U.S. patent application number 11/980237 was filed with the patent office on 2008-03-20 for binding molecules capable of neutralizing rabies virus and uses thereof.
This patent application is currently assigned to Crucell Holland B.V.. Invention is credited to Alexander Berthold Hendrik Bakker, Cornelis Adriaan De Kruif, Robert Arjen Kramer, Willem Egbert Marissen.
Application Number | 20080070799 11/980237 |
Document ID | / |
Family ID | 34968690 |
Filed Date | 2008-03-20 |
United States Patent
Application |
20080070799 |
Kind Code |
A1 |
Bakker; Alexander Berthold Hendrik
; et al. |
March 20, 2008 |
Binding molecules capable of neutralizing rabies virus and uses
thereof
Abstract
Provided are binding molecules that specifically bind rabies
virus and are able to neutralize the virus. Further provided are
nucleic acid molecules encoding the binding molecules, compositions
comprising the binding molecules and methods of identifying or
producing the binding molecules. The binding molecules can be used
in the diagnosis, prophylaxis and/or treatment of a condition
resulting from rabies virus. In certain embodiments, they can be
used in the post-exposure prophylaxis of rabies.
Inventors: |
Bakker; Alexander Berthold
Hendrik; (Hillegom, NL) ; Marissen; Willem
Egbert; (Woerden, NL) ; Kramer; Robert Arjen;
(Utrecht, NL) ; De Kruif; Cornelis Adriaan; (De
Bilt, NL) |
Correspondence
Address: |
TRASK BRITT
P.O. BOX 2550
SALT LAKE CITY
UT
84110
US
|
Assignee: |
Crucell Holland B.V.
|
Family ID: |
34968690 |
Appl. No.: |
11/980237 |
Filed: |
October 29, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11590126 |
Oct 31, 2006 |
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11980237 |
Oct 29, 2007 |
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PCT/EP05/52410 |
May 26, 2005 |
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11590126 |
Oct 31, 2006 |
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60575023 |
May 27, 2004 |
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Current U.S.
Class: |
506/9 ;
435/5 |
Current CPC
Class: |
A61P 31/12 20180101;
C07K 16/10 20130101; C07K 2317/34 20130101; A61P 31/14 20180101;
C12N 2760/20122 20130101; G01N 33/56983 20130101; C07K 2317/56
20130101; C07K 14/005 20130101; C07K 2317/622 20130101; A61K
2039/507 20130101; A61P 43/00 20180101; C07K 2317/21 20130101; C07K
2317/76 20130101; A61K 2039/505 20130101 |
Class at
Publication: |
506/009 ;
435/005 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70; C40B 30/04 20060101 C40B030/04 |
Foreign Application Data
Date |
Code |
Application Number |
May 27, 2004 |
EP |
PCT/EP04/50943 |
Jul 29, 2004 |
EP |
PCT/EP04/51661 |
Sep 23, 2004 |
EP |
PCT/EP04/52286 |
Nov 3, 2004 |
EP |
PCT/EP04/52772 |
Jan 25, 2005 |
EP |
PCT/EP05/50310 |
Mar 3, 2005 |
EP |
PCT/EP05/50953 |
Claims
1. A method of identifying a binding molecule potentially having
neutralizing activity against a rabies virus or a nucleic acid
molecule encoding a binding molecule potentially having
neutralizing activity against a rabies virus, wherein the method
comprises: contacting a collection of binding molecules on the
surface of replicable genetic packages with a cell expressing a
protein of a rabies virus under conditions conducive to binding,
wherein the collection of binding molecules is prepared from RNA
isolated from cells obtained from a subject that has been
vaccinated against rabies or that has been exposed to rabies virus,
separating and recovering binding molecules that bind to the rabies
virus from binding molecules that do not bind, isolating at least
one recovered binding molecule, and verifying if the binding
molecule isolated has neutralizing activity against rabies
virus.
2. The method according to claim 1, further comprising separating
and recovering binding molecules containing a variable heavy 3-30
germline gene.
3. The method according to claim 1, wherein the collection of
binding molecules on the surface of replicable genetic packages is
a single chain Fv library.
4. The method according to claim 1, wherein the subject is a human
individual who has been vaccinated against rabies.
5. The method according to claim 1, wherein the replicable genetic
package is selected from the group consisting of a phage particle,
a bacterium, yeast, a fungus, a spore of a microorganism, and a
ribosome.
6. The method according to claim 1, wherein the cell expressing a
protein of a rabies virus is a cell expressing rabies virus G
protein.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 11/590,126, filed Oct. 31, 2006, now U.S. Pat. No. ______,
which application is a continuation of International Patent Appln.
No. PCT/EP2005/052410 filed May 26, 2005, and published in English
as PCT Internat'l Publication No. WO 2005/118644 A2, on Dec. 15,
2005, which application claims priority to Internat'l Patent Appln.
No. PCT/EP2005/050953 filed Mar. 3, 2005, which application claims
priority to Internat'l Patent Appln. No. PCT/EP2005/050310, filed
Jan. 25, 2005, which application claims priority to Internat'l
Patent Appln. No. PCT/EP2004/052772 filed Nov. 3, 2004, which
application claims priority to Internat'l Patent Appln. No.
PCT/EP2004/052286 filed Sep. 23, 2004, which application claims
priority to Internat'l Patent Appln. No. PCT/EP2004/051661 filed
Jul. 29, 2004 which application claims priority to Internat'l
Patent Appln. No. PCT/EP2004/050943 filed May 27, 2004, and U.S.
Provisional Patent Appln. Ser. No. 60/575,023 filed May 27, 2004,
the contents of the entirety of each of which are incorporated
herein by this reference.
STATEMENT ACCORDING TO 37 C.F.R. .sctn. 1.52(e)(5)--SEQUENCE
LISTING SUBMITTED ON COMPACT DISC
[0002] Pursuant to 37 C.F.R. .sctn. 1.52(e)(1)(ii), a compact disc
containing an electronic version of the Sequence Listing has been
submitted concomitant with this application, the contents of which
are hereby incorporated herein by reference. A second compact disc
is being submitted herewith and is an identical copy of the first
compact disc. The discs are labeled "copy 1" and "copy 2,"
respectively, and each disc contains one file entitled "Sequence
Listing 2578-7990US.txt" which is 337 KB and was created on Oct.
30, 2006.
TECHNICAL FIELD
[0003] The invention relates generally to biotechnology and
medicine. In particular, the invention relates to binding molecules
directed against rabies, such as virus-neutralizing binding
molecules. The binding molecules are useful in the post-exposure
prophylaxis of rabies.
BACKGROUND
[0004] 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 invariably
fatal encephalitis. Annually, more than 70,000 human fatalities are
estimated, and millions of others require post-exposure
treatment.
[0005] 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).
[0006] 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.
[0007] Rabies can be treated or prevented by both passive and
active immunizations. Rabies post-exposure prophylaxis includes
prompt local wound care and administration of both passive
(anti-rabies immunoglobulins) and active (vaccines)
immunizations.
[0008] 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
post-exposure 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).
[0009] 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
post-exposure 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.
[0010] Currently, a significant need still exists for new human
rabies virus-neutralizing monoclonal antibodies having improved
post-exposure prophylactic potential, particularly antibodies
having different epitope-recognition specificities.
SUMMARY OF THE INVENTION
[0011] Described are human monoclonal antibodies that offer the
potential to be used in mixtures useful in the post-exposure
prophylaxis of a wide range of rabies viruses and
neutralization-resistant variants thereof.
[0012] Herebelow follow definitions of terms as used herein.
DEFINITIONS
[0013] 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 recognized by the intact
immunoglobulin. An antigen-binding fragment can comprise a peptide
or polypeptide comprising an amino acid sequence of at least two
contiguous amino acid residues, at least five contiguous amino acid
residues, at least ten 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 30
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 60 contiguous amino
residues, at least 70 contiguous amino acid residues, at least 80
contiguous amino acid residues, at least 90 contiguous amino acid
residues, at least 100 contiguous amino acid residues, at least 125
contiguous amino acid residues, at least 150 contiguous amino acid
residues, at least 175 contiguous amino acid residues, at least 200
contiguous amino acid residues, or at least 250 contiguous amino
acid residues of the amino acid sequence of the binding
molecule.
[0014] 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., IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4.
[0015] Antigen-binding fragments include, inter alia, Fab, F(ab'),
F(ab').sub.2, Fv, dAb, Fd, complementarity determining region (CDR)
fragments, single-chain antibodies (scFv), bivalent single-chain
antibodies, single-chain phage antibodies, diabodies, triabodies,
tetrabodies, peptides or polypeptides that contain at least a
fragment of an immunoglobulin that is sufficient to confer specific
antigen binding to the peptide or polypeptide, 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, N.Y.,
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 site, the binding sites
may be identical to one another or they may be different.
[0016] 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, or 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.
[0017] 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 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 post-translational modifications of proteins.
[0018] 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 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.
[0019] Conservative amino acid substitutions include the ones in
which the amino acid residue is replaced with an amino acid 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, 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 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.
[0020] A mutation in a nucleotide sequence can be a single
alteration made at a locus (a point mutation), such as 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.
[0021] Host: 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.
[0022] 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
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.
[0023] 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 has 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.
[0024] Nucleic acid molecule: The term "nucleic acid molecule" as
used in the 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 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, hair-pinned, 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.
[0025] 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 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.
[0026] 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.
[0027] 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
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-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 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
with related antigens. In certain embodiments, binding molecules or
fragments thereof that immunospecifically bind to an antigen do not
cross-react with other antigens.
[0028] 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.
[0029] 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) 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.
[0030] Provided are binding molecules capable of specifically
binding to and neutralizing rabies virus. Furthermore, provided are
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.
DESCRIPTION OF THE FIGURES
[0031] FIG. 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 two 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. FIG. 1A shows the comparison of
the nucleotide sequences. Numbers above amino acids indicate amino
acid numbers from rabies virus glycoprotein including signal
peptide. FIG. 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 FIG. 1 are
also represented by SEQ ID NOS:130 through 141 of the incorporated
SEQUENCE LISTING.
[0032] FIG. 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 two 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. FIG. 2A shows the comparison of
the nucleotide sequences. Numbers above amino acids indicate amino
acid numbers from rabies virus glycoprotein including the signal
peptide. FIG. 2B 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 FIG. 2 are
also represented by SEQ ID NOS:142 through 151.
[0033] FIG. 3 shows the vector PDV-C06.
[0034] FIG. 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
.mu.g/ml). Subsequently, CR-57bio binding was monitored in absence
and presence of scFvs.
[0035] FIG. 5 shows a competition ELISA of anti-rabies virus scFvs
and the anti-rabies virus antibody called CR-57. ELISA plates
coated with purified rabies virus G protein were incubated with
CR-57 (1 .mu.g/ml) before addition of excess scFvs. Subsequently,
scFv binding was monitored in absence and presence of CR-57.
[0036] FIG. 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.
[0037] FIG. 7 shows a competition FACS assay of anti-rabies virus G
protein IgGs and the anti-rabies virus antibody called CR-57. 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 streptavidin-PE. FACS signals are
shown as percentage of CR57bio binding alone.
[0038] FIG. 8 shows the comparison of the amino acid sequences of
CVS-11 and E98 escape viruses. Virus-infected cells were harvested
two 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. FIG. 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. FIG. 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.
[0039] FIG. 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.
[0040] FIG. 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 indicated. Amino
acid numbering is from the mature protein minus the signal peptide
sequence.
DETAILED DESCRIPTION OF THE INVENTION
[0041] In a first aspect, the invention encompasses binding
molecules capable of specifically binding to rabies virus. In
certain embodiments, the binding molecules of the invention also
have rabies virus-neutralizing activity. In certain embodiments,
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. In certain embodiments, 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.
[0042] 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.degree. 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., 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; UV light irradiation may also be
used. In certain embodiments, 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.
[0043] 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 peptides or
polypeptides derived from rabies virus or a cell transfected with a
rabies virus protein and/or peptide or polypeptide. 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 diagnostic 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.
[0044] In certain embodiments, 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 peptide or polypeptide, protein or
glycoprotein, or analog or fragment thereof, that is capable of
binding to a human binding molecule of the invention with
sufficiently high affinity to form a detectable antigen-binding
molecule complex.
[0045] 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').sub.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 peptides
or polypeptides 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.
[0046] 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 genome, which could result in differences in amino acid
composition of viral proteins. If such mutations occur in
structural viral proteins, the virus 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, 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 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 binding molecules may
further comprise at least one other therapeutic agent such as a
medicament suitable for the post exposure prophylaxis of
rabies.
[0047] Typically, binding molecules according to the invention can
bind to their binding partners, i.e., rabies virus or fragments
thereof such as rabies virus proteins, with an affinity constant
(K.sub.d-value) that is lower than 0.2*10.sup.-4 M, 1.0*10.sup.-5
M, 1.0*10.sup.-6 M, 1.0*10.sup.-7 M, preferably lower than
1.0*10.sup.-8 M, more preferably lower than 1.0*10.sup.-9 M, more
preferably lower than 1.0*10.sup.-10 M, even more preferably lower
than 1.0*10.sup.-11 M, and in particular lower than 1.0*10.sup.-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.sup.-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, using the
BIACORE system (Pharmacia Biosensor AB, Uppsala, Sweden).
[0048] 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 peptides or polypeptides of the rabies virus. In
certain embodiments, the binding molecules are capable of
specifically binding to the rabies virus G protein or fragment
thereof. The rabies virus proteins or peptides or polypeptides may
either be in soluble form or may bind to rabies virus bound or
attached to a carrier or substrate as described above. In certain
embodiments, cells transfected with the G protein may be used as
binding partner for the binding molecules.
[0049] In certain embodiments, 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%,
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 the 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.
[0050] 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 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. In
certain embodiments, the CDR3 region is a heavy chain CDR3
region.
[0051] 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 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 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.
[0052] 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 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:66, 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 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 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.
[0053] In a preferred embodiment, the binding molecules having
rabies virus-neutralizing activity of the invention are
administered in IgG format, preferably IgG1 format.
[0054] Another aspect of the invention includes functional variants
of binding molecules as defined herein. Molecules are considered to
be "functional variants of a binding molecule according to the
invention," if the variants are capable of competing for specific
binding to rabies virus or a fragment thereof with the parent
binding molecules; in other words, when the functional variants are
still capable of binding to rabies virus or a fragment thereof.
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
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, demethylation, formation of covalent
cross-links, formation of cysteine, 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.
[0055] Alternatively, functional variants can be binding molecules
as defined in the 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. In
certain embodiments, 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 residues from hypervariable loops. Functional
variants intended to fall within the scope of the 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.
[0056] In certain embodiments, 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.
[0057] 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 C3b receptors or by agglutinating rabies virus to make it more
easily phagocytosed.
[0058] In yet a further aspect, disclosed are 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 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 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.
[0059] The tags of the immunoconjugates may be therapeutic agents
and/or 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 materials, luminescent materials,
bioluminescent materials, radioactive materials, positron emitting
metals, and nonradioactive paramagnetic metal ions.
[0060] 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
(ELISAs), radioimmunoassays (RIAs), 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
.sup.33P, .sup.32P, .sup.35S, .sup.3H, and .sup.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.
[0061] When the binding molecules of the 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 invention are
known to the skilled artisan. When the binding molecules of the
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
complexes.
[0062] When the immunoconjugates 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 diethylenetriaminepentaacetic acid, to
ultrasound contrast agents or to X-ray contrast agents, or by
radioisotopic labeling.
[0063] Furthermore, the binding molecules, functional variants
thereof or immunoconjugates of the invention can also be 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, 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 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 invention can usefully be attached to the surface
of a microtiter plate for ELISA.
[0064] The binding molecules 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.
[0065] 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. In certain
embodiments, these antigens are antigens that 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.
[0066] 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.
[0067] In another aspect, provided is a nucleic acid molecule
encoding at least a binding molecule 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.
[0068] One of ordinary skill in the art will appreciate that
functional variants of these nucleic acid molecules are also
intended to be a part of the 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.
[0069] In certain embodiments, 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.
[0070] 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 comprising essentially an amino
acid sequence comprising amino acids 1-119 of SEQ ID NO:335.
[0071] 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 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 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 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 NO:335 and a variable light chain comprising
the amino acid sequence comprising amino acids 1-107 of SEQ ID
NO:337.
[0072] 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 nucleotide
sequence selected from the group consisting of SEQ ID NO:74, SEQ ID
NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ
ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84,
SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID
NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ
ID NO:94, SEQ ID NO:95, SEQ ID NO:96 and SEQ ID NO:97. In certain
embodiments, 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 NO:334.
[0073] In certain embodiments, 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 NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID
NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105,
SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID
NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID 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. In certain embodiments,
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.
[0074] In another aspect, provided are vectors, i.e., nucleic acid
constructs, comprising one or more nucleic acid molecules according
to the invention. Vectors can be derived from plasmids such as
inter alia F, R1, RP1, Co1, pBR322, TOL, Ti, etc.; cosmids; phages
such as lambda, lambdoid, M13, Mu, P1, P22, Q.sub..beta., T-even,
T-odd, T2, T4, T7, etc.; plant viruses such as inter alia alfalfa
mosaic virus, bromovirus, capillovirus, carlavirus, carnovirus,
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, bimaviridae, bunyaviridae,
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, poxyiridae, reoviridae, retroviruses,
rhabdoviridae, rhinoviruses, Semliki Forest virus, tetraviridae,
togaviridae, toroviridae, vaccinia virus, vescular stomatitis
virus, etc. Vectors can be used for 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 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, lipofectamine transfection or
electroporation. Vectors may be autonomously replicating or may
replicate together with the chromosome into which they have been
integrated. In certain embodiments, 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, zeocin,
thymidine kinase gene from Herpes simplex virus (HSV-TK), and
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.
[0075] Hosts containing one or more copies of the vectors mentioned
above are an additional subject of the invention. In certain
embodiments, the hosts are host cells. Host cells include, but are
not limited to, cells of mammalian, plant, 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 or 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 invention. Mammalian cells provide expressed
proteins with post-translational modifications that are most
similar to natural molecules of mammalian origin. Since the
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,
Wiltshire SP4 OJG, Great Britain on 29 Feb. 1996 under number
96022940 and marketed under the trademark PER.C6.RTM. (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.
[0076] In certain embodiments, the human producer cells comprise at
least a functional part of a nucleic acid sequence encoding an
adenovirus E1 region in expressible format. In certain embodiments,
the host cells are derived from a human retina and immortalized
with nucleic acids comprising adenoviral E1 sequences, such as the
cell line deposited at the European Collection of Cell Cultures
(ECACC), CAMR, Salisbury, Wiltshire SP4 OJG, Great Britain on 29
Feb. 1996 under number 96022940 and marketed under the trademark
PER.C6.RTM.. 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.RTM. 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.
[0077] 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 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
obtainable by the above described method are also a part of the
invention.
[0078] 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 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 are also a part of the invention.
[0079] In certain embodiments, binding molecules or functional
variants thereof, according to the invention, may be generated by
transgenic non-human mammals, such as, for instance, transgenic
mice or rabbits, that express human immunoglobulin genes. In
certain embodiments, 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 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, N.Y., and "Current
Protocols in Immunology," 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.
[0080] In a further aspect, provided is 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.
[0081] 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
certain embodiments, 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 peptides or polypeptides derived from
rabies virus, such as the G protein, fusion proteins comprising
these proteins or peptides or polypeptides, and the like. In
certain embodiments, cells transfected with rabies virus G protein
are used for selection procedures.
[0082] In yet a further aspect, provided is 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 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).
[0083] 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 Fvs, 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 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.
[0084] In yet a further aspect, described is 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, wherein 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 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.
[0085] 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 invention comprise this specific
V.sub.H germline gene.
[0086] 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 U.S. Pat. No. 5,696,108; Burton and
Barbas, 1994; de Kruif et al., 1995b; and "Phage Display: A
Laboratory Manual," edited by C. F. Barbas, D. R. Burton, J. K.
Scott and G. J. Silverman (2001), Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. All these references are herewith
incorporated herein in their entirety.
[0087] 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, 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*10.sup.9 antibody specificities and may be assembled
from 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 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 been exposed to rabies virus. In certain
embodiments, 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 invention.
[0088] 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 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 immobilizing target antigens such as antigens
from rabies 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-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 peptides or polypeptides, host cells
expressing one or more rabies virus proteins or peptides or
polypeptides 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 E. 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 peptides or polypeptides 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 peptides or
polypeptides. 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 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.RTM. process. MAbstract.RTM. is a registered
trademark of Crucell Holland B.V. See also, U.S. Pat. No.
6,265,150, which is incorporated herein by reference.)
[0089] In yet a further aspect, provided are 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 stabilizing molecules, such as albumin
or polyethylene glycol, or salts. In certain embodiments, 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.
[0090] In yet a further aspect, provided are compositions
comprising at least one nucleic acid molecule as defined in the
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.
[0091] Furthermore, included are pharmaceutical compositions
comprising at least one binding molecule according to the
invention, at least one functional variant or fragment thereof, at
least one immunoconjugate of the invention, at least one
composition of the invention, or combinations thereof. The
pharmaceutical composition of the invention may further comprise at
least one pharmaceutically acceptable excipient.
[0092] In certain embodiments, a pharmaceutical composition
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. The 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 certain embodiments, the binding
molecule comprises a heavy chain variable region comprising the
amino acid sequence SEQ ID NO:273. In certain embodiments, 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. In certain
embodiments, 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 Lyssavirus
genus or even with other viruses of the rhabdovirus family, while
exhibiting no cross-reactivity with other viruses or normal
cellular proteins. In certain embodiments, the binding molecule is
capable of neutralizing escape variants of the other binding
molecule in the cocktail.
[0093] Another aspect of the invention pertains to a pharmaceutical
composition comprising at least two rabies virus-neutralizing
binding molecules, preferably (human) binding molecules according
to the invention, wherein the binding molecules are capable of
reacting with different, non-competing epitopes of the rabies
virus. In certain embodiments, 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 amino acids 34-42 and amino acids
198-200 (see, Prehaud et al., 1988). Antigenic site III is a
continuous conformational epitope at amino acids 330-338 and
harbors two charged residues, K330 and R333, that affect viral
pathogenicity (see, Seif et al., 1985; Coulon et al., 1998; and
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 epitopes (see, Tordo, 1996;
Bunschoten et al., 1989; Luo et al., 1997; and Ni et al., 1995).
Benmansour et 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 currently known linear and
conformational-neutralizing epitopes on rabies glycoprotein (FIG.
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
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.
[0094] In a preferred embodiment, the pharmaceutical composition
comprises a first rabies virus-neutralizing binding molecule
comprising at least a CDR3 region, preferably heavy chain CDR3
region, comprising the amino acid sequence of SEQ ID NO: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 consisting of SEQ
ID NO:4, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 and
SEQ ID NO:22. In certain embodiments, the second rabies
virus-neutralizing binding molecule comprises at least a CDR3
region, preferably a heavy chain CDR3 region, comprising the amino
acid sequence of SEQ ID NO:14. In certain embodiments, 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, 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. In certain embodiments, the heavy and
light chain of the first rabies virus-neutralizing binding molecule
are encoded by SEQ ID NO:122 and SEQ ID NO:124, respectively, and
the heavy and light chain of the second rabies virus-neutralizing
binding molecule are encoded by SEQ ID NO:334 and SEQ ID NO:336,
respectively.
[0095] A pharmaceutical composition comprising two binding
molecules, wherein the pI of the binding molecules is divergent and
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, wherein 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 certain embodiments, the binding molecules are
binding molecules according to the invention and the pharmaceutical
composition is a pharmaceutical composition according to the
invention. In certain embodiments, the binding molecules are
monoclonal antibodies, e.g., human monoclonal antibodies such as
IgG1 antibodies. In certain embodiments, 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
certain embodiments, 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. In certain embodiments, the binding molecules
have the heavy chain CDR3 region of SEQ ID NO:14 and SEQ ID NO:25,
respectively.
[0096] In certain embodiments, 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 are being used, the
selection of binding molecules having a compatible p1 is also very
useful in order to obtain a composition, wherein each binding
molecule is sufficiently stable for storage, handling and
subsequent use.
[0097] In certain embodiments, the binding molecules present in the
pharmaceutical composition of the invention augment each other's
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 in vitro neutralization assays
or animal models such as hamsters.
[0098] Furthermore, the pharmaceutical composition according to the
invention may comprise at least one other therapeutic, prophylactic
and/or diagnostic agent. The further therapeutic and/or
prophylactic agents may be anti-viral agents such as ribavirin or
interferon-alpha.
[0099] 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.
[0100] 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 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.
[0101] Alternatively, the binding molecules, variant or fragments
thereof, immunoconjugates, nucleic acid molecules or compositions
of the 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. In certain embodiments, the pharmaceutically
acceptable excipient used in the invention is suitable to high drug
concentration, can maintain proper fluidity and, if necessary, can
delay absorption.
[0102] 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.
[0103] 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.
[0104] In a further aspect, the binding molecules, functional
variants, immunoconjugates, compositions, or pharmaceutical
compositions of the invention can be used as a medicament. Thus, a
method of treatment and/or prevention of a lyssavirus infection
using the human binding molecules, functional variants,
immunoconjugates, compositions, or pharmaceutical compositions of
the invention is another part of the invention. The lyssavirus can
be a virus from any of the known genotypes, but is preferably
rabies virus. The above-mentioned molecules or compositions can be
used in the post-exposure prophylaxis of rabies.
[0105] 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 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 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 include, but are not limited to,
purified chick embryo cell (PCEC) vaccine (RabAvert), human diploid
cell vaccine (HDCV; Imovax vaccine) or rabies vaccine adsorbed
(RVA).
[0106] The molecules are typically formulated in the compositions
and pharmaceutical compositions of the invention in a
therapeutically or diagnostically effective amount. 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.
[0107] In certain embodiments, a single bolus of the binding
molecules or pharmaceutical compositions of the invention are
administered. The molecules and pharmaceutical compositions
according to the 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.
[0108] In another aspect, disclosed is the use of binding molecules
or functional variants thereof, immunoconjugates described herein,
nucleic acid molecules described herein, compositions or
pharmaceutical compositions described herein 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. In certain embodiments,
the molecules mentioned above are used in the preparation of a
medicament for the post exposure prophylaxis of rabies.
[0109] 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 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 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.
[0110] Currently, HRIG products are used for post exposure
prophylaxis of rabies. An adult dose of HRIG of 1500 IU (75 kg
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. Provided is 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
certain embodiments, the pharmaceutical composition is stored in a
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. Furthermore, the kit may
comprise instructions for diluting the pharmaceutical composition
and/or instructions for administering the pharmaceutical
composition, whether diluted or not.
[0111] Further described is 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, peptides or
polypeptides or other antigenic fragments. In certain embodiments,
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. Such methods include, inter alia, homogeneous and
heterogeneous binding immunoassays, such as radioimmunoassays
(RIA), ELISA, immunofluorescence, immunohistochemistry, FACS,
BIACORE and Western blot analyses.
[0112] 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 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 enzyme-linked immuno assay (ELISA). In certain
embodiments, 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
molecules may identify one or more neutralizing epitopes. The
peptides/epitopes found can be used as vaccines and for the
diagnosis of rabies.
[0113] In a further aspect, provided is a method of screening a
binding molecule or a functional variant of a 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 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 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. In certain embodiments, 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 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 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.
[0114] 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 certain embodiments, 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 certain embodiments, 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
molecules 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 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
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.
[0115] 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. In certain
embodiments, 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% to 90% or even greater. The
identification of one or more 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
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 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.
[0116] 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 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 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 the cell, (c) isolating at least one
recovered 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 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.RTM. cell. However,
other (E1-immortalized) cell lines could also be used to express
the proteins such as BHK, CHO, NS0, HEK293, or 911 cells. In
certain embodiments, the binding molecule is human. The infectious
agent can be a virus, a bacterium, a yeast, a fungus or a parasite.
In certain embodiments, 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.
[0117] Another aspect of the invention pertains to a binding
molecule as defined herein having rabies virus-neutralizing
activity, wherein the human binding molecule comprises at least a
heavy chain CDR3 region comprising the amino acid sequence
comprising SEQ ID NO:25 and further wherein the human binding
molecule has a rabies virus-neutralizing activity of at least 2500
IU/mg protein. In certain embodiments, the 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.
[0118] In certain embodiments, the binding molecule comprises a
variable heavy chain comprising the amino acid sequence comprising
SEQ ID NO:273. In certain embodiments, 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.
[0119] A nucleic acid molecule encoding the binding molecules as
described above is also a part of the invention. In certain
embodiments, 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. In
certain embodiments, 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, 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 Feb. 1996 under number 96022940 and
marketed under the trademark PER.C6.RTM. (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
[0120] 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-J
[0121] 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 IgG1 expression vector named
pcDNA3002(Neo). The resulting vectors pgSO57C11 and pgSOJBC11 were
used for transient expression in cells from the cell line deposited
at the European Collection of Cell Cultures (ECACC), CAMR,
Salisbury, Wiltshire SP4 OJG, Great Britain on 29 Feb. 1996 under
number 96022940 and marketed under the trademark PER.C6.RTM.. The
nucleotide and amino acid sequences of the heavy and light chains
of these antibodies are shown in SEQ ID NOS:122 through 129,
respectively. Serial dilutions (0.5 ml) of rabies virus strain
CVS-11 (dilutions ranging from 10.sup.-1 to 10.sup.-8) were
incubated with a constant amount (.about.4 IU/ml) of antibody CR-57
or CR-JB (0.5 ml) for one hour at 37.degree. C./5% CO.sub.2 before
addition to wells containing mouse neuroblastoma cells (MNA cells)
or BSR cells (Baby Hamster Kidney-like cell line). After three 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.degree. C. until further use.
Subsequently, the cells were acetone-fixed for 20 minutes at
4.degree. C., and stained overnight at 37.degree. C./5% CO.sub.2
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 one
single focus with the exception of E57B1 (three foci). EJB escape
viruses were isolated from one focus (EJB3F), three foci (EJB2B,
four foci (EJB2C), five foci (EJB2E, 2F), or six 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% to 100% infection after 24 hours) for
use in a virus neutralization assay.
[0122] 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 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% to 100% infection. Virus/IgG mix was
incubated for one hour at 37.degree. C./5% CO.sub.2 before addition
to MNA cells. Twenty-four hours post-infection (at 34.degree. C./5%
CO.sub.2) the cells were acetone-fixed for 20 minutes at 4.degree.
C., and stained for minimally three 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 which
the virus infection is blocked by 50% in this assay. To calculate
the potency, an Internat'l 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 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.
[0123] EJB viruses were no longer neutralized by CR-JB or CR-57
(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 the
antibodies CR-57 and CR-JB (in a 1:1 IU/mg ratio) gave similar
results as observed with the single antibodies (data not
shown).
[0124] 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.
[0125] Both E57 and EJB escape viruses showed mutations in the same
region of the glycoprotein (see, FIGS. 1 and 2, respectively; see
for all the sequences described in FIGS. 1 and 2 SEQ ID NOS:130
through 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
[0126] 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.degree. 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 separation (TRIZOL.TM.) 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/.mu.l. Next, 1 .mu.g of RNA was converted
into cDNA as follows: To 10 .mu.l total RNA, 13 .mu.l DEPC-treated
ultrapure water and 1 .mu.l random hexamers (500 ng/.mu.l) were
added and the obtained mixture was heated at 65.degree. C. for five
minutes and quickly cooled on wet-ice. Then, 8 .mu.l 5.times.
First-Strand buffer, 2 .mu.l dNTP (10 mM each), 2 .mu.l DTT (0.1
M), 2 .mu.l Rnase-inhibitor (40 U/.mu.l) and 2 .mu.l
Superscript.TM. III MMLV reverse transcriptase (200 U/.mu.l) were
added to the mixture, incubated at room temperature for five
minutes and incubated for one hour at 50.degree. C. The reaction
was terminated by heat inactivation, i.e., by incubating the
mixture for 15 minutes at 75.degree. C.
[0127] The obtained cDNA products were diluted to a final volume of
200 .mu.l 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.
[0128] For each donor 5 to 10 .mu.l 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 through 7).
PCR reaction mixtures contained, besides the diluted cDNA products,
25 .mu.mol sense primer and 25 .mu.mol anti-sense primer in a final
volume of 50 .mu.l of 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM
MgCl.sub.2, 250 .mu.M dNTPs and 1.25 units Taq polymerase. In a
heated-lid thermal cycler having a temperature of 96.degree. C.,
the mixtures obtained were quickly melted for two minutes, followed
by 30 cycles of: 30 seconds at 96.degree. C., 30 seconds at
60.degree. C. and 60 seconds at 72.degree. C.
[0129] 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'-ACACTCTCCCCTGTTGAAGCTCTT-3' (see, SEQ ID NO:152) or
C-lambda constant region HuC.lamda.2 5'-TGAACATTCTGTAGGGGCCACTG-3'
(see, SEQ ID NO:153) and HuC.lamda.7 5'-AGAGCATTCTGCAGGGGCCACTG-3'
(see, SEQ ID NO:154) (the HuC.lamda.2 and HuC.lamda.7 anti-sense
primers were mixed to equimolarity before use), yielding four 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. One-tenth 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, FIG. 3 and SEQ ID NO:155).
This resulted in four times 63 products of approximately 350
basepairs that were pooled to a total of ten 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 .mu.g of
pooled fraction and 100 .mu.g PDV-C06 vector were digested with
SalI and NotI and purified from gel. Thereafter, a ligation was
performed overnight at 16.degree. C. as follows. To 500 ng PDV-C06
vector 70 ng pooled fraction was added in a total volume of 50
.mu.l ligation mix containing 50 mM Tris-HCl (pH 7.5), 10 mM
MgCl.sub.2, 10 mM DTT, 1 mM ATP, 25 .mu.g/ml BSA and 2.5 .mu.l T4
DNA Ligase (400 U/.mu.l). 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 .mu.l ultrapure water and per ligation
mix two times 2.5 .mu.l aliquots were electroporated into 40 .mu.l
of TG1 competent E. coli bacteria according to the manufacturer's
protocol (Stratagene). Transformants were grown overnight at
37.degree. C. in a total of 30 dishes (three dishes per pooled
fraction; dimension of dish: 240 mm.times.240 mm) containing 2TY
agar supplemented with 50 .mu.g/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 Qiagen.TM.
QIAFilter MAXI prep kit.
[0130] 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. One-tenth of each of the
isolated products was used in an identical PCR reaction as
described 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
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 columns. Next, the fractions
were digested with SfiI 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 NcoI 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 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.times.240 mm)
containing 2TY agar supplemented with 50 .mu.g/ml ampicillin and
4.5% glucose. All bacteria were harvested in 2TY culture medium
containing 50 .mu.g/ml ampicillin and 4.5% glucose, mixed with
glycerol to 15% (v/v) and frozen in 1.5 ml aliquots at -80.degree.
C. Rescue and selection of each library were performed as described
below.
Example 3
Selection of Phages Carrying Single Chain Fv Fragments Specifically
Recognizing Rabies Virus Glycoprotein
[0131] Antibody fragments were selected using antibody phage
display libraries, general phage display technology and
MAbstract.RTM. technology, essentially as described in U.S. Pat.
No. 6,265,150 and in WO 98/15833 (both of which are incorporated by
reference herein). The antibody phage libraries used were two
different semi-synthetic scFv phage libraries (JK1994 and WT2000)
and the immune scFv phage libraries (RAB-03-G01 and RAB-04-G01)
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 built 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 (incorporated by reference herein) were used in the
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 virus ERA strain), and/or transfected cells
expressing rabies virus G protein (rabies virus ERA strain).
[0132] 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-minute incubation at 4.degree. C., the virus sample was
centrifuged (36,000 rpm, 4.degree. C.) in a SW51 rotor. The
supernatant was collected and dialyzed overnight at 4.degree. C.
against 0.1 M Tris/EDTA. Subsequently, the glycoprotein was
collected from the dialysis chamber, aliquoted, and stored at
-80.degree. 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.
[0133] Whole inactivated rabies virus or rabies virus G protein
were diluted in phosphate buffered saline (PBS), 2 to 3 ml was
added to MaxiSorp Nunc-Immuno Tubes (Nunc) and incubated overnight
at 4.degree. C. on a rotating wheel. An aliquot of a phage library
(500 .mu.l, approximately 10.sup.13 cfu, amplified using CT helper
phage (see, WO 02/103012)) was blocked in blocking buffer (2%
Protifar in PBS) for one to two 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 two 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 ten minutes at room temperature with 1 ml
of 50 mM Glycine-HCl pH 2.2. Subsequently, the eluted phages were
mixed with 0.5 ml of 1 M Tris-HCl pH 7.5 to neutralize the pH. This
mixture was used to infect 5 ml of an XL1-Blue E. coli culture that
had been grown at 37.degree. C. to an OD 600 nm of approximately
0.3. The phages were allowed to infect the XL1-Blue bacteria for 30
minutes at 37.degree. C. Then, the mixture was centrifuged for ten
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 tetracycline, ampicillin and glucose. After
incubation overnight of the plates at 37.degree. 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.degree. C. to an OD 600 nm of .about.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.degree. 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.
[0134] 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
Feb. 1996, under number 96022940 and marketed under the trademark
PER.C6.RTM.. They are hereinafter referred to as PER.C6.RTM. cells.
Here, the blocked phage library (2 ml) was first added to
1*10.sup.7 subtractor cells (in DMEM/10% FBS) and incubated for one
hour at 4.degree. C. on a rotating wheel. The subtractor cells were
PER.C6.RTM. cells that expressed the Vesicular Stomatitis Virus
(VSV) glycoprotein ecto domain on their 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.RTM. cells were removed from the phage library.
The phage/cell mixture was centrifuged (five minutes at 4.degree.
C. at 500.times.g) to remove cell-bound phages, and the supernatant
was added to a new tube containing 3 ml of 1*10.sup.7 subtractor
cells. The subtraction step was repeated twice with the respective
supernatant. Subsequently, the subtracted phages were incubated for
1.5 hours at 4.degree. C. on a rotating wheel with the rabies virus
glycoprotein expressing transfected cells (PER.C6.RTM. cells
(3*10.sup.6 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.
[0135] 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
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 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
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 transfected cells, i.e., cell 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
Validation of the Rabies Virus Glycoprotein-Specific Single-Chain
Phage Antibodies
[0136] 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 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
Maxisorp.TM. ELISA plates. After coating, the plates were blocked
in PBS/1% Protifar for one hour at room 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, 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 SO57). 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
[0137] 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 NO:165, SEQ ID 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.
[0138] The VH and VL gene identity (see, I. M. Tomlinson, S. C.
Williams, O. Ignatovitch, S. J. Corbett, G. Winter, "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)
[0139] 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 added to each dilution at a
concentration that gives 80% to 100% infection. Virus/scFv mix was
incubated for one hour at 37.degree. C./5% CO.sub.2 before addition
to MNA cells. Twenty-four hours post-infection (at 34.degree. C./5%
CO.sub.2), the cells were acetone-fixed for 20 minutes at 4.degree.
C., and stained for minimally three 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).
[0140] 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
[0141] To identify antibodies that bind to non-overlapping,
non-competing epitopes, a rabies glycoprotein competition ELISA was
performed. Nunc-Immuno.TM. Maxisorp F96 plates (Nunc) were coated
overnight at 4.degree. C. with a 1:1000 dilution of purified rabies
virus glycoprotein (1 mg/ml; rabies virus ERA strain) in PBS (50
.mu.l). Uncoated protein was washed away before the wells were
blocked with 100 .mu.l PBS/1% Protifar for one hour at room
temperature. Subsequently, the blocking solution was discarded and
50 .mu.l of the non-purified anti-rabies virus scFvs in PBS/1%
Protifar (2.times. diluted) was added. Wells were washed five times
with 100 .mu.l of PBS/0.05% Tween-20. Then, 50 .mu.l biotinylated
anti-rabies virus competitor IgG, CR-57bio, was added to each well,
incubated for five minutes at room temperature, and the wells were
washed five times with 100 .mu.l of PBS/0.05% Tween-20. To detect
the binding of CR-57bio, 50 .mu.l of a 1:2000 dilution of
streptavidin-HRP antibody (Becton Dickinson) was added to the wells
and incubated for one hour at room temperature. Wells were washed
again as above and the ELISA was further developed by addition of
100 .mu.l of OPD reagens (Sigma). The reaction was stopped by
adding 50 .mu.l 1 M H.sub.2SO.sub.4 before measuring the OD at 492
nm.
[0142] The signal obtained with CR-57bio alone could be reduced to
background levels when co-incubated with scFv SO57, i.e., the scFv
form of CR-57 (for nucleotide and amino acid sequence of SO57 see
SEQ ID NOS: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 NOS:312 and 313, respectively). This indicates that the
scFvs SO57 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, FIG. 4).
[0143] Similar results were obtained with the following experiment.
First, the rabies virus antibody CR-57 was added 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, FIG. 5).
Example 8
Construction of Fully Human Immunoglobulin Molecules (Human
Monoclonal Anti-Rabies Virus Antibodies) from the Selected
Anti-Rabies Virus Single Chain Fvs
[0144] 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 expression vectors
pSyn-C03-HC.gamma.1 (see, SEQ ID NO:277) and pSyn-C04-C.lamda.
(see, SEQ ID NO:278), respectively. The V.sub.H and V.sub.L 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-HC.gamma.1 and pSyn-C04-C.lamda., respectively.
[0145] 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 oligonucleotides to append
restriction sites and/or sequences for expression in the IgG
expression vectors pSyn-C03-HC.gamma.1 and pSyn-C05-C.kappa. (see,
SEQ ID NO:279), respectively. The V.sub.H and V.sub.L genes were
amplified using the oligonucleotides as given in Table 12 and 13,
respectively, and the PCR products were cloned into the vectors
pSyn-C03-HC.gamma.1 and pSyn-C05-Ck, respectively. The
oligonucleotides are designed such that they correct any deviations
from the germline sequence that have 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.
[0146] The resulting expression constructs pgG104-001C03,
pgG104-008C03, pgG104-018C03, pgG104-040C03 and pgG104-126C03
encoding the anti-rabies virus human IgG1 heavy chains in
combination with the relevant pSyn-C04-V.lamda. construct encoding
the corresponding light chain were transiently expressed in 293T
cells and supernatants containing IgG1 antibodies were obtained.
The expression constructs pgG104-004C03, pgG104-010C03,
pgG104-021C03, pgG104-026C03, pgG104-031C03, pgG104-038C03,
pgG104-060C03, pgG104-073C03, pgG104-097C03, pgG104-098C03,
pgG104-103C03, pgG104-104C03, pgG104-108C03, pgG104-120C03,
pgG104-125C03, pgG104-140C03, pgG104-144C03, pgG104-146C03 and
pgG104-164C03 encoding the anti-rabies virus human IgG1 heavy
chains in combination with the relevant pSyn-C05-V.kappa. construct
encoding the corresponding light chain were transiently expressed
in 293T cells and supernatants containing IgG1 antibodies were
obtained.
[0147] 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 immunoglobulins (see, for instance WO
00/63403 which is incorporated by reference herein).
[0148] Additionally, for CR04-098, a single human IgG1 expression
vector named pgG104-098C10 was generated as described above for
vectors pgSO57C11 and pgSOJBC11 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 NOS:334 through
337, respectively. Vectors pgSO57C11 (see, Example 1) and
pgG104-098C10 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 Feb. 1996 under number
96022940 and marketed under the trademark PER.C6.RTM.. 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.
Example 9
Rabies Virus G Protein Competition ELISA with IgGs
[0149] 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 to
50 .mu.g/ml) of unlabeled anti-rabies virus G protein IgG for one
hour at room temperature. Then, 50 .mu.l of a different
biotinylated anti-rabies virus IgG (1 .mu.g/ml) is added to each
well, incubated for five minutes at room temperature, and
immediately washed five times with 100 .mu.l of PBS/0.05% Tween-20.
Subsequently, wells are incubated for one hour at room temperature
with 50 .mu.l 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.
[0150] Alternatively, wells coated with rabies virus G protein (ERA
strain) were incubated with 50 .mu.g/ml of unlabeled anti-rabies
virus G protein IgG for one hour at room temperature. Then, 50
.mu.l of biotinylated CR57 (0.5 to 5 .mu.g/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, FIG. 6; no competitor).
From FIG. 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
FIG. 6 can further be deduced that none of the anti-rabies virus G
protein IgGs competed significantly with CR-57, which is in
agreement with the scFv competition data as described in Example
7.
[0151] 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
.mu.l of unlabeled anti-rabies virus G protein IgG (50 .mu.g/ml)
for 20 minutes at 4.degree. C. After washing of the cells with PBS
containing 1% BSA, 20 .mu.l of biotinylated CR57 (0.5 to 5
.mu.g/ml; at subsaturated levels) were added to each well,
incubated for five minutes at 4.degree. C., and immediately washed
twice with 100 .mu.l of PBS containing 1% BSA. Subsequently, wells
were incubated for 15 minutes at 4.degree. C. with 20 .mu.l 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, FIG. 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, FIG. 7). The latter
did not compete in ELISA (see, FIG. 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, FIG. 7) in the
FACS-based competition assay indicating that they might compete
with 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.
Example 10
Additive/Synergistic Effects of Anti-Rabies IgGs in In Vitro
Neutralization of Rabies Virus (Modified RFFIT)
[0152] In order to determine whether the anti-rabies virus G
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 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 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.
[0153] Alternatively, additive or synergistic effects can be
determined by the following experiment. First, the potency of 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 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% neutralizing endpoint. Subsequently, the
combination index (CI) is determined for the antibody mixture using
the formula CI=(C1/Cx1)+(C2/Cx2)+(C1C2/Cx1Cx2) as described by Chou
et al. (1984). C1 and C2 are the amount (in .mu.g) of monoclonal
antibody 1 and monoclonal antibody 2 that lead to 50%
neutralization when used in combination and Cx1 and Cx2 are the
amount (in .mu.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
[0154] 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 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 to 60 minutes,
while completely covered in the solution. Finally, the cards were
washed extensively with excess of H.sub.2O and sonicated in disrupt
buffer containing 1% SDS/0.1% beta-mercaptoethanol in PBS (pH 7.2)
at 70.degree. C. for 30 minutes, followed by sonication in H.sub.2O
for another 45 minutes.
[0155] 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 credit card format polypropylene cards,
containing the covalently linked peptides, were incubated with the
antibodies (10 .mu.g/ml; diluted in blocking solution, which
contained 5% horse-serum (v/v) and 5% ovalbumin (w/v)) (4.degree.
C., overnight). After washing, the peptides were incubated with
anti-human antibody peroxidase (dilution 1/1000) (one hour,
25.degree. C.), and subsequently, after washing, the peroxidase
substrate 2,2'-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and
2 .mu.l/ml 3% H.sub.2O.sub.2 were added. Controls (for linear and
looped) were incubated with anti-human antibody peroxidase only.
After one 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.
[0156] The human anti-rabies virus G protein monoclonal antibodies
were tested for binding to the 15-mer linear and 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.
[0157] 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) and
ACKLKLCGVLGLRLM (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 recognized by antibody CR57. Binding was most prominent to
the peptide with the amino acid sequence KLCGVLGLRLMDGTW (SEQ ID
NO:322).
[0158] 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 AYTIFNKTLMEADAH (SEQ ID NO:327) and 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 sequences TIFNKTLMEADAHYK (SEQ ID
NO:329), IFNKTLMEADAHYKS (SEQ ID NO:330) and FNKTLMEADAHYKSV (SEQ
ID NO:331).
[0159] CRJB and the antibodies called CR04-040, CR04-098 and
CR04-103 (data not shown) did not recognize a region of linear
antigenic peptides.
[0160] 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.
[0161] 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.
[0162] 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 noncompeting epitopes.
Example 12
Determination of Neutralizing Potency of Anti-Rabies G Protein IgGs
Using an In Vitro Neutralization Assay (Modified RFFIT)
[0163] 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 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 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).
[0164] 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. 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
[0165] 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. CR04-021, 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
[0166] 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 one single focus with the exception of E98-2
(two foci) and E98-4 (four 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 plus monoclonal antibody (.about.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.
[0167] 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 CR04-126 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).
[0168] 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 position 336 of the rabies
glycoprotein (see, FIG. 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).
[0169] Moreover, Pepscan analysis of binding of CR57 to peptides
harboring 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.RTM. cells, as measured by flow cytometry (data not shown),
even though viruses containing this mutation were no longer
neutralized.
[0170] Furthermore, epitope mapping studies and affinity ranking
studies were performed using surface plasmon resonance analysis
using a BIAcore3000.TM. analytical system. Purified rabies
glycoprotein (ERA strain) was immobilized as a ligand on a research
grade CM5 four-flow channel (Fc) sensor chip (Biacore AB, Sweden)
using amine coupling. Ranking was performed at 25.degree. C. with
HBS-EP (Biacore AB, Sweden) as running buffer. 50 .mu.l of each
antibody was injected at a constant flow rate of 20 .mu.l/minute.
Then, running buffer was applied for 750 seconds followed by
regeneration of the CM5 chip with 5 .mu.l 2M NaOH, 5 .mu.l 45 mM
HCl and 5 .mu.l 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 for CR57 and CR04-098 as
determined by surface plasmon resonance analysis were 2.4 nM and
4.5 nM, 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.
[0171] 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
Assessment of Conservation of the Epitope Recognized by CR57 and
CR04-098
[0172] 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.
[0173] One hundred twenty-three 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, FIG. 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. TABLE-US-00001 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) (IU/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
[0174] TABLE-US-00002 TABLE 2 Human lambda chain variable region
primers (sense). Primer name Primer nucleotide sequence SEQ ID NO
HuV.lamda.1A 5'-CAGTCTGTGCTGACTCAGCCACC-3' SEQ ID NO:208
HuV.lamda.1B 5'-CAGTCTGTGYTGACGCAGCCGCC-3' SEQ ID NO:209
HuV.lamda.1C 5'-CAGTCTGTCGTGACGCAGCCGCC-3' SEQ ID NO:210
HuV.lamda.2 5'-CARTCTGCCCTGACTCAGCCT-3' SEQ ID NO:211 HuV.lamda.3A
5'-TCCTATGWGCTGACTCAGCCACC-3' SEQ ID NO:212 HuV.lamda.3B
5'-TCTTCTGAGCTGACTCAGGACCC-3' SEQ ID NO:213 HuV.lamda.4
5'-CACGTTATACTGACTCAACCGCC-3' SEQ ID NO:214 HuV.lamda.5
5'-CAGGCTGTGCTGACTCAGCCGTC-3' SEQ ID NO:215 HuV.lamda.6
5'-AATTTTATGCTGACTCAGCCCCA-3' SEQ ID NO:216 HuV.lamda.7/8
5'-CAGRCTGTGGTGACYCAGGAGCC-3' SEQ ID NO:217 HuV.lamda.9
5'-CWGCCTGTGCTGACTCAGCCMCC-3' SEQ ID NO:218
[0175] TABLE-US-00003 TABLE 3 Human kappa chain variable region
primers (sense). Primer name Primer nucleotide sequence SEQ ID NO
HuV.kappa.1B 5'-GACATCCAGWTGACCCAGTCTCC-3' SEQ ID NO:219
HuV.kappa.2 5'-GATGTTGTGATGACTCAGTCTCC-3' SEQ ID NO:220 HuV.kappa.3
5'-GAAATTGTGWTGACRCAGTCTCC-3' SEQ ID NO:221 HuV.kappa.4
5'-GATATTGTGATGACCCACACTCC-3' SEQ ID NO:222 HuV.kappa.5
5'-GAAACGACACTCACGCAGTCTCC-3' SEQ ID NO:223 HuV.kappa.6
5'-GAAATTGTGCTGACTCAGTCTCC-3' SEQ ID NO:224
[0176] TABLE-US-00004 TABLE 4 Human kappa chain variable region
primers extended with SalI restriction sites (sense), human kappa
chain J-region primers extended with NotI restriction sites
(anti-sense), human lambda chain variable region primers extended
with SalI restriction sites (sense) and human lambda chain J-region
primers extended with NotI restriction sites (anti-sense). Primer
name Primer nucleotide sequence SEQ ID NO HuV.kappa.1B-SalI
5'-TGAGCACACAGGTCGACGGACATCCAGWTGACCCAGTCTCC-3' SEQ ID NO:225
HuV.kappa.2-SalI 5'-TGAGCACACAGGTCGACGGATGTTGTGATGACTCAGTCTCC-3'
SEQ ID NO:226 HuV.kappa.3B-SalI
5'-TGAGCACACAGGTCGACGGAAATTGTGWTGACRCAGTCTCC-3' SEQ ID NO:227
HuV.kappa.4B-SalI 5'-TGAGCACACAGGTCGACGGATATTGTGATGACCCACACTCC-3'
SEQ ID NO:228 HuV.kappa.5-SalI
5'-TGAGCACACAGGTCGACGGAAACGACACTCACGCAGTCTCC-3' SEQ ID NO:229
HuV.kappa.6-SalI 5'-TGAGCACACAGGTCGACGGAAATTGTGCTGACTCAGTCTCC-3'
SEQ ID NO:230 HuJ.kappa.1-NotI
5'-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATTTCCACCTTGGTCCC-3' SEQ ID
NO:231 HuJ.kappa.2-NotI
5'-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATCTCCAGCTTGGTCCC-3' SEQ ID
NO:232 HuJ.kappa.3-NotI
5'-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATATCCACTTTGGTCCC-3' SEQ ID
NO:233 HuJ.kappa.4-NotI
5'-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATCTCCACCTTGGTCCC-3' SEQ ID
NO:234 HuJ.kappa.5-NotI
5'-GAGTCATTCTCGACTTGCGGCCGCACGTTTAATCTCCAGTCGTGTCCC-3' SEQ ID
NO:235 HuV.lamda.1A-SalI
5'-TGAGCACACAGGTCGACGCAGTCTGTGCTGACTCAGCCACC-3' SEQ ID NO:236
HuV.lamda.1B-SalI 5'-TGAGCACACAGGTCGACGCAGTCTGTGYTGACGCAGCCGCC-3'
SEQ ID NO:237 HuV.lamda.1C-SalI
5'-TGAGCACACAGGTCGACGCAGTCTGTCGTGACGCAGCCGCC-3' SEQ ID NO:238
HuV.lamda.2-SalI 5'-TGAGCACACAGGTCGACGCARTCTGCCCTGACTCAGCCT-3' SEQ
ID NO:239 HuV.lamda.3A-SalI
5'-TGAGCACACAGGTCGACGTCCTATGWGCTGACTCAGCCACC-3' SEQ ID NO:240
HuV.lamda.3B-SalI 5'-TGAGCACACAGGTCGACGTCTTCTGAGCTGACTCAGGACCC-3'
SEQ ID NO:241 HuV.lamda.4-SalI
5'-TGAGCACACAGGTCGACGCACGTTATACTGACTCAACCGCC-3' SEQ ID NO:242
HuV.lamda.5-SalI 5'-TGAGCACACAGGTCGACGCAGGCTGTGCTGACTCAGCCGTC-3'
SEQ ID NO:243 HuV.lamda.6-SalI
5'-TGAGCACACAGGTCGACGAATTTTATGCTGACTCAGCCCCA-3' SEQ ID NO:244
HuV.lamda.7/8-SalI 5'-TGAGCACACAGGTCGACGCAGRCTGTGGTGACYCAGGAGCC-3'
SEQ ID NO:245 HuV.lamda.9-SalI
5'-TGAGCACACAGGTCGACGCWGCCTGTGCTGACTCAGCCMCC-3' SEQ ID NO:246
HuJ.lamda.1-NotI
5'-GAGTCATTCTCGACTTGCGGCCGCACCTAGGACGGTGACCTTGGTCCC-3' SEQ ID
NO:247 HuJ.lamda.2/3-NotI
5'-GAGTCATTCTCGACTTGCGGCCGCACCTAGGACGGTCAGCTTGGTCCC-3' SEQ ID
NO:248 HuJ.lamda.4/5-NotI
5'-GAGTCATTCTCGACTTGCGGCCGCACYTAAAACGGTGAGCTGGGTCCC-3' SEQ ID
NO:249
[0177] TABLE-US-00005 TABLE 5 Distribution of the different light
chain products over the ten fractions. Light chain products Number
of alleles Fraction number alleles/fraction Vk1B/Jk1-5 19 1 and 2
9.5 Vk2/Jk1-5 9 3 9 Vk3B/Jk1-5 7 4 7 Vk4B/Jk1-5 1 5 5 Vk5/Jk1-5 1
Vk6/Jk1-5 3 V.lamda.1A/Jl1-3 5 6 5 V.lamda.1B/Jl1-3
V.lamda.1C/Jl1-3 V.lamda.2/Jl1-3 5 7 5 V.lamda.3A/Jl1-3 9 8 9
V.lamda.3B/Jl1-3 V.lamda.4/Jl1-3 3 9 5 V.lamda.5/Jl1-3 1
V.lamda.6/Jl1-3 1 V.lamda.7/8/Jl1-3 3 10 6 V.lamda.9/Jl1-3 3
[0178] TABLE-US-00006 TABLE 6 Human IgG heavy chain variable region
primers (sense). Primer name Primer nucleotide sequence SEQ ID NO
HuVH1B/ 5'-CAGRTGCAGCTGGTGCARTCTGG-3' SEQ ID NO:250 7A HuVH1C
5'-SAGGTCCAGCTGGTRCAGTCTGG-3' SEQ ID NO:251 HuVH2B
5'-SAGGTGCAGCTGGTGGAGTCTGG-3' SEQ ID NO:252 HuVH3B
5'-SAGGTGCAGCTGGTGGAGTCTGG-3' SEQ ID NO:253 HuVH3C
5'-GAGGTGCAGCTGGTGGAGWCYGG-3' SEQ ID NO:254 HuVH4B
5'-CAGGTGCAGCTACAGCAGTGGGG-3' SEQ ID NO:255 HuVH4C
5'-CAGSTGCAGCTGCAGGAGTCSGG-3' SEQ ID NO:256 HuVH5B
5'-GARGTGCAGCTGGTGCAGTCTGG-3' SEQ ID NO:257 HuVH6A
5'-CAGGTACAGCTGCAGCAGTCAGG-3' SEQ ID NO:258
[0179] TABLE-US-00007 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
sequence SEQ ID NO HuVH1B/7A-SfiI
5'-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGRTGCAGCTGGTGCARTCTGG-3' SEQ
ID NO:259 HuVH1C-SfiI
5'-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCSAGGTCCAGCTGGTRCAGTCTGG-3' SEQ
ID NO:260 HuVH2B-SfiI
5'-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGRTCACCTTGAAGGAGTCTGG-3' SEQ
ID NO:261 HuVH3B-SfiI
5'-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCSAGGTGCAGCTGGTGGAGTCTGG-3' SEQ
ID NO:262 HuVH3C-SfiI
5'-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGAGGTGCAGCTGGTGGAGWCYGG-3' SEQ
ID NO:263 HuVH4B-SfiI
5'-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTACAGCAG TGGGG-3' SEQ
ID NO:264 HuVH4C-SfiI
5'-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGSTGCAGCTGCAGGAGTCSGG-3' SEQ
ID NO:265 HuVH5B-SfiI
5'-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGARGTGCAGCTGGTGCAGTCTGG-3' SEQ
ID NO:266 HuVH6A-SfiI
5'-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGTACAGCTGCAGCAGTCAGG-3' SEQ
ID NO:267 HuJH1/2-XhoI 5'-GAGTCATTCTCGACTCGAGACGGTGACCAGGGTGCC-3'
SEQ ID NO:268 HuJH3-XhoI 5'-GAGTCATTCTCGACTCGAGACGGTGACCATTGTCCC-3'
SEQ ID NO:269 HuJH4/5-XhoI
5'-GAGTCATTCTCGACTCGAGACGGTGACCAGGGTTCC-3' SEQ ID NO:270 HuJH6-XhoI
5'-GAGTCATTCTCGACTCGAGACGGTGACCGTGGTCCC-3' SEQ ID NO:271
[0180] TABLE-US-00008 TABLE 8 Binding of single-chain (scFv) phage
antibodies to rabies virus G protein (ERA strain) and to FBS as
measured by ELISA. Rabies virus G protein FBS Name phage antibody
(OD 492 nm) (OD 492 nm) 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 SC04-097 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 (SO57) 0.650 0.055 control (02-007)
0.063 0.052
[0181] TABLE-US-00009 TABLE 9 Data of the single-chain Fvs capable
of binding rabies virus G protein. SEQ ID NO SEQ ID NO Name scFv of
nucl. of amino acid (libr.) sequence sequence HCDR3 (SEQ ID NO:)
V.sub.H-locus V.sub.L-locus sc04-001 157 158 GLYGELFDY 3-20 (DP32)
V13 (31-V2-13) (JK1994) (SEQ ID NO:1) sc04-004 159 160 DYLYPTTDFDY
3-23 (DP47) VkI (O12/O2-DPK9) (WT2000) (SEQ ID NO:2) sc04-008 161
162 MGFTGTYFDY 2-70 (DP28) V13 (3h-V2-14) (RAB-03-G01) (SEQ ID
NO:3) sc04-010 163 164 DGLDLTGTIQPFGY 3-30 (DP49) VkI (L11-DPK3)
(RAB-03-G01) (SEQ ID NO:4) sc04-018 165 166 VSVTTGAFNI 4-04 (DP70)
V11 (1c-V1-16) (RAB-03-G01) (SEQ ID NO:5) sc04-021 167 168
GSVLGDAFDI 3-30 (DP49) VkI (L8) (RAB-03-G01) (SEQ ID NO:6) sc04-026
169 170 TSNWNYLDRFDP 5-51 (DP73) VkII (A19/03-DPK15) (RAB-03-G01)
(SEQ ID NO:7) sc04-031 171 172 GSVLGDAFDI 3-30 (DP49) VkI (L5-DPK5)
(RAB-03-G01) (SEQ ID NO:8) sc04-038 173 174 GSVLGDAFDI 3-30 (DP49)
VkI (L5-DPK5) (RAB-03-G01) (SEQ ID NO:9) sc04-040 175 176 GSKVGDFDY
3-30 (DP49) V13 (3h-V2-14) (RAB-03-G01) (SEQ ID NO:10) sc04-060 177
178 EKEKYSDRSGYSYYYYYMDV 4-59 (DP71) VkI (O12/O2-DPK9) (RAB-04-G01)
(SEQ ID NO:11) sc04-073 179 180 DGLDLTGTIQPFGY 3-30 (DP49) VkI
(L12) (RAB-04-G01) (SEQ ID NO:12) sc04-097 181 182 TASNLGRGGMDV
3-23 (DP47) VkI (L8) (RAB-04-G01) (SEQ ID NO:13) sc04-098 183 184
VAVAGTHFDY 3-30 (DP49) VkI (A30) (RAB-04-G01) (SEQ ID NO:14)
sc04-103 185 186 VAVAGESFDS 3-30 (DP49) VkI (L5-DPK5) (RAB-04-G01)
(SEQ ID NO:15) sc04-104 187 188 IVVVTALDAFDI 3-30 (DP49) VkI (L12)
(RAB-04-G01) (SEQ ID NO:16) sc04-108 189 190 FMIVADDAFDI 3-30
(DP49) VkI (L1) (RAB-04-G01) (SEQ ID NO:17) sc04-120 191 192
GGKTGEFDY 3-30 (DP49) VkI (L8) (RAB-04-G01) (SEQ ID NO:18) sc04-125
193 194 IATAGTGFDY 3-30 (DP49) VkI (L8) (RAB-04-G01) (SEQ ID NO:19)
sc04-126 195 196 MGFTGTYFDY 2-70 (DP28) V13 (3h-V2-14) (RAB-04-G01)
(SEQ ID NO:20) sc04-140 197 198 VTNPGDAFDI 3-30 (DP49) VkI (L4/18a)
(RAB-04-G01) (SEQ ID NO:21) sc04-144 199 200 GGKTGEFDY 3-30 (DP49)
VkI (L8) (RAB-04-G01) (SEQ ID NO:22) sc04-146 201 202 GGKTGEFDY
3-30 (DP49) VkIII (L2-DPK21) (RAB-04-G01) (SEQ ID NO:23) sc04-164
203 204 GSVLGDAFDI 3-30 (DP49) VkI (L19-DPK6) (RAB-04-G01) (SEQ ID
NO:24) SO57 205 206 ENLDNSGTYYYFSGWFDP 1-69 (DP10) V12 (2e-V1-3)
(SEQ ID NO:25) SOJB 312 313 RQHISSFPWFDS 2-05 V13 (3h-V2-14) (SEQ
ID NO:276)
[0182] TABLE-US-00010 TABLE 10 Data of assay for rabies
virus-neutralizing activity of scFvs. 50% endpoint 50% endpoint
dilution Potency Name scFv dilution WHO standard (2 IU/ml) (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
10 270 0.1 SC04-140 135 270 1 SC04-144 810 270 6 SC04-146 405 270 3
SC04-164 45 270 0.3
[0183] TABLE-US-00011 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 (IU/ml)
[0184] TABLE-US-00012 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 (IU/ml)
[0185] TABLE-US-00013 TABLE 12 Oligonucleotides used for PCR
amplification of V.sub.H genes. Name and nucleotide sequence
V.sub.H gene SEQ ID NO: 5H-B: SC04-001 280
acctgtcttgaattctccatggccgaggtgcagctggtggagtctg 5H-C: SC04-021 281
acctgtcttgaattctccatggcccaggtgcagctggtggagtctgg SC04-031 SC04-125
SC04-164 5H-C-long: SC04-010 282
acctgtcttgaattctccatggcccaggtgcagctggtggagtctgggg SC04-038 SC04-040
SGO4-073 SC04-098 SC04-103 SC04-104 SC04-108 SC04-120 SC04-140
SC04-144 SC04-146 5H-F: SC04-018 283
acctgtcttgaattctccatggcccaggtgcagctgcaggagtccggccc SC04-060 5H-H:
SC04-026 284 acctgtcttgaattctccatggccgaggtgcagctggtgcagtctgg 5H-I:
SC04-004 285 acctgtcttgaattctccatggccgaggtgcagctgctggagtctgg
SC04-097 5H-M: SC04-008 286
acctgtcttgaattctccatggcccaggtgaccttgaaggagtctgg SC04-126 sy3H-A:
SC04-001 287 gcccttggtgctagcgctggagacggtcaccagggtgccctggcccc
SC04-004 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
gcccttggtgctagcgctggagacggtcacggtggtgccctggcccc sy3H-C-long:
SC04-060 289 gcccttggtgctagcgctggagacggtcacggtggtgcccttgccccagacgtc
sy3H-D: SC04-018 290
gcccttggtgctagcgctggacacggtcaccatggtgccctggcccc SC04-021 SC04-031
SC04-038 SC04-104 SC04-108 SC04-140 SC04-164 sy3H-E: SC04-103 291
gcccttggtgctagcgctggacacggtcaccagggtgccccggcccc
[0186] TABLE-US-00014 TABLE 13 Oligonucleotides used for PCR
amplification of V.sub.L genes. Name and nucleotide sequence
V.sub.L gene SEQ ID NO: 3L-B: SC04-001 292
ttttccttagcggccgcgactcacctaggacggtcagcttggtc 5K-B: SC04-031 293
acctgtctcgagttttccatggctgacatccagatgacccagtc SC04-060 SC04-073
SC04-098 SC04-103 SC04-104 SC04-108 SC04-164 5K-C: SC04-004 294
acctgtctcgagttttccatggctgacatccagatgacccagtctccatcctccc 5K-G:
SC04-026 295 acctgtctcgagttttccatggctgacatcgtgatgacccagtctcc 5K-K:
SC04-010 296 acctgtctcgagttttccatggctgccatccagatgacccagtctcc 5K-M:
SC04-021 297 acctgtctcgagttttccatggctgacatccagctgacccagtc SC04-097
SC04-120 SC04-125 SC04-144 5K-N: SC04-038 298
acctgtctcgagttttccatggctgacatccagatgactcagtc 5K-O: SC04-140 299
acctgtctcgagttttccatggctgccatccagctgacccagtc 5K-Q: SC04-146 300
acctgtctcgagttttccatggctgagatcgtgatgactcagtc 5L-E: SC04-008 301
acctgtctcgagttttccatggcttcctacgtgctgactcagccg 5L-F: SC04-018 302
acctgtctcgagttttccatggctcagtccgtgctgactcagcc 5L-G: SC04-040 303
acctgtctcgagttttccatggcttcctacgtgctgactcagcc SC04-126 sy3K-F:
SC04-004 304 gctgggggcggccacggtccgcttgatctccaccttggtccc SC04-010
SC04-021 SC04-031 SC04-098 SC04-104 SC04-125 SC04-140 SC04-144
SC04-164 sy3K-I: SC04-038 305
gctgggggcggccacggtccgcttgatctccagccgtgtccc SC04-097 SC04-103
SC04-108 SC04-146 sy3K-J: SC04-026 306
gctgggggcggccacggtccgcttgatctccagcttggtccc SC04-060 SC04-073
sy3K-K: SC04-120 307 gctgggggcggccacggtccgcttgatgtccaccttggtccc
sy3L-A: SC04-018 308 ccagcacggtaagcttcagcacggtcaccttggtgccagttcc
SC04-126 sy3L-C: SC04-040 309
ccagcacggtaagcttcagcacggtcagcttggtgcctccgcc sy3L-D: SC04-008 310
ccagcacggtaagcttcaacacggtcagctgggtccc sy5L-A: SC04-001 311
acctgtctcgagttttccatggcttcctccgagctgacccaggaccctgctg
[0187] TABLE-US-00015 TABLE 14 Binding of the human monoclonal
antibodies CR57, CRJB and CR04-010 (10 .mu.g/ml) to linear peptides
of the extracellular domain of glycoprotein G of rabies virus
strain ERA. Amino acid sequence of linear peptide CR57 CRJB
CR04-010 KFPIYTILDKLGPWS 71 97 1 FPIYTILDKLGPWSP 42 105 39
PIYTLLDKLGPWSPI 36 89 87 IYTILDKLGPWSPID 44 97 104 YTILDKLGPWSPIDI
48 114 91 TILDKLGPWSPIDIH 76 96 88 ILDKLGPWSPIDIHH 54 104 69
LDKLGPWSPIDIHHL 55 99 107 DKLGPWSPIDIHHLS 62 103 93 KLGPWSPIDIHHLSC
72 105 45 LGPWSPIDIHHLSCP 69 112 19 GPWSPIDIHHLSCPN 68 114 33
PWSPIDIHHLSCPNN 62 104 47 WSPIDIHHLSCPNNL 80 106 11 SPIDIHHLSCPNNLV
74 85 1 PIDIHHLSCPNNLVV 46 93 90 IDIHHLSCPNNLVVE 69 102 55
DIHHLSCPNNLVVED 38 96 78 IHHLSCPNNLVVEDE 37 85 113 HHLSCPNNLVVEDEG
56 76 117 HLSCPNNLVVEDEGC 65 119 111 LSCPNNLVVEDEGCT 69 117 127
SCPNNLVVEDEGCTN 83 114 91 CPNNLVVEDEGCTNL 77 97 49 PNNLVVEDEGCTNLS
78 107 97 NNLVVEDEGCTNLSG 72 99 97 NLVVEDEGCTNLSGF 75 119 55
LVVEDEGCTNLSGFS 76 103 52 VVEDEGCTNLSGFSY 73 107 91 VEDEGCTNLSGFSYM
74 103 31 EDEGCTNLSGFSYME 54 90 7 DEGCTNLSGFSYMEL 1 23 1
EGCTNLSGFSYMELK 51 114 129 GCTNLSGFSYMELKV 55 114 118
CTNLSGFSYMELKVG 47 110 137 TNLSGFSYMELKVGY 43 106 161
NLSGFSYMELKVGYI 61 115 170 LSGFSYMELKVGYIL 71 132 169
SGFSYMELKVGYILA 79 132 161 GFSYMELKVGYILAI 65 111 141
FSYMELKVGYILAIK 89 112 192 SYMELKVGYILAIKM 65 123 152
YMELKVGYILAIKMN 78 114 150 MELKVGYILAIKMNG 76 141 107
ELKVGYILAIKMNGF 87 132 76 LKVGYILAIKMNGFT 78 112 118
KVGYILAIKMNGFTC 78 118 68 VGYILAIKMNGFTCT 77 93 1 GYILAIKMNGFTCTG
75 90 1 YILAIKMNGFTCTGV 47 107 107 ILAIKMNGFTCTGVV 79 103 104
LAIKMNGFTCTGVVT 68 130 159 AIKMNGFTCTGVVTE 47 103 152
IKMNGFTCTGVVTEA 68 108 138 KMNGFTCTGVVTEAE 76 104 133
MNGFTCTGVVTEAEN 69 99 148 NGFTCTGVVTEAENY 69 101 138
GFTCTGVVTEAENYT 71 86 129 FTCTGVVTEAENYTN 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 TEAENYTNFVGYVTT
68 84 8 EAENYTNFVGYVTTT 69 117 142 AENYTNFVGYVTTTF 66 106 110
ENYTNFVGYVTTTFK 44 112 183 NYTNFVGYVTTTFKR 49 114 174
YTNFVGYVTTTFKRK 51 104 138 TNFVGYVTTTFKRKH 71 125 165
NFVGYVTTTFKRKHF 65 107 154 FVGYVTTTFKRKHFR 70 111 152
VGYVTTTFKRKHFRP 75 113 155 GYVTTTFKRKHFRPT 70 123 160
YVTTTFKRKHFRPTP 85 106 160 VTTTFKRKHFRPTPD 79 105 119
TTTFKRKHFRPTPDA 80 108 137 TTFKRKHFRPTPDAC 74 99 110
TFKRKHFRPTPDACR 96 111 108 FKRKHFRPTPDACRA 64 92 62 KRKHFRPTPDACRAA
65 93 1 RKHFRPTPDACRAAY 64 107 99 KHFRPTPDACRAAYN 73 112 124
HFRPTPDACRAAYNW 46 113 118 FRPTPDACRAAYNWK 43 112 148
RPTPDACRAAYNWKM 77 101 129 PTPDACRAAYNWKMA 99 125 143
TPDACRAAYNWKMAG 92 132 160 PDACRAAYNWKMAGD 61 124 147
DACRAAYNWKMAGDP 84 113 136 ACRAAYNWKMAGDPR 82 116 138
CRAAYNWKMAGDPRY 87 118 137 RAAYNWKMAGDPRYE 90 130 120
AAYNWKMAGDPRYEE 68 106 120 AYNWKMAGDPRYEES 96 94 77 YNWKMAGDPRYEESL
83 118 116 NWKMAGDPRYEESLH 58 101 69 WKMAGDPRYEESLHN 69 101 1
KMAGDPRYEESLHNP 62 102 84 MAGDPRYEESLHNPY 64 116 112
AGDPRYEESLHNPYP 40 101 125 GDPRYEESLHNPYPD 36 98 123
DPRYEESLHNPYPDY 57 110 118 PRYEESLHNPYPDYR 73 115 129
RYEESLHNPYPDYRW 69 112 125 YEESLHNPYPDYRWL 58 106 120
EESLHNPYPDYRWLR 76 123 141 ESLHNPYPDYRWLRT 92 132 125
SLHNPYPDYRWLRTV 78 111 137 LHNPYPDYRWLRTVK 79 106 142
HNPYPDYRWLRTVKT 86 108 146 NPYPDYRWLRTVKTT 85 102 151
PYPDYRWLRTVKTTK 65 93 103 YPDYRWLRTVKTTKE 72 97 97 PDYRWLRTVKTTKES
76 85 27 DYRWLRTVKTTKESL 54 111 105 YRWLRTVKTTKESLV 46 117 125
RWLRTVKTTKESLVI 40 110 120
WLRTVKTTKESLVII 41 104 125 LRTVKTTKESLVIIS 65 104 161
RTVKTTKESLVIISP 82 120 150 TVKTTKESLVIISPS 76 116 150
VKTTKESLVIISPSV 71 120 154 KTTKESLVIISPSVA 101 112 147
TTKESLVIISPSVAD 78 121 141 TKESLVIISPSVADL 86 112 132
KESLVIISPSVADLD 86 117 111 ESLVIISPSVADLDP 88 125 143
SLVIISPSVADLDPY 68 105 125 LVIISPSVADLDPYD 85 107 93
VIISPSVADLDPYDR 59 98 50 IISPSVADLDPYDRS 83 125 14 ISPSVADLDPYDRSL
50 119 91 SPSVADLDPYDRSLH 59 114 118 PSVADLDPYDRSLHS 44 114 118
SVADLDPYDRSLHSR 49 106 129 VADLDPYDRSLHSRV 71 113 141
ADLDPYDRSLHSRVF 70 121 141 DLDPYDRSLHSRVFP 111 152 127
LDPYDRSLHSRVFPS 99 142 106 DPYDRSLHSRVFPSG 90 120 134
PYDRSLHSRVFPSGK 86 120 130 YDRSLHSRVFPSGKC 364 818 127
DRSLHSRVFPSGKCS 98 142 141 RSLHSRVFPSGKCSG 87 141 156
SLHSRVFPSGKCSGV 69 111 141 LHSRVFPSGKCSGVA 78 114 129
HSRVFPSGKCSGVAV 97 118 111 SRVFPSGKCSGVAVS 100 125 24
RVFPSGKCSGVAVSS 69 110 106 VFPSGKCSGVAVSST 74 114 142
FPSGKCSGVAVSSTY 64 134 146 PSGKCSGVAVSSTYC 56 112 132
SGKCSGVAVSSTYCS 64 121 120 GKCSGVAVSSTYCST 92 143 145
KCSGVAVSSTYCSTN 88 130 130 CSGVAVSSTYCSTNH 110 165 143
SGVAVSSTYCSTNHD 79 110 115 GVAVSSTYCSTNHDY 79 114 108
VAVSSTYCSTNHDYT 85 114 118 AVSSTYCSTNHDYTI 71 105 102
VSSTYCSTNHDYTIW 78 107 121 SSTYCSTNHDYTIWM 76 107 121
STYCSTNHDYTIWMP 86 99 119 TYCSTNHDYTIWMPE 96 107 74 YCSTNHDYTIWMPEN
47 92 29 CSTNHDYTIWMPENP 52 106 86 STNHDYTIWMPENPR 60 112 107
TNHDYTIWMPENPRL 69 129 119 NHDYTIWMPENPRLG 71 119 130
HDYTIWMPENPRLGM 82 125 123 DYTIWMPENPRLGMS 93 127 123
YTIWMPENPRLGMSC 97 132 143 TIWMPENPRLGMSCD 69 106 134
IWMPENPRLGMSCDI 98 110 101 WMPENPRLGMSCDIF 88 113 120
MPENPRLGMSCDIFT 105 121 143 PENPRLGMSCDIFTN 83 111 104
ENPRLGMSCDIFTNS 71 118 111 NPRLGMSCDIFTNSR 90 113 138
PRLGMSCDIFTNSRG 72 112 105 RLGMSCDIFTNSRGK 88 106 113
LGMSCDIFTNSRGKR 76 110 114 GMSCDIFTNSRGKRA 54 120 101
MSCDIFTNSRGKRAS 46 110 106 SCDIFTNSRGKRASK 44 111 98
CDIFTNSRGKRASKG 42 104 117 DIFTNSRGKRASKGS 70 107 111
IFTNSRGKRASKGSE 77 125 87 FTNSRGKRASKGSET 83 111 119
TNSRGKRASKGSETC 68 108 110 NSRGKRASKGSETCG 92 100 119
SRGKRASKGSETCGF 64 93 90 RGKRASKGSETCGFV 75 104 115 GKRASKGSETCGFVD
92 124 118 KRASKGSETCGFVDE 92 106 129 RASKGSETCGFVDER 86 110 134
ASKGSETCGFVDERG 97 108 103 SKGSETCGFVDERGL 92 102 76
KGSETCGFVDERGLY 90 97 44 GSETCGFVDERGLYK 57 115 92 SETCGFVDERGLYKS
33 116 86 ETCGFVDERGLYKSL 64 120 138 TCGFVDERGLYKSLK 47 120 125
CGFVDERGLYKSLKG 72 115 120 GFVDERGLYKSLKGA 84 120 129
FVDERGLYKSLKGAC 86 121 124 VDERGLYKSLKGACK 50 108 110
DERGLYKSLKGACKL 90 119 54 ERGLYKSLKGACKLK 90 118 106
RGLYKSLKGACKLKL 90 121 121 GLYKSLKGACKLKLC 94 129 92
LYKSLKGACKLKLCG 93 136 141 YKSLKGACKLKLCGV 80 112 110
KSLKGACKLKLCGVL 129 113 110 SLKGACKLKLCGVLG 200 111 124
LKGACKLKLCGVLGL 340 90 23 KGACKLKLCGVLGLR 181 111 100
GACKLKLCGVLGLRL 123 134 129 ACKLKLCGVLGLRLM 148 117 142
CKLKLCGVLGLRLMD 410 111 147 KLKLCGVLGLRLMDG 273 120 114
LKLCGVLGLRLMDGT 918 145 148 KLCGVLGLRLMDGTW 3152 132 86
LCGVLGLRLMDGTWV 83 138 129 CGVLGLRLMDGTWVA 99 117 104
GVLGLRLMDGTWVAM 89 148 117 VLGLRLMDGTWVAMQ 90 141 127
LGLRLMDGTWVAMQT 102 115 97 GLRLMDGTWVAMQTS 104 138 120
LRLMDGTWVAMQTSN 103 114 118 RLMDGTWVAMQTSNE 100 113 130
LMDGTWVAMQTSNET 96 106 106 MDGTWVAMQTSNETK 97 97 110
DGTWVAMQTSNETKW 69 114 92 GTWVAMQTSNETKWC 58 113 82 TWVAMQTSNETKWCP
78 118 107 WVAMQTSNETKWCPP 50 114 116 VAMQTSNETKWCPPD 86 104 151
AMQTSNETKWCPPDQ 104 114 128 MQTSNETKWCPPDQL 104 132 125
QTSNETKWCPPDQLV 92 120 155 TSNETKWCPPDQLVN 97 111 90
SNETKWCPPDQLVNL 99 129 110
NETKWCPPDQLVNLH 90 128 107 ETKWCPPDQLVNLHD 105 120 118
TKWCPPDQLVNLHDF 85 125 125 KWCPPDQLVNLHDFR 89 113 121
WCPPDQLVNLHDFRS 101 119 99 CPPDQLVNLHDFRSD 93 137 127
PPDQLVNLHDFRSDE 107 120 56 PDQLVNLHDFRSDEI 35 106 63
DQLVNLHDFRSDEIE 54 117 97 QLVNLHDFRSDEIEH 60 113 106
LVNLHDFRSDEIEHL 47 104 100 VNLHDFRSDEIEHLV 83 129 98
NLHDFRSDEIEHLVV 83 113 110 LHDFRSDEIEHLVVE 93 115 121
HDFRSDEIEHLVVEE 69 107 150 DFRSDEIEHLVVEEL 99 103 110
FRSDEIEHLVVEELV 86 114 116 RSDEIEHLVVEELVR 100 138 104
SDEIEHLVVEELVRK 101 117 118 DEIEHLVVEELVRKR 94 123 143
EIEHLVVEELVRKRE 82 113 121 IEHLVVEELVRKREE 90 129 118
EHLVVEELVRKREEC 82 114 106 HLVVEELVRKREECL 82 123 46
LVVEELVRKREECLD 64 100 79 VVEELVRKREECLDA 62 108 97 VEELVRKREECLDAL
58 111 101 EELVRKREECLDALE 69 112 123 ELVRKREECLDALES 82 113 117
LVRKREECLDALESI 86 130 124 VRKREECLDALESIM 58 181 151
RKREECLDALESIMT 73 110 137 KREECLDALESIMTT 102 113 97
REECLDALESIMTTK 94 110 106 EECLDALESIMTTKS 82 120 133
ECLDALESIMTTKSV 91 112 125 CLDALESIMTTKSVS 101 146 155
LDALESIMTTKSVSF 97 116 152 DALESIMTTKSVSFR 104 120 188
ALESIMTTKSVSFRR 97 132 137 LESIMTTKSVSFRRL 48 114 130
ESIMTTKSVSFRRLS 62 111 114 SIMTTKSVSFRRLSH 54 130 97
IMTTKSVSFRRLSHL 43 101 111 MTTKSVSFRRLSHLR 59 116 125
TTKSVSFRRLSHLRK 66 118 111 TKSVSFRRLSHLRKL 83 125 123
KSVSFRRLSHLRKLV 108 124 129 SVSFRRLSHLRKLVP 64 123 117
VSFRRLSHLRKLVPG 90 111 105 SFRRLSHLRKLVPGF 92 110 96
FRRLSHLRKLVPGFG 90 108 111 RRLSHLRKLVPGFGK 92 143 118
RLSHLRKLVPGFGKA 93 123 148 LSHLRKLVPGFGKAY 96 139 150
SHLRKLVPGFGKAYT 113 132 132 HLRKLVPGFGKAYTI 99 111 102
LRKLVPGFGKAYTIF 83 118 82 RKLVPGFGKAYTIFN 47 115 86 KLVPGFGKAYTIFNK
47 114 123 LVPGFGKAYTIFNKT 54 112 139 VPGFGKAYTIFNKTL 58 114 138
PGFGKAYTIFNKTLM 78 113 157 GFGKAYTIFNKTLME 78 123 320
FGKAYTIFNKTLMEA 90 151 356 GKAYTIFNKTLMEAD 76 127 418
KAYTIFNKTLMEADA 101 123 554 AYTIFNKTLMEADAH 86 121 197
YTIFNKTLMEADAHY 104 147 161 TIFNKTLMEADAHYK 107 123 1405
IFNKTLMEADAHYKS 100 118 1828 FNKTLMEADAHYKSV 111 141 2736
NKTLMEADAHYKSVR 104 116 141 KTLMEADAHYKSVRT 91 98 123
TLMEADAHYKSVRTW 100 114 90 LMEADAHYKSVRTWN 73 107 97
MEADAHYKSVRTWNE 62 129 83 EADAHYKSVRTWNEI 58 97 106 ADAHYKSVRTWNEIL
56 100 100 DAHYKSVRTWNEILP 59 121 112 AHYKSVRTWNEILPS 112 160 125
HYKSVRTWNEILPSK 80 130 123 YKSVRTWNEILPSKG 66 137 116
KSVRTWNEILPSKGC 115 125 114 SVRTWNEILPSKGCL 106 138 118
VRTWNEILPSKGCLR 90 124 133 RTWNEILPSKGCLRV 120 127 120
TWNEILPSKGCLRVG 97 146 127 WNEILPSKGCLRVGG 102 136 117
NEILPSKGCLRVGGR 104 130 163 EILPSKGCLRVGGRC 104 112 128
ILPSKGCLRVGGRCH 79 113 107 LPSKGCLRVGGRCHP 77 119 100
PSKGCLRVGGRCHPH 69 138 91 SKGCLRVGGRCHPHV 72 121 103
KGCLRVGGRCHPHVN 68 130 115 GCLRVGGRCHPHVNG 85 125 123
CLRVGGRCHPHVNGV 102 132 134 LRVGGRCHPHVNGVF 104 143 133
RVGGRCHPHVNGVFF 86 143 99 VGGRCHPHVNGVFFN 120 136 120
GGRCHPHVNGVFFNG 86 119 119 GRCHPHVNGVFFNGI 117 113 117
RCHPHVNGVFFNGII 98 141 143 CHPHVNGVFFNGIIL 97 150 151
HPHVNGVFFNGIILG 104 138 164 PHVNGVFFNGIILGP 93 173 146
HVNGVFFNGIILGPD 97 123 114 VNGVFFNGIILGPDG 68 116 85
NGVFFNGIILGPDGN 66 117 97 GVFFNGIILGPDGNV 58 116 100
VFFNGIILGPDGNVL 55 132 108 FFNGIILGPDGNVLI 92 143 105
FNGIILGPDGNVLIP 61 139 130 NGIILGPDGNVLIPE 102 146 141
GIILGPDGNVLIPEM 107 132 123 IILGPDGNVLIPEMQ 85 118 93
ILGPDGNVLIPEMQS 125 134 119 LGPDGNVLIPEMQSS 100 134 150
GPDGNVLIPEMQSSL 86 154 157 PDGNVLIPEMQSSLL 87 129 139
DGNVLIPEMQSSLLQ 123 134 169 GNVLIPEMQSSLLQQ 96 120 168
NVLIPEMQSSLLQQH 72 120 150 VLIPEMQSSLLQQHM 92 104 142
LIPEMQSSLLQQHME 89 111 85 IPEMQSSLLQQHMEL 89 128 129
PEMQSSLLQQHMELL 62 133 93 EMQSSLLQQHMELLE 58 129 142
MQSSLLQQHMELLES 65 113 117 QSSLLQQHMELLESS 82 114 132
SSLLQQHMELLESSV 90 128 132 SLLQQHMELLESSVI 124 163 133
LLQQHMELLESSVIP 78 111 121 LQQHMELLESSVIPL 106 134 128
QQHMELLESSVIPLV 103 134 133 QHMELLESSVIPLVH 98 146 139
HMELLESSVIPLVHP 110 129 134 MELLESSVIPLVHPL 90 125 152
ELLESSVIPLVHPLA 90 133 155 LLESSVIPLVHPLAD 72 117 118
LESSVIPLVHPLADP 90 128 128 ESSVIPLVHPLADPS 104 138 143
SSVIPLVHPLADPST 73 104 93 SVIPLVHPLADPSTV 72 137 107
VIPLVHPLADPSTVF 69 141 123 IPLVHPLADPSTVFK 96 156 188
PLVHPLADPSTVFKD 93 112 138 LVHPLADPSTVFKDG 164 174 188
VHPLADPSTVFKDGD 98 138 125 HPLADPSTVFKDGDE 74 141 117
PLADPSTVFKDGDEA 99 125 90 LADPSTVFKDGDEAE 68 116 113
ADPSTVFKDGDEAED 147 152 110 DPSTVFKDGDEAEDF 98 147 137
PSTVFKDGDEAEDFV 104 143 141 STVFKDGDEAEDFVE 104 120 125
TVFKDGDEAEDFVEV 107 124 96 VFKDGDEAEDFVEVH 100 106 93
FKDGDEAEDFVEVHL 65 76 119 KDGDEAEDFVEVHLP 72 93 76 DGDEAEDFVEVHLPD
85 123 91 GDEAEDFVEVHLPDV 46 124 113 DEAEDFVEVHLPDVH 68 136 123
EAEDFVEVHLPDVHN 76 117 114 AEDFVEVHLPDVHNQ 123 138 123
EDFVEVHLPDVHNQV 90 141 123 DFVEVHLPDVHNQVS 96 141 118
FVEVHLPDVHNQVSG 92 143 102 VEVHLPDVHNQVSGV 106 141 123
EVHLPDVHNQVSGVD 91 150 139 VHLPDVHNQVSGVDL 110 114 137
HLPDVHNQVSGVDLG 104 150 129 LPDVHNQVSGVDLGL 104 154 154
PDVHNQVSGVDLGLP 106 129 115 DVHNQVSGVDLGLPN 117 133 113
VHNQVSGVDLGLPNW 100 119 38 HNQVSGVDLGLPNWG 76 106 38
NQVSGVDLGLPNWGK 78 138 98 Average 91.9 119.5 130.9 StDV 157.9 37.6
169.8
[0188] TABLE-US-00016 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
[0189] TABLE-US-00017 TABLE 16 Neutralizing potencies of
anti-rabies virus G protein IgGs against E57 escape viruses. E57A2
E57A3 E57B1 E57B2 E57B3 E57C3 Name IgG (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 CR04-125 708
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
[0190] TABLE-US-00018 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.
[0191] TABLE-US-00019 TABLE 18 Occurrence of amino acid residues in
the minimal binding region of CR57 within genotype 1 rabies
viruses. Wild-type K L C G V L K (99.6%)* L (100%) C (100%) G
(98.7%) V (99.6%) L (70.7%) R (0.4%) E (0.9%) I (0.4%) P (26.7%) R
(0.4%) S (2.6%) *Percentage of occurrence of each amino acid is
shown within 229 rabies virus isolates.
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