of VH
genes.
Name and nucleotide sequence Vu gene SEQ ID NO:
5H-B: SC04-001 280
acctgtcttgaattctccatggccgaggtgcagct
ggtggagtctg
5H-C: SC04-021 281
acctgtcttgaattctccatggcccaggtgcagct SC04-031
ggtggagtctgg SC04-125
SC04-164
5H-C-long: SC04-010 282
acctgtcttgaattctccatggcccaggtgcagct SC04-038
ggtggagtctgggg SC04-040
SC04-073
SC04-098
SC04-103
SC04-104
SC04-108
SC04-120
SC04-140
SC04-144
SC04-146
5H-F: SC04-018 283
acctgtcttgaattctccatggcccaggtgcagct SC04-060
gcaggagtccggccc
5H-H: SC04-026 284
acctgtcttgaattctccatggccgaggtgcagct
ggtgcagtctgg
5H-I: SC04-004 285
acctgtcttgaattctccatggccgaggtgcagct SC04-097
gctggagtctgg
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5H-M: SC04-008 286
acctgtcttgaattctccatggcccaggtgacctt SC04-126
gaaggagtctgg
sy3H-A: SC04-001 287
gcccttggtgctagcgctggagacggtcaccaggg SC04-004
tgccctggcccc SC04-008
SC04-010
SC04-026
SC04-040
SC04-073
SC04-098
SC04-120
SC04-125
SC04-126
SC04-144
SC04-146
sy3H-C: SC04-097 288
gcccttggtgctagcgctggagacggtcacggtgg
tgccctggcccc
sy3H-C-long: SC04-060 289
gcccttggtgctagcgctggagacggtcacggtgg
tgcccttgccccagacgtc
sy3H-D: SC04-018 290
gcccttggtgctagcgctggacacggtcaccatgg SC04-021
tgccctggcccc SC04-031
SC04-038
SC04-104
SC04-108
SC04-140
SC04-164
sy3H-E: SC04-103 291
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gcccttggtgctagcgctggacacggtcaccaggg
tgccccggcccc
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Table 13: Oligonucleotides used for PCR amplification of VL
genes.
Name and nucleotide sequence VL gene SEQ ID NO:
3L-B: SC04-001 292
ttttccttagcggccgcgactcacctaggacggtc
agcttggtc
5K-B: SC04-031 293
acctgtctcgagttttccatggctgacatccagat SC04-060
gacccagtc SC04-073
SC04-098
SC04-103
SC04-104
SC04-108
SC04-164
5K-C: SC04-004 294
acctgtctcgagttttccatggctgacatccagat
gacccagtctccatcctccc
5K-G: SC04-026 295
acctgtctcgagttttccatggctgacatcgtgat
gacccagtctcc
5K-K: SC04-010 296
acctgtctcgagttttccatggctgccatccagat
gacccagtctcc
5K-M: SC04-021 297
acctgtctcgagttttccatggctgacatccagct SC04-097
gacccagtc SC04-120
SC04-125
SC04-144
5K-N: SC04-038 298
acctgtctcgagttttccatggctgacatccagat
gactcagtc
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5K-O: SCQ4-140 299
acctgtctcgagttttccatggctgccatccagct
gacccagtc
5K-Q: SC04-146 300
acctgtctcgagttttccatggctgagatcgtgat
gactcagtc
5L-E: SC04-008 301
acctgtctcgagttttccatggcttcctacgtgct
gactcagccg
5L-F: SC04-018 302
acctgtctcgagttttccatggctcagtccgtgct
gactcagcc
5L-G: SC04-040 303
acctgtctcgagttttccatggcttcctacgtgct SC04-126
gactcagcc
sy3K-F: SC04-004 304
gctgggggcggccacggtccgcttgatctccacct SC04-010
tggtccc SC04-021
SC04-031
SC04-098
SC04-104
SC04-125
SC04-140
SC04-144
SC04-164
sy3K-I: SC04-038 305
gctgggggcggccacggtccgcttgatctccagcc SC04-097
gtgtccc SC04-103
SC04-108
SC04-146
sy3K-J: SC04-026 306
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gctgggggcggccacggtccgcttgatctccagct SC04-060
tggtccc SC04-073
sy3K-K: SC04-120 307
gctgggggcggccacggtccgcttgatgtccacct
tggtccc
sy3L-A: SC04-018 308
ccagcacggtaagcttcagcacggtcaccttggtg SC04-126
ccagttcc
sy3L-C: SC04-040 309
ccagcacggtaagcttcagcacggtcagcttggtg
cctccgcc
sy3L-D: SC04-008 310
ccagcacggtaagcttcaacacggtcagctgggtc
cc
sy5L-A: SC04-001 311
acctgtctcgagttttccatggcttcctccgagct
gacccaggaccctgctg
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Table 14: Binding of the human monoclonal antibodies CR57,
CRJB and CR04-010 (10 pg/ml) to linear peptides of the
extracellular domain of glycoprotein G of rabies virus strain
ERA.
Amino acid sequence CR57 CRJB CR04-010
of linear peptide
FPIYTILDKLGPWS 71 97 1
PIYTILDKLGPWSP 42 105 39
IYTILDKLGPWSPI 36 89 87
IYTILDKLGPWSPID 44 97 104
TILDKLGPWSPIDI 48 114 91
ILDKLGPWSPIDIH 76 96 88
ILDKLGPWSPIDIHH 54 104 69
r DKLGPWSPIDIHHL 55 99 107
KLGPWSPIDIHHLS 62 103 93
LGPWSPIDIHHZSC 72 105 45
GPWSPIDIHHLSCP 69 112 19
PWSPIDIHHLSCPN 68 114 33
PWSPIDIHHLSCPNN 62 104 47
SPIDIHHLSCPNNL 80 106 11
SPIDIHHLSCPNNLV 74 85 1
IDIHHLSCPNNLVV 46 93 90
IDIHHLSCPNNLVVE 69 102 55
IHHLSCPNNLVVED 38 96 78
IHHLSCPNNLVVEDE 37 85 113
HLSCPNNLVVEDEG 56 76 117
V LSCPNNLVVEDEGC 65 119 111
SCPNNLVVEDEGCT 69 117 127
SCPNNLVVEDEGCTN 83 114 91
PNNLVVEDEGCTNL 77 97 49
PNNLVVEDEGCTNLS 78 107 97
LVVEDEGCTNLSG 72 99 97
VE 75 119 55
VVEDEGCTNLSGFS 76 103 52
DEGCTNLSGFSY 73 107 91
DEGCTNLSGFSYM 74 103 31
DEGCTNLSGFSYME 54 90 7
EGCTNLSGFSYMEL 1 23 1
GCTNLSGFSYMELK 51 114 129
CTNLSGFSYMELKV 55 114 118
CTNLSGFSYMELKVG 47 110 137
NLSGFSYMELKVGY 43 106 161
LSGFSYMELKVGYI 61 115 170
SGFSYMELKVGYIL L 71 132 169
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SGFSYMELKVGYILA 79 132 161
GFSYMELKVGYILAI 65 111 141
SYMELKVGYILAIK 89 112 192
SYMELKVGYILAIKM 65 123 152
YMELKVGYILAIKMN 78 114 150
LKVGYILAIKMNG 76 141 107
LKVGYILAIKMNGF 87 132 76
LKVGYIIAI 78 112 118
GYILAIKMNGFTC 78 118 68
GYILAIKMNGFTCT 77 93 1
GYILAIKMNGFTCTG 75 90 1
ILAIKMNGFTCTGV 47 107 107
ILAIKMNGFTCTGVV 79 103 104
JLAIKMNGFTCTGVVT 68 130 159
IKMNGFTCTGVVTE 47 103 152
IKMNGFTCTGVVTEA 68 108 138
KMNGFTCTGVVTEAE 76 104 133
GFTCTGVVTEAEN 69 99 148
1GFTCTGVVTEAENY 69 101 138
FTCTGVVTEAENYT 71 86 129
TCTGVVTEAENYTN 83 125 154
TCTGVVTEAENYTNF 92 112 129
CTGVVTEAENYTNFV 76 123 150
TGVVTEAENYTNFVG 85 110 154
GVVTEAENYTNFVGY 86 111 110
VVTEAENYTNFVGYV 87 106 114
VTEAENYTNFVGYVT 79 90 73
EAENYTNFVGYVTT 68 84 8
EAENYTNFVGYVTTT 69 117 142
NYTNFVGYVTTTF 66 106 110
NYTNFVGYVTTTFK 44 112 183
YTNFVGYVTTTFKR 49 114 174
TNFVGYVTTTFKRK 51 104 138
TNFVGYVTTTFKRKH 71 125 165
FVGYVTTTFKRKHF 65 107 154
GYVTTTFKRKHFR 70 111 152
GYVTTTFKRKHFRP 75 113 155
YVTTTFKRKHFRPT 70 123 160
YV'TTTFKRKHFRPTP 85 106 160
TTTFKRKHFRPTPD 79 105 119
TTTFKRKHFRPTPDA 80 108 137
TTFKRKHFRPTPDAC 74 99 110
TFKRKHFRPTPDACR 96 111 108
KRKHFRPTPDACRA 64 92 62
HFRPTPDACRAA 65 93 1
HFRPTPDACRAAY 64 107 99
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FRPTPDACRAAYN 73 112 124
FRPTPDACRAAYNW 46 113 118
FRPTPDACRAAYNWK 43 112 148
PTPDACRAAYNWKM 77 101 129
PTPDACRAAYNWKMA .99 125 143
TPDACRAAYNWKMAG 92 132 160
PDACRAAYNWKMPGD 61 124 147
ACRAAYNWKMAGDP 84 113 136
CRAAYNWKMAGDPR 82 116 138
CRAAYNWKMAGDPRY 87 118 137
P,AAYNWKt4AGDPRYE 90 130 120
YNWKMAGDPRYEE 68 106 120
YNWKMAGDPRYEES 96 94 77
GDPRYEESL 83 118 116
GDPRYEESLH 58 101 69
GDPRYEESLHN 69 101 1
GDPRYEESLHNP 62 102 84
GDPRYEESLHNPY 64 116 112
GDPRYEESLHNPYP 40 101 125
GDPRYEESLHNPYPD 36 98 123
PRYEESLHNPYPDY 57 110 118
RYEESLHNPYPDYR 73 115 129
YEESLHNPYPDYRW 69 112 125
ESLHNPYPDYRWL 58 106 120
ESLHNPYPDYRWLR 76 123 141
SLHNPYPDYRWLRT 92 132 125
SLHNPYPDYRWLRTV 78 111 137
LHNPYPDYRWLRTVK 79 106 142
PYPDYRWLRTVKT 86 108 146
PYPDYRWLRTVKTT 85 102 151
XPDYRWLRTVKTTK 65 93 103
PDYRWLRTVKTTKE 72 97 97
PDYRWLRTVKTTKES 76 85 27
YRWLRTVKTTKESL 54 111 105
WLRTVKTTKESLV 46 117 125
WLRTVKTTKESLVI 40 110 120
TVKTTKESLVII 41 104 125
TVKTTKESLVIIS 65 104 161
TVKTTKESLVIISP 82 120 150
TVKTTKESLVIISPS 76 116 150
TTKESLVIISPSV 71 120 154
TTKESLVIISPSVA 101 112 147
TTKESLVIISPSVAD 78 121 141
KESLVIISPSVADL 86 112 132
SLVIISPSVADLD 86 117 111
SLVIISPSVADLDP 88 125 143
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SLVIISPSVADLDPY 68 105 125
VIISPSVADLDPYD 85 107 93
IISPSVADLDPYDR 59 98 50
IISPSVADLDPYDRS 83 125 14
ISPSVADLDPYDRSL 50 119 91
3PSVADLDPYDRSLH 59 114 118
SVADLDPYDRSLHS 44 114 118
SVADLDPYDRSLHSR 49 106 129
ADLDPYDRSLHSRV 71 113 141
LDPYDRSLHSRVF 70 121 141
LDPYDRSLHSRVFP 111 152 127
DPYDRSLHSRVFPS 99 142 106
PYDRSLHSRVFPSG 90 120 134
YDRSLHSRVFPSGK 86 120 130
DRSLHSRVFPSGKC 364 818 127
RSLHSRVFPSGKCS 98 142 141
SLHSRVFPSGKCSG 87 141 156
SLHSRVFPSGKCSGV 69 111 141
HSRVFPSGKCSGVA 78 114 129
SRVFPSGKCSGVAV 97 118 111
SRVFPSGKCSGVAVS 100 125 24
VFPSGKCSGVAVSS 69 110 106
FPSGKCSGVAVSST 74 114 142
PSGKCSGVAVSSTY 64 134 146
SGKCSGVAVSSTYC 56 112 132
3GKCSGVAVSSTYCS 64 121 120
KCSGVAVSSTYCST 92 143 145
CSGVAVSSTYCSTN 88 130 130
SGVAVSSTYCSTNH 110 165 143
GVAVSSTYCSTNHD 79 110 115
VAVSSTYCSTNHDY 79 114 108
AVSSTYCSTNHDYT 85 114 118
VSSTYCSTNHDYTI 71 105 102
SSTYCSTNHDYTIW 78 107 121
SSTYCSTNHDYTIWM 76 107 121
TYCSTNHDYTIWMP 86 99 119
YCSTNHDYTIWMPE 96 107 74
CSTNHDYTIWMPEN 47 92 29
CSTNHDYTIWMPENP 52 106 86
STNHDYTIWMPENPR 60 112 107
TNHDYTIWMPENPRL 69 129 119
HDYTIWMPENPRLG 71 119 130
DYTIWMPENPRLGM 82 125 123
YTIWMPENPRLGMS 93 127 123
TIWMPENPRLGMSC 97 132 143
TIWMPENPRLGMSCD 69 106 134
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IWMPENPRLGMSCDI 98 110 101
PENPRLGMSCDIF 88 113 120
PENPRLGMSCDIFT 105 121 143
PENPRLGMSCDIFTN 83 111 104
NPRLGMSCDIFTNS 71 118 111
PRLGMSCDIFTNSR 90 113 138
PRLGMSCDIFTNSRG 72 112 105
GMSCDIFTNSRGK 88 106 113
LGMSCDIFTNSRGKR 76 1.10 114
GMSCDIFTNSRGKRA 54 120 101
1SCDIFTNSRGKRAS 46 110 106
SCDIFTNSRGKRASK 44 111 98
CDIFTNSRGKRASKG 42 104 117
DIFTNSRGKRASKGS 70 107 Ill
IFTNSRGKRASKGSE 77 125 87
FTNSRGKRASKGSET 83 111 119
TNSRGKRASKGSETC 68 108 110
SRGKRASKGSETCG 92 100 119
SRGKRASKGSETCGF 64 93 90
GKRASKGSETCGFV 75 104 115
GKRASKGSETCGFVD 92 124 118
RASKGSETCGFVDE 92 106 129
P,ASKGSETCGFVDER 86 110 134
SKGSETCGFVDERG 97 108 103
SKGSETCGFVDERGL 92 102 76
GSETCGFVDERGLY 90 97 4'4
GSETCGFVDERGLYK 57 115 92
SETCGFVDERGLYKS 33 116 86
TCGFVDERGLYKSL 64 120 138
TCGFVDERGLYKSLK 47 120 125
CGFVDERGLYKSLKG 72 115 120
GFVDERGLYKSLKGA 84 120 129
FVDERGLYKSLKGAC 86 121 124
DERGLYKSLKGACK 50 108 110
DERGLYKSLKGACKL 90 119 54
RGLYKSLKGACKLK 90 118 106
GLYKSLKGACKLKL 90 121 121
LYKSLKGACKLKLC 94 129 92
LYKSLKGACKLKLCG 93 136 141
KSLKGACKLKLCGV 80 112 110
SLKGACKLKLCGVL 129 113 110
SLKGACKLKLCGVLG 'OIO. 1l1 124
KGACKLKLCGVLGL 13`4'0 90 23
GACKLKLCGVLGLR 183.r 111 100
GACKLKLCGVLGLRL 123; 134 129
CKLKLCGVLGLRLM _ 117 142
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CKLKLCGVLGLRLMD 10 111 147
LKLCGVLGLRLMDG x=73 120 114
KLCGVLGLRLMDGT 145 148
LCGVLGLRLMDGTW 3152 132 86
LCGVLGLRLMDGTWV 83 138 129
CGVLGLRLMDGTWVA 99 117 104
GVLGLRLMDGTWVAM 89 148 117
LGLRLMDGTWVAMQ 90 141 127
LGLRLMDGTWVAMQT 102 115 97
LRLMDGTWVAMQTS 104 138 120
RLMDGTWVAMQTSN 103 114 118
LMDGTWVAMQTSNE 100 113 130
LMDGTWVAMQTSNET 96 106 106
4DGTWVAMQTSNETK 97 97 110
DGTWVAMQTSNETKW 69 114 92
TWVAMQTSNETKWC 58 113 82
TWVAMQTSNETKWCP 78 118 107
AMQTSNETKWCPP 50 114 116
AMQTSNETKWCPPD 86 104 151
QTSNETKWCPPDQ 104 114 128
4QTSNETKWCPPDQL 104 132 125
TSNETKWCPPDQLV 92 120 155
SNETKWCPPDQLVN 97 111 90
SNETKWCPPDQLVNL 99 129 110
ETKWCPPDQLVNLH 90 128 107
TKWCPPDQLVNLHD 105 120 118
KWCPPDQLVNLHDF 85 125 125
CPPDQLVNLHDFR 89 113 121
CPPDQLVNLHDFRS 101 119 99
CPPDQLVNLHDFRSD 93 137 127
PPDQLVNLHDFRSDE 107 120 56
PDQLVNLHDFRSDEI 35 106 63
DQLVNLHDFRSDEIE 54 117 97
LVNLHDFRSDEIEH 60 113 106
LVNLHDFRSDEIEHL 47 104 100
LHDFRSDEIEHLV 83 129 98
LHDFRSDEIEHLVV 83 113 110
LHDFRSDEIEHLVVE 93 115 121
1DFRSDEIEHLVVEE 69 107 150
DFRSDEIEHLVVEEL 99 103 110
FRSDEIEHLVVEELV 86 114 116
SDEIEHLVVEELVR 100 138 104
SDEIEHLVVEELVRK 101 117 118
DEIEHLVVEELVRKR 94 123 143
IEHLVVEELVRKRE 82 113 121
IEHLVVEELVRKREE 90 129 118
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HLVVEELVRKREEC 82 114 106
LVVEELVRKREECL 82 123 46
LVVEELVRKREECLD 64 100 79
VVEELVRKREECLDA 62 108 97
EELVRKREECLDAL 58 111 101
ELVRKREECLDALE 69 112 123
LVRKREECLDALES 82 113 117
LVRKREECLDALESI 86 130 124
VRKREECLDALESIM 58 181 151
REECLDALESIMT 73 110 137
KREECLDALESIMTT 102 113 97
EECLDALESIMTTK 94 110 106
ECLDALESIMTTKS 82 120 133
CLDALESIMTTKSV 91 112 125
CLDALESIMTTKSVS 101 146 155
LDALESIMTTKSVSF 97 116 152
DALESIMTTKSVSFR 104 120 188
LESIMTTKSVSFRR 97 132 137
ESIMTTKSVSFRRL 48 114 130
SIMTTKSVSFRRLS 62 111 114
SIMTTKSVSFRRLSH 54 130 97
IMTTKSVSFRRLSHL 43 101 111
4TTKSVSFRRLSHLR 59 116 125
TTKSVSFRRLSHLRK 66 118 111
TKSVSFRRLSHLRKL 83 125 123
SVSFRRLSHLRKLV 108 124 129
SVSFRRLSHLRKLVP 64 123 117
SFRRLSHLRKLVPG 90 111 105
SFRRLSHLRKLVPGF 92 110 96
FRRLSHLRKLVPGFG 90 108 111
LSHLRKLVPGFGK 92 143 118
P,LSHLRKLVPGFGKA 93 123 148
LSHLRKLVPGFGKAY 96 139 150
SHLRKLVPGFGKAYT 113 132 132
LRKLVPGFGKAYTI 99 111 102
RKLVPGFGKAYTIF 83 118 82
LVPGFGKAYTIFN 47 115 86
LVPGFGKAYTIFNK 47 114 123
VPGFGKAYTIFNKT 54 112 139
PGFGKAYTIFNKTL 58 114 138
PGFGKAYTIFNKTLM 78 113 157
GFGKAYTIFNKTLME 78 123 -=2G
FGKAYTIFNKTLMEA 90 151 '156
GKAYTIFNKTLMEAD 76 127 YTIFNKTLMEADA 101 123 554 YTIFNKTLMEADAH 86 121 '7'97..
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YTIFNKTLMEADAHY 104 147
TIFNKTLMEADAHYK 107 123 14;O
IFNKTLMEADAHYKS 100 1182!
FNKTLMEADAHYKSV 111 141 2736
KTLMEADAHYKSVR 104 116 141
TLMEADAHYKSVRT 91 98 123
TLMEADAHYKSVRTW 100 114 90
LMEADAHYKSVRTWN 73 107 97
ADAHYKSVRTWNE 62 129 83
ADAHYKSVRTWNEI 58 97 106
DAHYKSVRTWNEIL 56 100 100
DAHYKSVRTWNEILP 59 121 112
YKSVRTWNEILPS 112 160 125
YKSVRTWNEILPSK 80 130 123
KSVRTWNEILPSKG 66 137 116
SVRTWNEILPSKGC 115 125 1.14
SVRTWNEILPSKGCL 106 138 118
TWNEILPSKGCLR 90 124 133
TWNEILPSKGCLRV 120 127 120
TWNEILPSKGCLRVG 97 146 127
EILPSKGCLRVGG 102 136 117
EILPSKGCLRVGGR 104 130 163
ILPSKGCLRVGGRC 104 112 128
ILPSKGCLRVGGRCH 79 113 107
LPSKGCLRVGGRCHP 77 119 100
PSKGCLRVGGRCHPH 69 138 91
SKGCLRVGGRCHPHV 72 121 103
GCLRVGGRCHPHVN 68 130 115
GCLRVGGRCHPHVNG 85 125 123
CLRVGGRCHPHVNGV 102 132 134
RVGGRCHPHVNGVF 104 143 133
VGGRCHPHVNGVFF 86 143 99
GGRCHPHVNGVFFN 120 136 120
GGRCHPHVNGVFFNG 86 119 119
GRCHPHVNGVFFNGI 117 113 117
CHPHVNGVFFNGII 98 141 143
CHPHVNGVFFNGIIL 97 150 151
PHVNGVFFNGIILG 104 138 164
PHVNGVFFNGIILGP 93 173 146
VNGVFFNGIILGPD 97 123 114
GVFFNGIILGPDG 68 116 85
GVFFNGIILGPDGN 66 117 97
GVFFNGIILGPDGNV 58 116 100
FFNGIILGPDGNVL 55 132 108
FFNGIILGPDGNVLI 92 143 105
FNGIILGPDGNVLIP 61 139 130
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GIILGPDGNVLIPE 102 146 141
IILGPDGNVLIPEM 107 132 123
IILGPDGNVLIPEMQ 85 118 93
ILGPDGNVLIPEMQS 125 134 119
GPDGNVLIPEMQSS 100 134 150
PDGNVLIPEMQSSL 86 154 157
PDGNVLIPEMQSSLL 87 129 139
DGNVLIPEMQSSLLQ 123 134 169
GNVLIPEMQSSLLQQ 96 120 168
NVLIPEMQSSLLQQH 72 120 150
LIPEMQSSLLQQHM 92 104 142
IPEMQSSLLQQHME 89 111 85
IPEMQSSLLQQHMEL 89 128 129
PEMQSSLLQQHMELL 62 133 93
MQSSLLQQHMELLE 58 129 142
QSSLLQQHMELLES 65 113 117
SSLLQQHMELLESS 82 114 132
SSLLQQHMELLESSV 90 128 132
LLQQHMELLESSVI 124 163 133
LLQQHMELLESSVIP 78 111 121
QQHMELLESSVIPL 106 134 128
QHMELLESSVIPLV 103 134 133
HMELLESSVIPLVH 98 146 139
LLESSVIPLVHP 110 129 134
LLESSVIPLVHPL 90 125 152
LLESSVIPLVHPLA 90 133'"'' 155
LESSVIPLVHPLAD 72 117 118
ESSVIPLVHPLADP 90 128 128
SSVIPLVHPLADPS 104 138 143
SVIPLVHPLADPST 73 104 93
VIPLVHPLADPSTV 72 137 107
IPLVHPLADPSTVF 69 141 123
IPLVHPLADPSTVFK 96 156 188
PLVHPLADPSTVFKD 93 112 138
VHPLADPSTVFKDG 164 174 188
HPLADPSTVFKDGD 98 138 125
PLADPSTVFKDGDE 74 141 117
PLADPSTVFKDGDEA 99 125 90
PSTVFKDGDEAE 68 116 113
rDPSTVFKDGDEAED 147 152 110
PSTVFKDGDEAEDF 98 147 137
PSTVFKDGDEAEDFV 104 143 141
STVFKDGDEAEDFVE 104 120 125
VFKDGDEAEDFVEV 107 124 96
KDGDEAEDFVEVH 100 106 93
KDGDEAEDFVEVHL 65 76 119
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DGDEAEDFVEVHLP 72 93 76
GDEAEDFVEVHLPD 85 123 91
GDEAEDFVEVHLPDV 46 124 113
DEAEDFVEVHLPDVH 68 136 123
AEDFVEVHLPDVHN 76 117 114
DFVEVHLPDVHNQ 123 138 123
DFVEVHLPDVHNQV 90 141 123
FVEVHLPDVHNQVS 96 141 118
FVEVHLPDVHNQVSG 92 143 102
VHLPDVHNQVSGV 106 141 123
VHLPDVHNQVSGVD 91 150 139
HLPDVHNQVSGVDL 110 114 137
LPDVHNQVSGVDLG 104 150 129
PDVHNQVSGVDLGL 104 154 154
PDVHNQVSGVDLGLP 106 129 115
VHNQVSGVDLGLPN 117 133 113
NQVSGVDLGLPNW 100 119 38
NQVSGVDLGLPNWG 76 106 38
QVSGVDLGLPNWGK 78 138 98
Average 91.9 119.5 130.9
StDV 157.9 37.6 169.8
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Table 15: Neutralizing potencies of anti-rabies virus G
protein IgGs.
Name IgG IU/mg
CR04-001 89
CR04-004 5
CR04-008 1176
CR04-010 3000
CR04-018 1604
CR04-021 1500
CR04-026 <2
CR04-031 272
CR04-038 2330
CR04-040 3041
CR04-060 89
CR04-073 6116
CR04-097 <1
CR04-098 7317
CR04-103 3303
CR04-104 4871
CR04-108 4871
CR04-120 4938
CR04-125 4718
CR04-126 2655
CR04-140 478
CR04-144 6250
CR04-146 ND
CR04-164 4724
CR57 3800
CRJB 605
ND = not determined
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Table 16: Neutralizing potencies of anti-rabies virus G
protein IgGs against E57 escape viruses.
Name IgG E57A2 E57A3 E57B1 E57B2 E57B3 E57C3
(IU/mg) (IU/mg) (IU/mg) (IU/mg) (IU/mg) (IU/mg)
CR04-008 0* 0 0 0 0 0
CR04-010 8127 1355 5418 1355 2709 4064
CR04-018 1604 0 1604 0 59 535
CR04-021 450 2 150 8 50 50
CR04-038 9437 1573 9437 1049 6291 1573
CR04-040 8209 2736 24628 1368 5473 912
CR04-073 8256 1835 11008 1835 3669 1835
CR04-098 9878 3293 9878 3293 3293 3293
CR04-103 8917 2972 17835 2972 5945 2972
CR04-104 3288 2192 6576 2192 4384 1096
CR04-108 3288 731 4384 731 2192 731
CR04-120 1111 14 741 82 247 41
CRO4-125 708 39 236 79 157 79
CR04-126 88 0 18 0 18 0
CR04-144 5625 2813 11250 2813 5625 1875
CR04-164 4252 472 4252 472 945 709
* 0 indicates no 50% endpoint at a dilution of 1:100 of the
antibody
Table 17. Neutralizing potency of CR-57 against E98 escape
viruses.
E98-2 E98-4 E98-5 E98-6 E98-7
(IU/mg) (IU/mg) (IU/mg) (IU/mg) (IU/mg)
CR-57 2813 8438 4219 2813 8438
CR04-098 0* 0 0 0 0
* Zero indicates no 50% endpoint at a dilution of 1:1000 of
the antibody.
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Table 18: Occurrence of amino acid residues in the minimal
binding region of CR57 within genotype 1 rabies viruses.
Wild K L C G V' L
type
K L C G V L
(99.6%)* (100%) (100%) (98.7%) (99.6%) (70.7%)
R E I P
(0.4%) (0.9%) (0.4%) (26.7%)
R S
(0.4%) (2.6%)
*Percentage of occurrence of each amino acid is shown within
229 rabies virus isolates.
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