HK1159651B - Binding molecules capable of neutralizing rabies virus and uses thereof - Google Patents

Description

binding molecules capable of neutralizing rabies virus and uses thereof

Divisional application of application No. 200580017233.1 entitled "binding molecule capable of neutralizing rabies virus and use thereof", filed on 27.11.2006

Technical Field

The present invention relates to the field of medicine. The invention particularly relates to binding molecules that neutralize rabies virus. The binding molecules are useful in the post-exposure prevention of rabies.

Background

Rabies is a viral infection distributed almost worldwide that affects mainly wild animals and livestock, but also humans, leading to devastating, hardly changeable fatal encephalitis. It is estimated that more than 70000 people are lost each year, and millions of others require post-exposure treatment.

Rabies virus is a bullet-shaped, enveloped, single-stranded RNA virus classified in the rhabdoviridae and lyssavirus genera. The genome of rabies virus encodes 5 viral proteins: RNA-dependent RNA polymerase (L), nucleoprotein (N), phosphorylated protein (P), matrix protein (M) located inside the viral protein envelope, and outer surface glycoprotein (G).

The G protein (62-67kDa) is a type I glycoprotein consisting of 505 amino acids, with 2-4 potential N glycosylation sites, only one or two of which are virus strain dependent glycosylated. The G protein forms a protuberance covering the outer surface of the virion envelope, which is known to induce virus-neutralizing antibodies.

Rabies can be treated or prevented by both passive and active immunization. Post-rabies-exposure prophylaxis involves the rapid treatment of local wounds and administration of passive (anti-rabies immunoglobulin) and active immunization (vaccine) measures.

Currently, anti-Rabies Immunoglobulin (RIG) is prepared from serum samples of human beings immunized with rabies virus (HRIG) or horses immunized with rabies virus (ERIG). The disadvantage of ERIG and HRIG is that a sufficient amount is not available, which is too expensive in the case of HRIG. In addition, use of ERIG can lead to adverse reactions such as anaphylactic shock. The possibility of contamination with known or unknown pathogens is an additional concern associated with HRIG. To overcome these disadvantages, it has been proposed to use monoclonal antibodies capable of neutralizing rabies virus in post-exposure prophylaxis. Murine monoclonal antibodies that neutralize rabies virus are known in the art (see Schumacher et al, 1989). However, the use of murine antibodies in vivo has been limited due to problems associated with administration of murine antibodies to humans, such as short serum half-life, inability to trigger certain human effector functions, and elicitation of an undesirable significant immune response to murine antibodies in humans (human anti-mouse antibody (HAMA) response).

Recently, neutralizing human rabies virus 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 that are 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 escape of virus resistant variants.

There is still a great need for new neutralizing monoclonal antibodies against human rabies virus with improved post-exposure prophylactic potential, in particular antibodies with different epitope recognition specificities. The present invention provides such human monoclonal antibodies which are useful in a mixture for post-exposure prophylaxis of a plurality of rabies viruses and neutralizing resistant variants thereof.

Description of the drawings

FIG. 1 shows an amino acid sequence alignment of the rabies virus strain CVS-11 with the E57 escape virus (escope virus). Virus-infected cells were harvested 2 days post infection and total RNA was isolated. cDNA was generated and used for DNA sequencing. Regions containing mutations are shown, with mutations indicated in bold. FIG. 1A shows a nucleotide sequence alignment. The numbers above the amino acids indicate the amino acid numbers from the rabies virus glycoprotein including the signal peptide. FIG. 1B shows an amino acid sequence alignment. A schematic representation of rabies virus glycoprotein is shown above. The black boxes indicate the signal peptide and the grey boxes the transmembrane domain. The sequence in FIG. 1 is also represented by SEQ ID No: 130-141.

FIG. 2 shows an amino acid sequence alignment of the rabies virus strain CVS-11 with the EJB escape virus. Virus-infected cells were harvested 2 days post infection and total RNA was isolated. cDNA was generated and used for DNA sequencing. Regions containing mutations are shown in bold type. FIG. 2A shows a nucleotide sequence alignment. The numbers above the amino acids indicate the amino acid numbers from the rabies virus glycoprotein including the signal peptide. Figure 2B shows an amino acid sequence alignment. A schematic representation of rabies virus glycoprotein is shown above. The black boxes indicate the signal peptide and the grey boxes the transmembrane domain. The sequence in FIG. 2 is also represented by SEQ ID No: 142 and 151.

FIG. 3 shows the vector PDV-C06.

FIG. 4 shows a competition ELISA of anti-rabies virus scFv with biotinylated anti-rabies virus antibody called CR-57. ELISA plates coated with purified rabies virus G protein were incubated with the respective scFv before addition of CR-57bio (0.5. mu.g/ml). Subsequently, CR-57bio binding was monitored with and without scFv.

FIG. 5 shows a competition ELISA for anti-rabies scFv with an anti-rabies 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 scFv. Subsequently, scFv binding was monitored with and without CR-57.

FIG. 6 shows a competition ELISA for anti-rabies G protein IgG and anti-rabies antibody called CR-57. The G protein (ERA strain) was incubated with unlabeled IgG (shown on the X-axis). Biotinylated CR57(CR57bio) was added, allowed to bind to the G protein, and developed using streptavidin-HRP. ELISA signals are shown as percent CR57bio binding alone.

FIG. 7 shows a competition FACS analysis of anti-rabies G protein IgG with an anti-rabies antibody called CR-57. C6 cells expressing G protein (ERA strain) were incubated with unlabeled IgG (shown on the X-axis). Biotinylated CR57(CR57bio) was added to bind to G protein expressing cells, followed by development using streptavidin-PE. FACS signals are shown as percent CR57bio binding alone.

FIG. 8 shows an amino acid sequence alignment of CVS-11 and E98 escape viruses. Virus-infected cells were harvested 2 days post infection and total RNA was isolated. cDNA was generated and used for DNA sequencing. The figure shows the region containing the point mutation, the mutation is in bold. FIG. 8A shows a nucleotide sequence alignment. The numbers above the nucleotides represent the mutated nucleotides of the rabies virus glycoprotein open reading frame without the signal peptide sequence (in bold). Figure 8B shows an amino acid sequence alignment. The numbers above the amino acids indicate the mutated amino acids of the rabies virus glycoprotein without the signal peptide sequence (in bold).

FIG. 9 shows a phylogenetic tree of 123 rabies street viruses (123 rabies virus G glycoprotein sequences, Neighbor joining, Kimura-2-parameter method, boot value (bootstrap) 500). Bold type indicates viruses with N > D mutations observed in E98 escape viruses.

Fig. 10 shows the neutralizing epitope on rabies glycoprotein. A schematic representation of the rabies virus glycoprotein is shown, depicting the antigenic site comprising the novel CR57 epitope. The signal peptide (19 amino acids) and transmembrane domain are indicated by black boxes. Disulfide bonds are shown. Amino acid numbering is obtained for the mature protein minus the signal peptide sequence.

Description of the invention

The following sets forth definitions of terms used in connection with the present invention.

Definition of

Binding molecules

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 antigen binding and/or variable domains of a fragment of an immunoglobulin comprising a binding partner that competes with the intact immunoglobulin for specific binding to the immunoglobulin (e.g., rabies virus or a fragment thereof, e.g., G protein). Regardless of structure, an antigen-binding fragment binds to the same antigen recognized by an intact immunoglobulin. An antigen-binding fragment may comprise a peptide or polypeptide comprising at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, or at least 250 consecutive amino acid residues of the amino acid sequence of the binding molecule.

The term "binding molecule" as used herein includes all immunoglobulin classes and subclasses known in the art. Binding molecules can be classified into 5 major classes of intact antibodies according to the amino acid sequence of their heavy chain constant domain: IgA, IgD, IgE, IgG and IgM, several of these classes can be further divided into subclasses (isotypes), e.g., IgA1, IgA2, IgG1, IgG2, IgG3 and IgG 4.

Antigen binding fragments include Fab, F (ab')₂Fv, dAb, Fd, Complementarity Determining Region (CDR) fragments, single chain antibodies (scFv), bivalent single chain antibodies, single chain phage antibodies, diabodies, triabodies, tetrabodies, (poly) peptides containing at least enough immunoglobulin fragments to confer specific antigen binding to the (poly) peptide, and the like. Such fragments may be produced synthetically or by enzymatic or chemical cleavage of intact immunoglobulins or genetically engineered by recombinant DNA techniques. Methods of production are well known in the art, for example Antibodies: a Laboratory Manual, Edited by: E.Harlow and D, Lane (1988), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, which are incorporated herein by reference. The 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 the same or different.

The binding molecule may be naked or unconjugated, but may also be part of an immunoconjugate. Naked or unconjugated binding molecules refer to binding molecules that are unconjugated, operably linked or otherwise physically or functionally associated with effector (effector) moieties or labels such as toxic substances, radioactive substances, liposomes, enzymes. It is understood that naked or unconjugated binding molecules do not exclude binding molecules that have been stabilized, multimerized, humanized, or manipulated in any other way, other than attachment to an effector moiety or label. Thus, all post-translationally modified naked and unconjugated binding molecules are included, including modifications made in the environment of the cell that produces the native binding molecule, produced by the cell that produces the recombinant binding molecule, and introduced artificially after the binding molecule was originally prepared. Of course, the term naked or unconjugated binding molecule does not exclude the ability of the binding molecule to form a functional binding with effector cells and/or molecules upon administration to the body, some of which interaction is necessary for the biological effect to be exerted. Thus, the lack of a relevant effector group or label serves to define a naked or unconjugated binding molecule in vitro rather than in vivo.

Complementarity Determining Region (CDR)

The term "complementarity determining region" as used herein refers to a sequence within the variable region of a binding molecule, such as an immunoglobulin, which typically provides, to a large extent, an antigen binding site that is complementary in morphology and charge distribution to an epitope recognized by the antigen. The CDR regions may be specific for linear epitopes, discontinuous epitopes, or conformational epitopes of the protein or protein fragment, presented on the protein in its native conformation or, in some cases, as a denatured protein (e.g., solubilized in SDS). Epitopes may also consist of post-translational modifications of proteins.

Functional variants

The term "functional variant" as used herein refers to a binding molecule comprising a nucleotide and/or amino acid sequence which is altered by one or more nucleotides and/or amino acids compared to the nucleotide and/or amino acid sequence of the parent binding molecule and which is still able to compete with the parent binding molecule for binding to a binding partner, e.g. rabies virus or a fragment thereof. In other words, modifications in the amino acid and/or nucleotide sequence of the parent binding molecule do not significantly affect or alter the binding properties of the binding molecule encoded by or comprising the nucleotide sequence, i.e. the binding molecule is still able to recognize and bind its target. The functional variants 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 can contain natural as well as non-natural nucleotides and amino acids.

Conservative amino acid substitutions include those in which an 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 person that other classes of amino acid residue families than the above-mentioned class of amino acids used can also be applied. Furthermore, variants may have non-conservative amino acid substitutions, such as the replacement of an amino acid with an amino acid residue having a different structure or chemical property. Similar minor changes may also include amino acid deletions or insertions or both. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing immunological activity can be found using computer programs well known in the art.

Mutations in a nucleotide sequence may be single changes (point mutations) at a locus, such as transition or transversion mutations, or insertion, deletion or alteration of multiple nucleotides at a locus. In addition, one or more changes may be made at a number of loci within the nucleotide sequence. The mutation may be performed by any suitable method known in the art.

Host computer

The term "host" as used herein refers to an organism or cell into which a vector, such as a cloning vector or an expression vector, has been introduced. The organism or cell may be a prokaryotic or eukaryotic organism or cell. It is understood that the term refers not only to the particular organism or cell, but also to the progeny of such an organism or cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such generations may in fact be different from the parent organism or cell, but are still included within the scope of the term "host" as used herein.

Human being

The term "human" when used in reference to a binding molecule described herein refers to a molecule that is directly derived from a human or a molecule that is based on a human sequence. Binding molecules are still considered human in this specification when they are derived from or based on human sequences and subsequently modified. In other words, the term human, when used in reference to binding molecules, is intended to encompass binding molecules having variable and constant regions derived from human germline immunoglobulin sequences based on either variable or constant regions that are present or absent or present in modified form in human or human lymphocytes. Thus, a human binding molecule may comprise amino acid residues not encoded by human germline immunoglobulin sequences, including substitutions and/or deletions (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). The term "based on" as used herein means that the nucleic acid sequence can be copied exactly from the template or be synthetically produced with fewer mutations, e.g., by error-prone PCR methods or with exact matches to the template or with fewer modifications. Semi-synthetic molecules based on human sequences are also considered herein as human.

Monoclonal antibodies

The term "monoclonal antibody" as used herein refers to a single molecular component, i.e., a preparation of antibody molecules having a single amino acid sequence, i.e., primary structure. Monoclonal antibodies exhibit a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody exhibiting a single binding specificity having variable and constant regions derived from either human germline immunoglobulin sequences or from fully synthetic sequences. The method of preparing monoclonal antibodies is not relevant to the present invention.

Nucleic acid molecules

The term "nucleic acid molecule" as used herein refers to multimeric forms of nucleotides, including RNA, cDNA, sense and antisense strands of genomic DNA, and synthetic forms and mixed polymers of the above molecules. A nucleotide refers to a ribonucleotide, a deoxynucleotide, or a modified form of either type of nucleotide. The term also includes single-stranded and double-stranded forms of DNA. In addition, a polynucleotide may include naturally occurring nucleotides or modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages, or both. The nucleic acid molecule may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled 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., methylphosphonates, 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 term also includes any topological conformation, including single-stranded, double-stranded, partially double-stranded, triple-stranded, hairpin, circular, and locked conformations. The term also includes synthetic molecules that mimic the ability of polynucleotides to bind to a given sequence through hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, molecules in which peptide bonds replace phosphate bonds in the backbone of the molecule. Unless otherwise indicated, reference to a nucleic acid sequence encompasses its complement. Thus, reference to a nucleic acid molecule having a particular sequence is understood to encompass its complementary strand and its complementary sequence. Complementary strands can also be used, for example, in antisense therapeutics, hybridization probes, and PCR primers.

Pharmaceutically acceptable excipients

By "pharmaceutically acceptable excipient" is meant any inert substance that is combined with an active molecule, such as a drug, formulation or binding molecule, to prepare a suitable or convenient dosage form. A "pharmaceutically acceptable excipient" is an excipient that is non-toxic to the recipient (recipient) at the dosage and concentration employed, or at least whose toxicity is acceptable for its intended use, and is compatible with the other ingredients of the formulation in which the drug, formulation or binding molecule is contained.

Post exposure prophylaxis

"post-exposure prophylaxis (PEP)" is for humans who may be exposed to rabies animals. Possible exposures include bite exposures (i.e., any penetration of the skin by the teeth), including animal bite and non-bite exposures. Non-bite exposures include exposure to large amounts of aerosolized rabies virus in a laboratory or cave and surgical recipients transplanted with rabies-killed patient corneas. Open wounds, abrasions, contamination of mucous membranes, or theoretically abrasions by rabies saliva or other potentially infectious materials (e.g., nerve tissue) also constitute non-bite exposures. Other self-exposure, such as touching and exposure to blood, urine or feces of rabies animals, does not constitute an exposure and is not an indication of prophylaxis. PEP should be performed immediately after exposure. Post exposure prophylaxis is not required if not exposed. In all post-exposure prophylaxis regimens, active and passive immunization should be performed simultaneously, except for those who have been previously immunized.

Specific binding

The term "specific binding" as used herein in describing the interaction of a binding molecule, e.g., an antibody, with its binding partner, e.g., an antigen, means that the interaction is dependent on the presence of a particular structure, e.g., an antigenic determinant or epitope, on the binding partner. In other words, an antibody preferentially binds or recognizes a binding partner even when the binding partner is present in a mixture of other molecules or organisms. This binding is mediated by covalent or non-covalent interactions or both. In still other words, the term "specifically binds" refers to immunospecifically binding to one antigen or fragment thereof and not immunospecifically binding to other antigens. Binding molecules that immunospecifically bind to an antigen can bind other peptides or polypeptides with lower affinity, as determined by Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), BIACORE, or other methods known in the art. A binding molecule or fragment thereof that immunospecifically binds an antigen can cross-react with the relevant antigen. Preferably, the binding molecule or fragment thereof that immunospecifically binds an antigen does not cross-react with other antigens.

A therapeutically effective amount

The term "therapeutically effective amount" refers to an amount of a binding molecule herein that is effective for prophylaxis following rabies exposure.

Carrier

The term "vector" refers to 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, the vector is capable of transporting the nucleic acid molecule to which it is linked. Cloning vectors as well as expression vectors are encompassed by the term "vector" as used herein. Vectors include, but are not limited to, plasmids, cosmids, Bacterial Artificial Chromosomes (BACs) and Yeast Artificial Chromosomes (YACs) and vectors derived from bacteriophages or plant or animal (including human) viruses. The vector contains an origin of replication recognized by the intended host, and in the case of an expression vector, a promoter and other regulatory regions recognized by the host. The vector containing the second nucleic acid molecule is introduced into the cell, for example, by transformation, transfection or by using bacterial or viral entry mechanisms. Other means of introducing nucleic acids into cells are known in the art, for example, electroporation or particle bombardment means are commonly used for plant cells, among others. The method of introducing the nucleic acid into the cell depends on factors such as the cell type. This is not critical to the present 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 may replicate in bacteria). Other vectors may integrate into the host genome upon introduction into the host and thereby replicate with the host genome.

Summary of The Invention

The present invention provides binding molecules that specifically bind to and neutralize rabies virus. Furthermore, the invention relates to nucleic acid molecules which encode at least the binding region of these binding molecules. The invention further provides the use of a binding molecule of the invention for the post-exposure prophylaxis of a subject at risk of developing a disease derived from rabies virus.

Detailed Description

The first aspect of the invention comprises a binding molecule capable of specifically binding to rabies virus. Preferably, the binding molecules of the invention also have rabies virus neutralizing activity. Preferably, the binding molecules of the invention are human binding molecules. Alternatively, they may be binding molecules of other animals. Rabies virus is part of the lyssavirus genus. The lyssavirus genus comprises 11 genotypes in total: rabies virus (genotype 1), Lagos yamavirus (genotype 2), Mokola virus (genotype 3), Duvenhage virus (genotype 4), european yamavirus 1 (genotype 5), european yamavirus 2 (genotype 6), australian yamavirus (genotype 7), Aravan virus (genotype 8), Khujand virus (genotype 9), Irkut virus (genotype 10) and West Caucasian virus (genotype 11). In addition to binding to rabies virus, the binding molecules of the invention can also bind to other genotypes of the lyssavirus genus. Preferably, the binding molecule is also capable of neutralizing other genotypes of the lyssavirus genus. Furthermore, the binding molecules of the invention are even capable of binding and/or neutralizing viruses of the Rhabdoviridae family other than the lyssavirus genus. This family includes the genera cytorhabdovirus (cytorhabdovirus), Ephemerovirus, lyssavirus, Nuclear Rhabdovirus (nucleorhabdovirus), Rhabdoviral (rhabdovirus) and Vesiculovirus.

The binding molecules are capable of specifically binding to the rabies virus in native form or in inactivated/attenuated form. Inactivation of rabies virus can be performed by treatment in the following manner: beta-propiolactone (BPL) treatment (White and Chappel, 1982), heating at 56 ℃ for more than 30 minutes, gamma irradiation, treatment with either ethylenimine or ethylenimine, or treatment with ascorbic acid and copper sulfate for 72 hours (Madhusudana et al, 2004). Common virus inactivation methods known in the art may also be used, such as pasteurization (moist heat), dry heat treatment, steam heat treatment, low pH treatment, organic solvent/detergent treatment, nanofiltration; UV light irradiation may also be used. Preferably, the inactivation is performed by treatment with beta-propiolactone (BPL). Methods for testing whether rabies virus is still infectious or partially or fully inactivated are well known to those skilled in the art and can be found in Laboratory technologies in rabes, Edited by: F. x Meslin, M.M.Kaplan and H.Koprowski (1996), 4th edition, Chapter 36, WorldHealth Organization, Geneva.

The binding molecule is also capable of specifically binding to one or more fragments of rabies virus, such as one or more protein and/or (poly) peptide preparations derived from rabies virus or cells transfected with rabies virus proteins and/or (poly) peptides. For therapeutic and/or prophylactic methods, such as post-exposure prophylaxis of rabies virus, the binding molecule preferably specifically binds a surface accessible protein of rabies virus, such as the M protein (see Ameyama et al 2003) or the G protein. For diagnostic purposes, the human binding molecule is also capable of specifically binding to proteins that are not presented on the surface of rabies virus. The amino acid sequences of surface accessible and internal proteins of various known rabies strains can be found in the EMBL database and/or other databases.

Preferably, the fragment comprises at least an antigenic determinant recognized by a human binding molecule of the invention. The term "antigenic determinant" as used herein is a moiety, such as a rabies virus (poly) peptide, (glyco) protein, or an analogue or fragment thereof, that is capable of binding the human binding molecule of the invention with sufficiently high affinity to form a detectable antigen-binding molecule complex.

The binding molecules of the present invention may be intact immunoglobulin molecules, such as polyclonal or monoclonal antibodies, particularly human monoclonal antibodies, or they may be antigen binding fragments, including but not limited to Fab, F (ab')₂Fv, dAb, Fd, Complementarity Determining Region (CDR) fragments, single chain antibodies (scFv), bivalent single chain antibodies, single chain phage antibodies, diabodies, triabodies, tetrabodies, and (poly) peptides comprising at least a fragment of an immunoglobulin sufficient to confer specific antigen binding to rabies virus or a fragment thereof. The binding molecules of the invention may be used in non-isolated form or in isolated form. Furthermore, the binding molecules of the invention may be used alone or in a mixture comprising at least one human binding molecule (or a variant or fragment thereof). In other words, the binding molecules may be used in combination, for example 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 treatment to achieve a desired prophylactic, therapeutic or diagnostic effect.

RNA viruses, such as rabies virus, utilize their own RNA polymerase during viral replication. These RNA polymerases tend to be error prone. This leads to the formation of so-called "quasispecies" during viral infection. Each quasispecies has a unique RNA genome, resulting in differences in the amino acid composition of viral proteins. If such mutations occur in the viral structural proteins, the virus escapes the host immune system due to changes in T or B cell epitopes. This is more likely to occur when an individual is treated with two binding molecules, such as human monoclonal antibodies, than when an individual is treated with a polyclonal antibody cocktail (HRIG). Thus, a prerequisite for the treatment of rabies with a mixture of two human monoclonal antibodies is that the two antibodies recognize non-overlapping, non-competing epitopes on their target antigens, i.e., the rabies virus glycoprotein. Thereby minimizing the chance of rabies escaping virus. As a result, the binding molecules of the invention preferably react with different, non-overlapping, non-competing epitopes of rabies virus, such as an epitope on the G protein of rabies virus. The mixture of binding molecules may further comprise at least one other therapeutic agent, such as a drug suitable for the prevention of rabies following exposure.

Typically, the binding molecules of the invention can bind to their binding partners, i.e. rabies virus or fragments thereof such as rabies virus protein, with an affinity constant (Kd value) of less than 0.2X 10^-4M、1.0×10^-5M、1.0×10^-6M、1.0×10^-7M, preferably less than 1.0X 10^-8M, more preferably less than 1.0X 10^-9M, more preferably less than 1.0X 10^-10M, more preferably less than 1.0X 10^-11M, particularly preferably less than 1.0X 10^-12And M. The affinity constant may vary depending on the antibody isotype. For example, affinity binding for an IgM isotype refers to at least about 1.0X 10^-7Binding affinity of M. The affinity constant can be determined, for example, using the surface plasmon resonance method, i.e.the optical phenomenon of real-time biospecific interaction is analyzed by detecting changes in the protein concentration within the biosensor matrix, for example using the BIACORE system (pharmacia Biosensor AB, Uppsala, Sweden).

The binding molecules of the invention may bind to rabies virus in purified/isolated form or in non-purified/non-isolated form. The binding molecule may bind to rabies virus in solubilized form, e.g. in a sample, or may bind to rabies virus bound to or attached to a carrier or substrate, e.g. microtiter plates, membranes, beads, etc. The carrier or substrate may be made of glass, plastic (e.g. polystyrene), polysaccharides, nylon, nitrocellulose or teflon or the like. The surface of such a support may be solid or porous and may be of any convenient shape for use. Alternatively, the binding molecule may also bind to a rabies virus fragment, such as a protein or (poly) peptide of rabies virus. In one embodiment, the binding molecule is capable of specifically binding to rabies virus G protein or a fragment thereof. The rabies virus protein or (poly) peptide may be in solubilized form or be rabies virus bound to or attached to the above-mentioned carrier or substrate. In another embodiment, cells transfected with G protein may be used as binding partners for the binding molecules.

In a preferred embodiment of the invention, the binding molecules of the invention neutralize the infectivity of rabies virus. This can be achieved by preventing the attachment of rabies virus to receptors on its host cell, such as the murine p75 neurotrophin receptor, the neuronal cell adhesion molecule (CD56) and the acetylcholine receptor, or by inhibiting the release of RNA into the cytoplasm or preventing RNA transcription or translation. In a particular embodiment, the binding molecule of the invention prevents the rabies virus from infecting the host cell by at least 99%, 95%, 90%, 85%, 80%, 75%, 70%, 60%, 50%, 45%, 40%, 45%, 35%, 30%, 25%, 20%, or at least 10% compared to the rabies virus infecting the host cell in the absence of the binding molecule. Neutralization can be carried out, for example, by Laboratory techniques in rabies, Edited by: F. x Meslin, M.M.Kaplan and H.Koproxski (1996), 4th edition, Chapters 15-17, World Health Organization, Geneva. Furthermore, the human binding molecule of the invention may be a complement fixation binding molecule (complement fixation molecule) that can contribute to the cleavage of the enveloped rabies virus. The human binding molecules of the invention may also act as opsonins and increase phagocytosis of rabies virus by facilitating its uptake by Fc or C3b receptors or by agglutinating rabies virus making it more susceptible to phagocytosis.

In a preferred embodiment, the binding molecule of the invention comprises at least one 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. in one embodiment, the CDR3 region is a heavy chain CDR3 region.

In another embodiment, the binding molecule of the invention comprises a variable heavy chain comprising essentially the 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 molecule of the invention comprises a variable heavy chain comprising essentially the amino acid sequence of SEQ ID NO: 335 amino acid sequence from 1 st to 119 th amino acid.

In another embodiment, the binding molecule of the invention comprises a polypeptide comprising SEQ id no: 26 and a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 50, a variable light chain comprising the amino acid sequence set forth in SEQ ID NO: 27 and a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 51, a variable light chain comprising the amino acid sequence set forth in SEQ ID NO: 28 and a variable heavy chain comprising the amino acid sequence set forth in SEQ id no: 52, a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 29 and a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 53, a variable light chain comprising the amino acid sequence set forth in SEQ ID NO: 30 and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 54, a variable light chain comprising the amino acid sequence set forth in SEQ id no: 31 and a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 55, a variable light chain comprising the amino acid sequence set forth in SEQ ID NO: 32 and a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 56, a variable light chain comprising the amino acid sequence set forth in SEQ ID NO: 33 and a variable heavy chain comprising the amino acid sequence of seq id NO: 57, a variable light chain comprising the amino acid sequence set forth in SEQ ID NO: 34 and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 58, a variable light chain comprising the amino acid sequence set forth in SEQ ID NO: 35 and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 59, a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 36 and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 60, a variable light chain comprising the amino acid sequence set forth in SEQ ID NO: 37 and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 61, a variable light chain comprising the amino acid sequence set forth in SEQ ID NO: 38 and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 62, a variable light chain comprising the amino acid sequence set forth in SEQ ID NO: 39 and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 63, a variable light chain comprising the amino acid sequence set forth in SEQ ID NO: 40 and a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 64, a variable light chain comprising the amino acid sequence set forth in SEQ ID NO: 41 and a variable heavy chain comprising the amino acid sequence shown in SEQ id no: 65, a variable light chain comprising the amino acid sequence set forth in SEQ ID NO: 42 and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 66, a variable light chain comprising the amino acid sequence set forth in SEQ ID NO: 43 and a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 67, a variable light chain comprising the amino acid sequence set forth in SEQ id no: 44 and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 68, a variable light chain comprising the amino acid sequence set forth in SEQ ID NO: 45 and a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 69, a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 46 and a variable heavy chain comprising the amino acid sequence shown in SEQ id no: 70, a variable light chain comprising the amino acid sequence set forth in SEQ ID NO:47 and a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO:71, a variable light chain comprising the amino acid sequence set forth in SEQ ID NO: 48 and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 72, a variable light chain comprising the amino acid sequence set forth in SEQ id no: 49 and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 73, or a variable light chain of the amino acid sequence shown in seq id no. In a preferred embodiment, the human binding molecule of the invention comprises a polypeptide comprising SEQ ID NO: 335 and a variable heavy chain comprising the amino acid sequence of amino acids 1-119 of SEQ ID NO: 337 at amino acids 1-107.

In a preferred embodiment, the binding molecule having rabies virus neutralizing activity of the invention is administered in the form of IgG, preferably in the form of IgG 1.

Another aspect of the invention includes functional variants of the binding molecules. A variant molecule is considered to be a functional variant of a binding molecule of the invention if it competes with the parent binding molecule for specific binding to rabies virus or a fragment thereof. In other words, the functional variant is still able to bind to rabies virus or a fragment thereof. The functional variant should also still have rabies virus neutralizing activity. Functional variants include, but are not limited to, derivatives that are substantially similar to the primary structural sequence, but contain, for example, in vitro or in vivo modifications that are chemical and/or biochemical modifications not found in the parent binding molecule. Such modifications include 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 phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, covalent cross-link formation, cystine formation, pyroglutamate formation, formylation, gamma carboxylation, glycosylation, GPI-anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, PEGylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation (selenoylation), sulfation, transfer RNA mediated addition of amino acids to proteins such as arginylation, ubiquitination, and the like.

Alternatively, a functional variant may be a binding molecule of the invention comprising an amino acid sequence comprising a substitution, insertion, deletion or combination thereof of one or more amino acids compared to the amino acid sequence of the parent binding molecule. Furthermore, functional variants may comprise truncations of the amino-or carboxy-terminal amino acid sequence or both. The functional variants of the invention may have the same or different (higher or lower) binding affinity as the parent binding molecule, but still bind to and still neutralize rabies virus or fragments thereof. For example, the functional variants of the invention may have increased or decreased binding affinity for rabies virus or fragments thereof, or have higher or lower rabies virus neutralizing activity compared to the parental binding molecule. Preferably, the amino acid sequence of the variable regions is modified, including but not limited to framework regions, hypervariable regions, in particular the CDR3 regions. Typically, the light and heavy chain variable regions comprise three hypervariable regions, including three CDRs, and more conserved regions called Framework Regions (FRs). The hypervariable region comprises the amino acid residues of the CDRs and the amino acid residues of the hypervariable loops. Functional variants within the scope of the present invention have at least about 50% -99%, preferably at least about 60% -99%, more preferably at least about 7% -99%, more preferably at least about 80% -99%, most preferably at least about 90% -99%, particularly at least about 95% -99%, and especially at least about 97% -99% amino acid sequence homology with the parent binding molecule described herein. The amino acid sequences can be optimally aligned and similar or identical amino acid residues determined using computer algorithms known to those skilled in the art, such as Gap or Bestfit.

In another embodiment, functional variants are produced when the parent binding molecule contains a glycosylation site in its sequence that results in glycosylation of the binding molecule based on expression in eukaryotic cells, and thus possibly abolishing binding to the antigen. The resulting functional variants no longer contain glycosylation sites, but still bind rabies virus and still have neutralizing activity.

Functional variants can be obtained by altering the parent binding molecule or part thereof by general molecular biological methods known in the art, including but not limited to error-prone PCR, oligonucleotide-directed mutagenesis, and site-directed mutagenesis. In addition, the functional variants may have complementary immobilization activity, which can facilitate the lysis of enveloped rabies virus and/or act as opsonins and increase the phagocytosis of rabies virus, by facilitating its uptake by Fc or C3b receptors or by agglutinating rabies virus making it more susceptible to phagocytosis.

In another aspect, the invention encompasses immunoconjugates, i.e. molecules comprising at least one binding molecule as described herein or a functional variant thereof and further comprising at least one label, e.g. a detectable moiety/agent. The invention also encompasses mixtures of the immunoconjugates of the invention or mixtures of at least one immunoconjugate of the invention with another molecule, such as a therapeutic agent or another binding molecule or immunoconjugate. In another embodiment, the immunoconjugate of the invention may comprise one or more labels. These labels may be the same or different from each other and may be non-covalently bound/conjugated to the binding molecule. The label may also be directly bound/conjugated to the binding molecule by covalent bonds including, but not limited to, disulfide bonds, hydrogen bonds, electrostatic bonds, recombinant fusion, and conformational bonds. Alternatively, the label is bound/conjugated to the binding molecule by one or more linking compounds. Techniques for conjugating labels to binding molecules are well known to those skilled in the art.

The label of the immunoconjugate of the invention may be a therapeutic agent, but is preferably a detectable moiety/agent. Immunoconjugates comprising the detectable agents are useful, for example, for assessing whether a subject is infected with rabies virus, or for monitoring the occurrence or progression of rabies virus infection as part of a clinical testing procedure, e.g., to determine the efficacy of a provided treatment regimen. However, they may also be used for other detection and/or analysis 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.

The label used to label the binding molecule for detection and/or analysis and/or diagnosis depends on the specific detection/analysis/diagnosis technique and/or the method used, such as immunohistochemical staining of a (tissue) sample, flow cytometry detection, scanning laser cytometric detection, fluorescence immunoassay, enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), bioassays (e.g. neutralization analysis), Western blot, etc. For immunohistochemical staining of tissue samples, preferred markers are enzymes that catalyze the production and local deposition of detectable products. Enzymes typically conjugated to binding molecules to allow immunohistochemical development are well known in the art and include, but are not limited to, acetylcholinesterase, alkaline phosphatase, beta-galactosidase, glucose oxidase, horseradish peroxidase, and urease. Typical substrates for producing and depositing visually detectable products are also well known in the art. Subsequently, the immunoconjugates of the invention may be labeled with colloidal gold, or they may be labeled with a radioisotope, e.g.³³P、³²P、³⁵S、³H and¹²⁵I. the binding molecules of the invention may be attached directly or indirectly to the radionuclide via a chelator by methods well known in the art.

When the binding molecules of the invention are used in flow cytometry detection, scanning laser cytometry detection or fluorescence immunoassay, they may be labeled with a fluorophore. Numerous fluorophores useful for fluorescently labeling the binding molecules of the present invention are known to those skilled in the art. 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 a suitable prosthetic group complex.

When the immunoconjugates of the invention are used in vivo diagnostics, the binding molecules may also be detectable by conjugation with, for example, a Magnetic Resonance Imaging (MRI) contrast agent such as gadolinium diethylenetriaminepentaacetate, an ultrasound contrast agent or an X-ray contrast agent or by radiolabelling with a radioisotope.

Furthermore, the binding molecules, functional variants or immunoconjugates thereof of the invention can also be attached to a solid support, in particular for use in vitro immunoassays or in the purification of rabies virus or fragments thereof. Such solid supports may be porous or non-porous, planar or non-planar, including but not limited to glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene supports. The human binding molecule may also be conjugated to a filtration medium, such as NHS-activated Sepharose or CNBr-activated Sepharose, for example, for immunoaffinity chromatography. They may also be attached to paramagnetic microspheres, typically by biotin-streptavidin interactions. The microspheres can be used to isolate rabies virus or fragments thereof from a sample containing rabies virus or fragments thereof. As another example, the human binding molecules of the invention can be attached to the surface of a microtiter plate for ELISA.

The binding molecules of the invention or functional variants thereof may be fused to a marker sequence such as a peptide to facilitate purification. Examples of tag sequences include, but are not limited to, a hexahistidine tag, a Hemagglutinin (HA) tag, a myc tag, or a flag tag.

Alternatively, the antibody may be conjugated to a second antibody to form an antibody heteroconjugate. In another aspect, the human binding molecules of the invention may be conjugated/attached to one or more antigens. Preferably, these antigens are antigens recognized by the immune system of the subject to whom the binding molecule-antigen conjugate is administered. The antigens may be the same or different from each other. Conjugation methods for attaching antigens to binding molecules are well known in the art and include, but are not limited to, the use of cross-linking agents. The human binding molecule binds to rabies virus, and the antigen attached to the human binding molecule will trigger a powerful T cell attack on the conjugate, eventually leading to destruction of the rabies virus.

In addition to the chemical generation of immunoconjugates, either directly or indirectly by conjugation, for example via a linker, immunoconjugates can also be generated as fusion proteins comprising the human binding molecules of the invention and a suitable label. Fusion proteins can be produced by methods known in the art, for example, by recombinant production by constructing in frame a nucleic acid molecule comprising a nucleotide sequence encoding a human binding molecule and a nucleotide sequence encoding a suitable label, and then expressing the nucleic acid molecule.

In another aspect of the invention there is provided a nucleic acid molecule encoding at least one binding molecule of the invention or a functional variant thereof. Such nucleic acid molecules can be used as intermediates for cloning, for example during affinity maturation as described above. In a preferred embodiment, the nucleic acid molecule is isolated or purified.

The skilled person realizes that functional variants of these nucleic acid molecules are also part of the present invention. Functional variants are nucleic acid sequences that can be directly translated using standard genetic code to provide the same amino acid sequence as translated from a parent nucleic acid molecule.

Preferably, the nucleic acid molecule encodes a binding molecule 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.

more preferably, the nucleic acid molecule encodes a human binding molecule comprising a variable heavy chain comprising 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 molecule encodes a binding molecule comprising a variable heavy chain comprising substantially the amino acid sequence of seq id NO: 335 amino acid sequence from 1 st to 119 th amino acid.

In another embodiment, the nucleic acid molecule encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 26 and a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 50 or a variable light chain comprising the amino acid sequence set forth in SEQ ID NO: 27 and a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 51, or a variable light chain encoding an amino acid sequence comprising SEQ ID NO: 28 and a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 52, or a variable light chain encoding an amino acid sequence comprising SEQ id no: 29 and a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 53, or a variable light chain encoding an amino acid sequence comprising SEQ ID NO: 30 and a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 54, or a variable light chain encoding an amino acid sequence comprising SEQ ID NO: 31 and a variable heavy chain comprising the amino acid sequence shown in SEQ id no: 55, or a variable light chain encoding an amino acid sequence comprising SEQ ID NO: 32 and a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 56, or a variable light chain encoding an amino acid sequence comprising SEQ ID NO: 33 and a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 57, or a variable light chain encoding an amino acid sequence comprising seq id NO: 34 and a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 58, or a variable light chain encoding an amino acid sequence comprising SEQ ID NO: 35 and a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 59, or a variable light chain encoding an amino acid sequence comprising SEQ ID NO: 36 and a variable heavy chain comprising the amino acid sequence set forth in SEQ id no: 60, or a variable light chain encoding an amino acid sequence comprising SEQ ID NO: 37 and a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 61, or a variable light chain encoding an amino acid sequence comprising SEQ ID NO: 38 and a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 62, or a variable light chain encoding an amino acid sequence comprising SEQ ID NO: 39 and a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 63, or a variable light chain encoding an amino acid sequence comprising SEQ ID NO: 40 and a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 64, or a variable light chain encoding an amino acid sequence comprising SEQ ID NO: 41 and a variable heavy chain comprising the amino acid sequence of seq id NO: 65, or a variable light chain encoding an amino acid sequence comprising SEQ ID NO: 42 and a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 66, or a variable light chain encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 43 and a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 67, or a variable light chain encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 44 and a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 68, or a variable light chain encoding an amino acid sequence comprising SEQ ID NO: 45 and a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 69, or encodes a light chain comprising the amino acid sequence shown in SEQ ID NO: 46 and a variable heavy chain comprising the amino acid sequence of seq id NO: 70, or a variable light chain encoding an amino acid sequence comprising SEQ ID NO:47 and a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO:71 or a variable light chain encoding an amino acid sequence comprising SEQ ID NO: 48 and a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 72, or a variable light chain encoding an amino acid sequence comprising SEQ ID NO: 49 and a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 73 with a variable light chain having the amino acid sequence shown in seq id no. In a preferred embodiment, the nucleic acid molecule encodes a human binding molecule comprising a variable heavy chain and a variable light chain, the variable heavy chain comprising an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 335 and said variable light chain comprises an amino acid sequence comprising SEQ id no: 337 at amino acids 1-107.

In a particular embodiment of the invention, the nucleic acid molecule encoding the variable heavy chain of the binding molecule of the invention essentially comprises 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. preferably, the nucleic acid molecule encoding the variable heavy chain of the binding molecule of the invention essentially comprises a nucleotide sequence comprising the nucleotide sequence of SEQ id no: 334 from nucleotide 1 to nucleotide 357.

In another particular embodiment of the invention, said nucleic acid molecule encoding the variable light chain of a binding molecule of the invention essentially comprises a nucleotide sequence selected from 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. preferably, the nucleic acid molecule encoding the variable light chain of the binding molecule of the present invention essentially comprises a nucleotide sequence comprising the amino acid sequence of SEQ ID NO: 336 from nucleotide 1 to nucleotide 321.

Another aspect of the invention provides a vector, i.e.a nucleic acid construct, comprising one or more nucleic acid molecules of the invention. The vectors may be derived from plasmids, e.g. F, R1, RP1, Col, pBR322, TOL, Ti, etc.; sticking particles; bacteriophages, e.g. lambda, lambda-like, M13, Mu, P1, P22, Q_βT-even, T-odd, T2, T4, T7, etc.; plant viruses such as alfalfa mosaic virus, brome mosaic virus, hairy virus (capillovirus), carnation potexvirus (caravirous), carnation mottle virus (carmovirus), caulivirus, linear virus (clostervirus), cowpea mosaic virus (comovirus), cryptovirus (cryptovirus), squash mosaic virus (cuummovirus), carnation ringspot virus (dianhovirus), fava bean wilting virus (fabovirus), fijivirus (fijivirus), eumycete corymbovirus (furovirus), geminivirus (geminivirus), barley virus (hordeivirus), equiaxel unstable cyclovirus (ilvirous), yellow dwarf virus (luteirus), maize virus, lecytovirus (trichovirus), pseudomorphism virus (potato virus), potato virus (potato virus), potato virus (potato virus), potato virus, Rice stripe virus group (tenuivirus), tobacco mosaic virus (tobamovirus), tobacco rattle virus group (tobravirus), tomato spotted wilt virus (tomato spotted wilt virus), tomato bushy stunt virus (tombusvirus), turnip yellow mosaic virus (tymovirus), and the like; or animal viruses, such as adenoviruses, arenaviridae, baculoviridae, Birnaviridae, Bunyaviridae (bunyaviridae), Caliciviridae (calciviridae), Cardioviridae (cardioviruses), Coronaviridae (capaviridae), Conaviridae, Cogaviridae, Saciviridae, Epstein-Barr, Enterovirus, Filoviridae, Flaviviridae, Foot-and-Mouthdisease viruses, hepadnaviridae, Hepatiridae, immunodeficiency viruses, influenza viruses, filoviridae, iridoviridae, orthomyxoviridae, papovaviruses, paramyxoviridae, parvoviridae, picornaviridae, polioviruses, polydnaviridae, poxviridae, reoviridae, retroviruses, rhabdoviridae, rhinoviruses, Semliki Forest viruses, tetraviridae, togaviridae, toroviridae, vaccinia viruses, herpetiformis.Stomatitis virus, and the like. The vectors may be used for cloning and/or expressing the human binding molecules of the invention, and may even be used for gene therapy. Vectors comprising one or more nucleic acid molecules of the invention operably linked to one or more expression regulatory nucleic acid molecules are also encompassed within the scope of the invention. The choice of vector will depend on the recombinant method and the host used. Introduction of the vector into the host cell can be carried out by calcium phosphate transfection, viral infection, DEAE-dextran mediated transfection, lipofectamine transfection or electroporation. The vector may be autonomously replicating or may be replicating together with the chromosome into which it is introduced. Preferably, the vector contains one or more selectable markers. The choice of marker may depend on the host cell chosen, although this is not critical to the invention and is well known to those skilled in the art. Such markers include, but are not limited to, kanamycin, neomycin, puromycin, hygromycin, zeocin, thymidine kinase gene from herpes simplex virus (HSV-TK), mouse dihydrofolate reductase gene (dhfr). The invention also includes vectors comprising one or more nucleic acid molecules encoding human binding molecules operably linked to one or more nucleic acid molecules encoding proteins or peptides useful for isolating the binding molecules. 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.

Another aspect of the invention relates to a host containing one or more copies of the vector described above. Preferably, the host is a host cell. 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, gram-positive bacteria such as some species of Bacillus (Bacillus), Streptomyces (Streptomyces) and Staphylococcus (Staphylococcus), or gram-negative bacteria such as some species of Escherichia (Escherichia), e.g. Escherichia coli (e.coli) and Pseudomonas (Pseudomonas). In fungal cells, yeast cells are preferably used. Expression in yeast can be achieved by using yeast strains such as Pichia pastoris (Pichia pastoris), Saccharomyces cerevisiae (Saccharomyces cerevisiae) and Hansenula polymorphaSaccharomyces cerevisiae (Hansenula polymorpha). In addition, insect cells such as fly cells and Sf9 cells can be used as host cells. In addition, the host cell may be a plant cell. Transformed (transgenic) plants or plant cells produced by known methods, for example Agrobacterium-mediated gene transfer, leaf disc transformation, protoplast transformation by polyethylene glycol-induced DNA transfer, electroporation, sonication, microinjection or bolistic gene transfer. In addition, a suitable expression system may be a baculovirus system. The present invention preferably uses an expression system of mammalian cells such as Chinese Hamster Ovary (CHO) cells, COS cells, BHK cells or Bowes melanoma cells. Mammalian cells provide expressed proteins with post-translational modifications that most closely resemble the native mammalian molecules. Since the present invention relates to molecules for administration to the human body, a fully human expression system is particularly preferred. Thus, more preferably the host cell is a human cell. Examples of human cells are HeLa, 911, AT1080, a549, 293 and HEK293T cells. Preferred mammalian cells are human retinal cells such as 911 cells or a cell line deposited at the European Collection of animal cell cultures (ECACC; CAMR, Salisbury, Wiltshire SP4OJG, Great Britain) at 29.2.1996, deposit number 96022940 as PER. C6Trade mark sales (per.c6 is a registered trade mark of Crucell Holland b.v.). C6 "in this application refers to the cell deposited under accession number 96022940, or a progenitor cell, an upstream or downstream passage, and progeny derived from the deposited cell progenitor, as well as derivatives of any of the foregoing.

In a preferred embodiment, the human producer cell comprises at least a functional part of a nucleic acid sequence encoding the adenoviral E1 region in an expressible form. In a more preferred embodiment, the host cell is derived from human retina and immortalized with a nucleic acid comprising an adenoviral E1 sequence, such as the cell line deposited at the European Collection of animal cell cultures (ECACC; CAMR, Salisbury, Wiltshire SP4OJG, GreatBoritain) at 29.2.1996 under accession number 96022940, under the trademark "cell linePer.c6. Production of recombinant proteins in host cells can be performed according to methods well known in the art. C6 under the trade mark perThe use of cells sold as production platforms for proteins of interest has been described in WO00/63403, which is incorporated herein by reference in its entirety.

Methods of producing binding molecules or functional variants of the invention are another part of the invention. The method comprises the following steps: a) culturing the host of 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 molecule or functional variant thereof may be recovered from the cell-free extract, but is preferably recovered from the culture medium. Methods for recovering proteins, such as binding molecules, from cell-free extracts or culture media are well known to those skilled in the art. Binding molecules obtainable by the above-described methods or functional variants thereof are also part of the present invention.

Alternatively, the binding molecules of the invention or functional variants thereof may be produced synthetically by conventional peptide synthesizers after expression in a host, such as a host cell, or in cell-free translation systems using RNA nucleic acids derived from DNA molecules of the invention. Binding molecules or functional variants thereof obtainable by the above-described synthetic production methods or cell-free translation systems are also part of the present invention.

In another embodiment, the binding molecule of the invention or a functional variant thereof may be produced by a transgenic non-human mammal, such as a transgenic mouse or rabbit expressing human immunoglobulin genes. Preferably, the transgenic non-human mammal has a genome comprising a human heavy chain transgene and a human light chain transgene encoding all or part of a human binding molecule as described above. The transgenic non-human mammal may be immunized with a purified or concentrated preparation of rabies virus or fragments thereof. Protocols for immunizing non-human mammals are well known in the art. See Using Antibodies: a Laboratory Manual, Edited by: harlow, D.Lane (1998), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York and Current Protocols in Immunology, Edited by: j.e.col igan, a.m.kruisbeam, d.h.margulies, e.m.shevach, w.strober (2001), john wiley & Sons inc.

In another aspect, the present invention provides a method of identifying a binding molecule, such as a human monoclonal antibody or fragment thereof of the invention or a nucleic acid molecule of the invention, which is capable of specifically binding to rabies virus, said method comprising the steps of: a) contacting the collection of binding molecules on the surface of the replicable genetic package with the rabies virus or fragments thereof under conditions conducive to binding, b) selecting at least once a replicable genetic package that binds to the rabies virus or fragments thereof, and c) isolating and recovering the replicable genetic package that binds to the rabies virus or fragments thereof.

The selecting step may be performed in the presence of rabies virus. The rabies virus may be isolated or non-isolated, e.g. present in the serum and/or blood of an infected individual. In another embodiment, the rabies virus is inactivated. Alternatively, the selection step may be carried out in the presence of rabies virus fragments such as rabies virus extracellular portion, one or more (poly) peptides derived from rabies virus such as G protein, fusion proteins comprising these proteins or (poly) peptides and the like. In another embodiment, cells transfected with rabies virus G protein are used in the selection procedure.

In a further aspect, the invention provides a method for obtaining a binding molecule or nucleic acid molecule of the invention, wherein the method comprises the steps of: a) performing the above-described method for identifying a binding molecule, such as a human monoclonal antibody or fragment thereof according to the invention or a nucleic acid molecule according to the invention, and b) isolating the binding molecule and/or the nucleic acid encoding the binding molecule from the recovered replicable genetic package. Once a new monoclonal antibody has been identified or identified using the above-described method for identifying a binding molecule or a nucleic acid molecule encoding a binding molecule, DNA encoding an scFv or Fab may be isolated from bacterial or replicable genetic packages, combined with standard molecular biology techniques to produce a construct encoding a bivalent scFv or all human immunoglobulins (e.g., IgG, IgA, or IgM) of the desired specificity. These constructs can be transfected into appropriate cell lines to produce fully human monoclonal antibodies (see Huls et al, 1999; Boel et al, 2000).

A replicable genetic package as used herein may be a prokaryotic or eukaryotic organism, including cells, spores, bacteria, viruses, (bacterial) bacteriophages and polysomes. Preferred replicable genetic packages are bacteriophages. Human binding molecules such as single chain Fv's are displayed on replicable genetic packages, i.e., they are attached to a group or molecule located on the outer surface of the replicable genetic package. A replicable genetic package is a screenable unit containing a screened human binding molecule linked to a nucleic acid molecule encoding the binding molecule. The nucleic acid molecule should be replicable in vivo (e.g., vector) or in vitro (e.g., by PCR, transcription and translation). Replication in vivo can be spontaneous (for cells), with the aid of host factors (for viruses) or with the aid of both host and helper viruses (for phagemids). A replicable genetic package displaying a collection of human binding molecules is formed by introducing a displayed nucleic acid molecule encoding an exogenous binding molecule into the genome of the replicable genetic package to form a fusion protein with an endogenous protein that is normally expressed on the outer surface of the replicable genetic package. Expression, transport to the outer surface and assembly of the fusion protein results in the display of the exogenous binding molecule on the outer surface of the replicable genetic package. Yet another aspect of the invention relates to a human binding molecule capable of binding to rabies virus or a fragment thereof and obtainable by the above-described identification method.

Another aspect of the invention relates to a method of identifying a binding molecule potentially having rabies virus neutralizing activity, the method comprising the steps of: (a) contacting the collection of binding molecules on the surface of the replicable genetic package with rabies virus under conditions conducive to binding, (b) separating and recovering the binding molecules that bind to rabies virus from the unbound binding molecules, (c) separating at least one of the recovered binding molecules, (d) testing whether the separated binding molecules have rabies virus neutralizing activity, characterized in that the rabies virus is inactivated in step a. The inactivated rabies virus may be purified before being inactivated. Purification can be carried out using purification methods well known in the art suitable for viruses, for example by centrifugation over a glycerol pad (glycerol cushion). The inactivated rabies virus in step a may be immobilized on a suitable material prior to use. Alternatively, the rabies virus in step a may still be active. In another embodiment, a fragment of rabies virus, such as a polypeptide of rabies virus, e.g. the G protein, is used in step a. In another embodiment, cells transfected with rabies virus G protein are used to select binding molecules potentially having rabies virus neutralizing activity. As shown herein, when cells expressing rabies virus G protein are included in the selection method, the number of neutralizing antibodies selected is higher compared to the selection method in which only purified rabies virus G protein and/or inactivated rabies virus is used.

In another embodiment, the above method of identifying a binding molecule potentially having neutralizing activity against rabies virus further comprises the step of isolating and recovering and optionally isolating the human binding molecule comprising the variable heavy chain 3-30 germline gene. The skilled person can identify specific germline genes by methods known in the art, for example by nucleotide sequencing. The step of isolating and recovering the binding molecule comprising the variable heavy chain 3-30 germline gene can be performed before or after step c. As shown below, most of the rabies virus neutralizing human monoclonal antibodies of the invention comprise this specific V_HA germline gene.

Phage display methods for identifying and obtaining (neutralizing) binding molecules, such as antibodies, are well known to those skilled in the art. Such as those described in U.S. patent nos. 5,696,108; burton andBarbas, 1994; de Kruif et al, 1995 b; and Phage Display: a laboratory manual. edited by: CF Barbas, DR Burton, JK Scott and GJ Silverman (2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. These documents are incorporated herein by reference in their entirety.

To construct phage display libraries, the human monoclonal antibody heavy and light chain variable region genes are assembled for expression on the surface of phage, preferably filamentous phage particles, for example in single chain fv (scFv) or Fab format (see de Kruif et al, 1995 b). Large libraries of phage expressing antibody fragments typically contain 1.0X 10⁹The above antibodies are specific and can be assembled from immunoglobulin V regions expressed in B lymphocytes of immunized or unimmunized individuals. In a particular embodiment of the invention, the phage library of human binding molecules, preferably the scFv phage library, is prepared from RNA isolated from cells obtained from a subject that has been immunized against rabies or exposed to rabies virus. The RNA may be isolated from bone marrow or peripheral blood, preferably from peripheral blood lymphocytes. The subject may be an animal vaccinated or exposed to rabies virus, but is preferably a human that has been vaccinated or exposed to rabies virus. Preferably the human subject has been immunized. Another aspect of the invention includes a collection of human binding molecules, such as a scFv phage library, on the surface of a replicable genetic package as described above.

Alternatively, phage display libraries can 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, variable regions assembled in vitro contain synthetically generated, randomized or partially randomized DNA sequences in those regions of the molecule that are important for antibody specificity, e.g., CDR regions. Rabies virus specific phage antibodies can be selected from libraries by immobilizing a target antigen, such as an antigen from rabies virus, on a solid phase, followed by exposure of the target antigen to the phage library to causeAllowing binding to phage expressing antibody fragments specific for the antigen bound to the solid phase. Unbound phage are removed by washing and bound phage are eluted from the solid phase to infect E.coli and subsequently propagated. Multiple rounds of selection and propagation are typically required to sufficiently enrich for phage that specifically bind to the target antigen. Prior to exposing the phage library to the target antigen, subtraction can be performed, if desired, by first exposing the phage library to non-target antigens bound to a solid phase. The phage may also be selected for binding to a complex antigen, such as a mixture of rabies virus proteins or (poly) peptides, a host cell expressing one or more rabies virus proteins or (poly) peptides, or the (inactivated) rabies virus itself. Antigen-specific phage antibodies can be selected from the library by incubating the solid phase having the inactivated rabies virus preparation bound thereto with a phage antibody library to allow, for example, the scFv or Fab portion of the phage to bind to the protein/polypeptide of the rabies virus preparation. After incubation and several washes to remove unbound and loosely attached phage, phage that have bound to the scFv or Fab portion of the preparation are eluted and used to infect E.coli to amplify the new specificity. Typically, one or more rounds of selection are required to isolate the phage of interest from a large number of unbound phage. Alternatively, the known rabies virus proteins or (poly) peptides can be expressed in host cells, which can be used to select phage antibodies specific for the protein or (poly) peptide. Phage display methods using these host cells can be extended and improved by subtracting irrelevant binders during screening by adding an excess of host cells that do not contain the target molecule or non-target molecules similar to but not identical to the target molecule, thereby significantly increasing the chance of finding the relevant binding molecule (this method is called MAbstract)Method, MAbstractIs a registered trademark of Crucella B.V., see also incorporated hereinAs described in U.S. patent No.6,265,150, incorporated herein by reference).

In another aspect, the invention provides a composition comprising at least one binding molecule of the invention, at least one functional variant or fragment thereof, at least one immunoconjugate of the invention, or a combination thereof. The composition may further comprise a stabilizing molecule, such as albumin or polyethylene glycol, or a salt. Preferably, the salts used are those that retain the desired biological activity of the human binding molecule without any undesirable toxic effects. If desired, the human binding molecules of the invention may be coated in or with a material to protect them from the action of acids or other natural or unnatural conditions that can inactivate the binding molecules.

In yet another aspect, the invention provides a composition comprising at least one nucleic acid molecule of the invention. The composition may comprise an aqueous solution such as an aqueous solution containing a salt (e.g., NaCl or a salt as described above), a detergent (e.g., SDS), and/or other suitable ingredients.

Furthermore, the present invention relates to a pharmaceutical composition comprising at least one binding molecule, at least one functional variant or fragment thereof, at least one immunoconjugate, at least one composition or a combination thereof of the invention. The pharmaceutical composition of the present invention further comprises at least one pharmaceutically acceptable excipient.

In a preferred embodiment, the pharmaceutical composition of the invention comprises at least one additional binding molecule, i.e. the pharmaceutical composition may be a cocktail/mixture of binding molecules. The pharmaceutical composition may comprise at least two binding molecules of the invention, or at least one binding molecule of the invention and at least one further anti-rabies virus binding molecule. The additional binding molecule preferably comprises a CDR3 region, the CRD3 region comprising SEQ ID NO: 25, or a pharmaceutically acceptable salt thereof. Comprises a polypeptide comprising SEQ ID NO: 25 may be a chimeric or humanized monoclonal antibody or a functional fragment thereof, but is preferably a human monoclonal antibody or a functional fragment thereof. In one embodiment, the binding molecule comprises a heavy chain variable region comprising SEQ ID NO: 273 of a polypeptide of the formula. In another embodiment, the binding molecule comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 275. In another embodiment, the binding molecule comprises a polypeptide comprising SEQ ID NOs: 123 and SEQ ID NO: 125, or a light chain of the amino acid sequence shown in seq id no. The binding molecule in the pharmaceutical composition should be capable of reacting with a different, non-competing epitope of 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 high affinity and should have a broad specificity. Preferably, they neutralize as many fixed strains of rabies virus and street virus as possible. More preferably, they also exhibit neutralizing activity against other genotypes of the lyssavirus genus or even other viruses of the rhabdoviridae family, without cross-reactivity with other viruses or normal cellular proteins. Preferably, the binding molecule is capable of neutralizing escape variants of other binding molecules in the mixture.

Another aspect of the invention relates to a pharmaceutical composition comprising at least two rabies virus neutralizing binding molecules, preferably human binding molecules of the invention, characterized in that the binding molecules are reactive with different, non-competing epitopes of rabies virus. In one embodiment, the pharmaceutical composition comprises a first rabies virus neutralizing binding molecule reactive with an epitope located at antigenic site I of the rabies virus G protein and a second rabies virus neutralizing binding molecule reactive with an epitope located at antigenic site III of the rabies virus G protein. The antigenic structure of rabies virus glycoprotein was originally described by Lafon et al (1983). Antigenic sites were identified using a panel of mouse mabs and their respective mAb-resistant viral variants. Thereafter, antigenic sites have been mapped by identifying amino acid mutations in glycoproteins of mAb resistant variants (see Seif et al, 1985; Prehaud et al, 1988; and Benmansour et al, 1991). Most rabies neutralizing mabs are directed to antigenic site II (see Benmansour et al, 1991), a discontinuous conformational epitope comprising amino acids 34-42 and amino acid 198-. Antigenic site III is a continuous conformational epitope at amino acids 330-338 and has two charged residues K330 and R333, affecting the pathogenicity of the virus (see Seif et al, 1985; couron et al, 1998; and Dietzschold et al, 1983). Only one mAb, 509-6, defines the conformational antigenic site I, at amino acid 231 (see Benmansour et al, 1991; and Lafon et al, 1983). Antigenic site IV is known to have overlapping linear epitopes (see Tordo, 1996; Bunschelten et al., 1989; Luo et al., 1997; and Ni et al., 1995). Benmansour et al (1991) also describes the presence of a small site located at position 342-343, which, although closely adjacent to antigenic site III, is distinct therefrom. Sequence alignment of the CR-57 epitope with the linear and conformational neutralizing epitopes on the currently known rabies virus glycoproteins (FIG. 10) indicated 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 the nucleotide and amino acid sequences of the escape viral glycoprotein of CR04-098, the epitope recognized by this antibody appears to be located in the same region as the continuous conformation antigenic site III.

In a preferred embodiment, the pharmaceutical composition comprises a first rabies virus neutralizing binding molecule and a second rabies virus neutralizing binding molecule, said first binding molecule comprising at least one CDR3 region, preferably a heavy chain CDR3 region, comprising the amino acid sequence of SEQ id no: 25, and a second binding molecule comprising at least one CDR3 region, preferably a heavy chain CDR3 region, comprising an amino acid sequence selected from the group consisting of seq id nos: 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. more preferably, the second rabies virus neutralizing binding molecule comprises at least one CDR3 region, preferably a heavy chain CDR3 region, comprising the amino acid sequence of SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof. Preferably, the first rabies virus neutralizing binding molecule comprises a polypeptide comprising SEQ ID NO: 123 and SEQ ID NO: 125, and a second rabies virus neutralizing binding molecule comprising the amino acid sequence set forth in SEQ ID NO: 335 and SEQ ID NO: 337, or a light chain of the amino acid sequence shown. Preferably, the heavy and light chains of the first rabies virus neutralizing binding molecule consist of SEQ ID NO: 122 and SEQ ID NO: 124, and the heavy and light chains of the second rabies virus neutralizing binding molecule are encoded by SEQ ID NO: 334 and SEQ ID NO: 336.

Pharmaceutical compositions comprising two binding molecules, wherein the pI of the binding molecules differ, have difficulty when selecting a suitable buffer for optimal stabilization of both binding molecules. When the pH of the buffer of the composition is adjusted to increase the stability of one binding molecule, this will decrease the stability of the other binding molecule. Reduced or even unstable stability of the binding molecule can lead to its precipitation or aggregation, or to its spontaneous degradation, leading to loss of functionality of the binding molecule. Thus, a further aspect of the invention provides a pharmaceutical composition comprising at least two binding molecules, preferably human binding molecules, characterized in that the binding molecules differ from each other in isoelectric point (pI) by 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 by less than (including) 0.25pI units. The pI can be determined experimentally, e.g., using isoelectric focusing, or calculated based on the amino acid sequence of the binding molecule. In one embodiment, the binding molecule is a binding molecule of the invention and the pharmaceutical composition is a pharmaceutical composition of the invention. Preferably, the binding molecule is a monoclonal antibody, for example a human monoclonal antibody such as an IgG1 antibody. Preferably, the binding molecule is capable of binding and/or neutralizing an infectious agent, such as a virus, bacterium, yeast, fungus or parasite. In one embodiment, the binding molecule is capable of binding to and/or neutralizing a lyssavirus, such as rabies virus. In a particular embodiment, the calculated pI of both binding molecules is between 8.0 and 9.5, preferably between 8.1 and 9.2, more preferably between 8.2 and 8.5. Preferably, the binding molecules have the amino acid sequences of SEQ ID NOs: 14 and SEQ ID NO: 25, and a heavy chain CDR3 region.

In another embodiment, the invention provides a mixture 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 packaging display technology and at least one binding molecule is obtainable by hybridoma technology. When using different techniques, it is very beneficial to select binding molecules with compatible pI to obtain a composition, wherein each binding molecule is sufficiently stable to facilitate storage, handling and subsequent use.

In another embodiment, the binding molecules present in the pharmaceutical composition of the invention enhance the neutralizing activity of each other, i.e. act synergistically when they are combined. In other words, the pharmaceutical composition may exhibit synergistic neutralizing activity of rabies virus and even lyssavirus. The term "synergistic" as used herein refers to the addition of the combined effects of the binding molecules when used in combination over the effects of their individual applications. The ranges and ratios of the ingredients of the pharmaceutical compositions of the present invention should be determined based on their respective potency and tested in vitro neutralization assays or animal models such as hamsters.

In addition, the pharmaceutical composition of the present invention may comprise at least one other therapeutic, prophylactic and/or diagnostic agent. The additional therapeutic and/or prophylactic agent may be an antiviral agent such as ribavirin or interferon-alpha.

The binding molecules or pharmaceutical compositions of the invention may be tested in a suitable animal model system prior to use in humans. Such animal model systems include, but are not limited to, mice, rats, hamsters, monkeys, and the like.

Typically, pharmaceutical compositions must be sterile and stable under the conditions of manufacture and storage. The human binding molecules, variants or fragments thereof, immunoconjugates, nucleic acid molecules or compositions of the invention can be in powder form, reconstituted in a suitable pharmaceutically acceptable excipient prior to or at the time of administration (recinstitution). In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Alternatively, the binding molecules, variants or fragments thereof, immunoconjugates, nucleic acid molecules or compositions of the invention can be in solution and suitable pharmaceutically acceptable excipients can be added and/or mixed prior to or at the time of administration to provide a unit dose of an injectable form. Preferably, the pharmaceutically acceptable excipients used in the present invention are suitable for high drug concentrations, maintain adequate flowability, and delay absorption if desired.

The choice of the optimal route of administration of the pharmaceutical composition is influenced by a number of factors, including the physico-chemical properties of the active molecule in the composition, the exigencies of the clinical situation and the relationship between the plasma concentration of the active molecule and the desired therapeutic effect. For example, if desired, the human binding molecules of the invention may be formulated with carriers that will protect them from rapid release, such as controlled release formulations, including implants, transdermal patches and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. In addition, the human binding molecules may be coated with or co-administered with a material or mixture that prevents inactivation of the human binding molecules. For example, the human binding molecule can be administered to a subject in a suitable carrier, such as a liposome or diluent.

The route of administration can generally be divided into two main modes of administration, oral and parenteral. The preferred mode of administration of the human binding molecules and pharmaceutical compositions of the invention is by intramuscular injection into and around the wound and in the gluteal region. The formulation of the human binding molecules and pharmaceutical compositions depends on the route of administration.

In another aspect, the binding molecule, functional variant, immunoconjugate, composition or pharmaceutical composition of the invention may be used as a medicament. Thus, in a further aspect of the invention there is provided a method of treatment and/or prophylaxis of rabies virus infection using the human binding molecule, functional variant, immunoconjugate, composition or pharmaceutical composition of the invention. The lyssavirus may be of any known genotype, but is preferably rabies virus. The above molecules or compositions may be used in the post-exposure prevention of rabies.

The above molecules or compositions may be used in combination with other molecules useful in the diagnosis, prevention and/or treatment of rabies virus. They may be used in vitro, ex vivo or in vivo. For example, the human binding molecules, functional variants, immunoconjugates or pharmaceutical compositions of the invention can be co-administered with a rabies vaccine. Alternatively, the vaccine may be administered before or after administration of the molecule or composition of the invention. Administration of the molecules or compositions and vaccines of the invention 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 adsorbed Rabies Vaccine (RVA).

The molecules in the compositions and pharmaceutical compositions of the invention are typically formulated in a therapeutically or diagnostically effective amount. The dosage regimen may be adjusted to provide the optimum desired response (e.g., therapeutic response). A suitable dosage range may be, for example, 0.1-100IU/kg body weight, preferably 1.0-50IU/kg body weight, more preferably 10-30IU/kg body weight, e.g. 20IU/kg body weight.

Preferably, a single bolus (bolus) is administered of a binding molecule or pharmaceutical composition of the invention. The molecules and pharmaceutical compositions of the present invention are preferably sterile. Methods for rendering such molecules and compositions sterile are well known in the art. The post-exposure prophylactic dosing regimen was to administer 5 doses of rabies vaccine intramuscularly on days 0, 3, 7, 14 and 28 post-exposure in individuals who had not previously been vaccinated against rabies virus. The human binding molecule or pharmaceutical composition of the invention should be administered in and around the wound on day 0 or as soon as possible after exposure, with the remaining volume administered intramuscularly at a site remote from the inoculation. The non-vaccinated individual is administered an anti-rabies human binding molecule, but it is clear to the skilled person that vaccinated individuals in need of such treatment may also be administered an anti-rabies human binding molecule.

In another aspect, the invention relates to the use of a binding molecule or a functional variant, an immunoconjugate, a nucleic acid molecule, a composition or a pharmaceutical composition of the invention for the manufacture of a medicament for the diagnosis, prevention, treatment or combination of diseases resulting from rabies virus infection. The lyssavirus may be of any known genotype, but is preferably rabies virus. Preferably, the above molecules are used in the manufacture of a medicament for the prevention of rabies following exposure.

The invention also provides kits comprising at least one binding molecule, at least one functional variant thereof, at least one immunoconjugate, at least one nucleic acid molecule, at least one composition, at least one pharmaceutical composition, at least one vector, at least one host, or a combination thereof, of the invention. Optionally, the above-mentioned components of the kit of the invention are packaged in suitable containers and labeled for diagnosis, prevention and/or treatment of a given disease. The above components may be stored in unit-dose or multi-dose containers, for example sealed ampoules, vials, bottles, syringes and test tubes, in aqueous, preferably sterile solutions or in lyophilized, preferably sterile, preparations for reconstitution. The container may be made of a variety of materials, such as glass or plastic, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). The kit further comprises a plurality of containers comprising pharmaceutically acceptable buffers such as phosphate buffered saline, Ringer's solution, and dextrose solution. The kit may further include other materials that are commercially and user-desired, including other buffers, diluents, filters, needles, syringes, media for one or more suitable hosts. Instructions for use are typically included in the kits in commercial packages of therapeutic, prophylactic or diagnostic products, the instructions containing information regarding, for example, the indications, use, dosage, manufacture, administration, contraindications and/or care of use of such therapeutic, prophylactic or diagnostic products.

Currently, HRIG products are used for rabies prevention after exposure. An adult dose of 1500IU of HRIG was provided in a volume of only 10ml (75kg individuals, 20 IU/kg). More concentrated HRIG products are not possible because the currently available 10ml dose contains 1-1.5g total IgG. Whereas current HRIG products have two disadvantages. Firstly, it is generally not anatomically suitable to administer the recommended full dose in and around bite wounds, and secondly, the volume of HRIG currently administered is associated with significant pain. The present invention provides a solution to these disadvantages and provides a pharmaceutical composition comprising a full adult dose in a volume of about 2ml or less, if desired. Such pharmaceutical compositions may comprise, for example, two binding molecules capable of neutralizing rabies virus, preferably CR57 and CR 04-098. The pharmaceutical composition further comprises a pharmaceutically acceptable excipient in a volume of about 2 ml. Larger volumes are possible, but are undesirable in view of the pain associated with injecting larger volumes. Less than 2ml is also possible. The pharmaceutical composition comprises the full adult dose (IU) required for successful post-exposure prophylaxis. In one embodiment, the pharmaceutical composition is stored in a 10ml vial, for example a 10ml ready-to-use vial with a stopper (type I glass). By providing a 10ml vial, the pharmaceutical composition can be selectively diluted to a larger volume in the event that the individual presents a larger wound surface area. The invention also provides a kit comprising at least one container (e.g., a vial) comprising a pharmaceutical composition. The kit may further comprise a second container having a diluent suitable for diluting the pharmaceutical composition to a larger volume. Suitable diluents include, but are not limited to, pharmaceutically acceptable excipients for pharmaceutical compositions and saline solutions. In addition, the kit may comprise instructions for diluting the pharmaceutical composition and/or instructions for administering the pharmaceutical composition diluted or undiluted.

The invention further relates to a method for detecting rabies virus in a sample, said method comprising the steps of: a) contacting the sample with a diagnostically effective amount of a binding molecule, functional variant or immunoconjugate of the invention, and b) determining whether said 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 of a (potentially) infected subject. The subject of the (potential) infection may be a human, but animals suspected to be carriers of rabies virus may also be tested for the presence or absence of rabies virus using the human binding molecules, functional variants or immunoconjugates of the invention. The sample is first manipulated to make it more suitable for the detection method. Manipulation means the processing of a sample suspected of containing and/or containing rabies virus, whereby the rabies virus is decomposed into antigenic components such as proteins, (poly) peptides or other antigenic fragments. Preferably, the binding molecule, functional variant or immunoconjugate of the invention is contacted with the sample under conditions such that an immune complex is formed between the human binding molecule and rabies virus or an antigenic component thereof that may be present in the sample. If an immune complex is formed, this indicates the presence of rabies virus in the sample, which is then detected and determined by suitable means. Such methods include homogeneous (homogeneous) and heterogeneous (heterogeneous) binding immunoassays, such as Radioimmunoassay (RIA), ELISA, immunofluorescence, immunohistochemistry, FACS, BIACORE and Western blot analysis.

Furthermore, the binding molecules of the invention can be used to identify epitopes of rabies virus proteins such as the G protein. The epitope may be linear, but may also be structural and/or conformational. In one embodiment, the binding of the binding molecules of the invention to a series of overlapping peptides, such as 15-mer peptides, of rabies virus proteins, such as rabies virus G protein, can be analyzed using PEPSCAN (see WO 84/03564, WO 93/09872, sloottra et al 1996). The binding of human binding molecules to each peptide can be detected in a PEPSCAN-based enzyme-linked immunosorbent assay (ELISA). In another embodiment, a random peptide library comprising peptides of rabies virus proteins can be screened for peptides that bind to the human binding molecules of the invention. In the above assay, the use of human binding molecules that neutralize rabies virus can identify one or more neutralizing epitopes. The peptides/epitopes found can be used as vaccines or for the diagnosis of rabies.

In another aspect, the invention provides a method of screening for a binding molecule or functional variant of a binding molecule that specifically binds to a different, preferably non-overlapping, epitope of rabies virus than the epitope bound by the binding molecule or functional variant of the invention, the method comprising the steps of: a) contacting the screened binding molecule or functional variant, the binding molecule or functional variant of the invention with rabies virus or a fragment thereof (e.g. rabies virus G protein), b) determining whether the screened binding molecule or functional fragment competes with the binding molecule or functional variant of the invention for specific binding to rabies virus or a fragment thereof. If no competition is detected, the binding molecule or functional variant being screened binds to a different epitope. In a particular embodiment of the above screening method, the human binding molecule or functional variant thereof can be screened to identify a human binding molecule or functional variant thereof that binds to an epitope different from the epitope recognized by a binding molecule comprising the CDR3 region (the CDR3 region comprising the amino acid sequence of SEQ ID NO: 25). Preferably, the epitopes are non-overlapping or non-competing. The skilled person is aware that the above screening methods can also be used to identify binding molecules or functional variants thereof that bind to the same epitope. In a further step, it can be determined whether the screened binding molecule which is unable to competitively specifically bind to rabies virus or fragments thereof has neutralizing activity. It is also possible to determine whether the screened binding molecules capable of competing specifically for binding to rabies virus or fragments thereof have neutralizing activity. The anti-rabies virus neutralizing binding molecules or functional variants thereof found in the screening method are also a further part of the invention. In the screening method, "specifically binds to the same epitope" includes specifically binding to an epitope that is substantially or substantially the same as the epitope bound by the human binding molecule of the present invention. The ability to block or compete with the human binding molecules of the invention for binding to rabies virus typically indicates that the screened binding molecules bind to an epitope or binding site on rabies virus that overlaps structurally with the binding site on rabies virus that is immunospecifically recognized by the binding molecules of the invention. Alternatively, this may indicate that the selected binding molecule binds to an epitope or binding site that is sufficiently adjacent to the binding site that the binding molecule of the invention immunospecifically recognizes to sterically exclude or inhibit the binding of the binding molecule of the invention to rabies virus or a fragment thereof.

Typically, competitive inhibition is determined by an assay in which an antigenic composition, i.e. a composition comprising rabies virus or fragments thereof (e.g. G protein), is mixed with a reference binding molecule and a screened binding molecule. In one embodiment, the reference binding molecule may be one of the human binding molecules of the invention and the binding molecule screened may be another human binding molecule of the invention. In another embodiment, the reference binding molecule may be a binding molecule comprising a CDR3 region, the CDR3 region comprising the amino acid sequence of SEQ ID NO: 25, the binding molecule screened may be one of the human binding molecules of the invention. In another embodiment, the reference binding molecule may be one of the human binding molecules of the invention and the binding molecule screened may be a binding molecule comprising a CDR3 region, the CDR3 region comprising the amino acid sequence of SEQ ID NO: 25. Typically, the selected binding molecules are present in excess. ELISA-based protocols are suitable for use in this simple competitive study. In certain embodiments, the reference binding molecule can be mixed with varying amounts of the screening binding molecule (e.g., 1: 10, 1: 20, 1: 30, 1: 40, 1: 50, 1: 60, 1: 70, 1: 80, 1: 90, or 1: 100) in advance for a period of time before being used in the antigen composition. In other embodiments, the reference binding molecule and the different amounts of the screened binding molecules may be conveniently mixed during exposure to the antigen composition. In any case, by using species or isotype secondary antibodies, the skilled person can only detect the bound reference binding molecule, the binding of which can be reduced by the presence of the screened binding molecule recognizing substantially the same epitope. In conducting a binding molecule competition study between the reference binding molecule and any screened binding molecule (regardless of species or isotype), the skilled artisan may first label the reference binding molecule with a detectable label, such as biotin, an enzyme, a radiolabel, or other label that may be subsequently identified. In these cases, the labeled reference binding molecules are pre-mixed or incubated with the selected binding molecules in different 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 the labeled reference binding molecules are analyzed for reactivity (optionally after an appropriate time) and compared to control values obtained in the case where there is no incubation of potentially competing binding molecules. The assay may again be any immunological assay based on antibody hybridization, and the reference binding molecule may be detected by detecting its label, for example using streptavidin in the case of biotinylated reference binding molecules or by using a chromogenic substrate together with an enzymatic label (e.g. 3, 3 '5, 5' -Tetramethylbenzidine (TMB) substrate with peroxidase), or by simply detecting a radioactive label. Screened binding molecules that bind to the same epitope as the reference binding molecule can effectively compete for binding and thus significantly reduce binding of the reference binding molecule, as indicated by the reduction in binding label. Binding molecules that bind different non-competing epitopes showed no reduction in labeling. The reactivity of the (labelled) reference binding molecule in the absence of binding molecules not associated with competition is a control high value. The control low value is the value obtained by incubating the labeled reference binding molecule with an unlabeled reference binding molecule of substantially the same type, when competition occurs and binding of the labeled reference binding molecule is reduced. In the test assay, a significant decrease in reactivity of the labeled reference binding molecule in the presence of the screened binding molecule indicates the presence of a binding molecule that recognizes the same epitope, i.e., a binding molecule that is "cross-reactive" with the labeled reference binding molecule. The binding molecule binds to a different, non-competing epitope if its reactivity is not reduced.

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, and antibodies, antibody fragments, and other binding agents that bind to an epitope or binding site in close proximity to the epitope bound by the reference binding molecule, such that competitive binding occurs between the screened binding molecule and the reference binding molecule. Preferably, the competitive binding molecules of the invention inhibit specific binding of the reference binding molecule to the selected target by at least 10%, preferably at least 25%, 50%, most preferably at least 75% -90% or even more when present in excess. The identification of one or more competing binding molecules that bind to about the same, substantially the same, or the same epitope as the binding molecule of the invention is a simple technical procedure. Since the competitive binding molecule is identified by comparison with the reference binding molecule, it is to be understood that identifying a competitive binding molecule that binds to the same or substantially the same epitope as the reference binding molecule does not in any case necessitate actually determining the epitope to which the reference binding molecule and the competitive binding molecule bind. Alternatively, binding molecules that bind to different, non-competitive epitopes identified by these competition assays include, but are not limited to, antibodies, antibody fragments, and other binding agents.

In another aspect, the present invention provides a method of identifying a binding molecule, or a nucleic acid molecule encoding such a binding molecule, potentially having activity for neutralising an infectious agent causing a disease in a living organism, wherein the method comprises the steps of: a) contacting a collection of binding molecules on the surface of a replicable genetic package with at least one cell expressing on its surface a protein of an infectious agent causing a disease in a living subject under conditions conducive for binding, b) separating and recovering the binding molecules bound to the cells expressing on their surface the protein of an infectious agent causing a disease in a living subject from the binding molecules not bound to said cells, c) separating the at least one recovered binding molecule, d) testing whether the separated binding molecules have the activity of neutralizing the infectious agent causing a disease in a living subject. The cells expressing on their surface a protein of an infectious agent causing a disease in a living body may be cells transfected with the protein. It is known to those skilled in the art that antigens of infectious agents other than proteins can also be successfully used in this method. In a particular embodiment, the cell is per.c6A cell. However, other (E1-immortalized) cell lines can also be used to express proteins, such as BHK, CHO, NS0, HEK293 or 911 cells. In one embodiment, the binding molecule is a human binding molecule. The infectious agent may be a virus, a bacterium, a yeast, a fungus or a parasite. In one embodiment, the protein is a protein normally expressed on the surface of an infectious agent, or comprises at least a portion of a protein accessible on the surface. In a particular embodiment, the collection of binding molecules on the surface of the replicable genetic package is subjected to subtraction/counter-selection (transfected/counterselected) with cells for expression of the infective factor protein, i.e. the cells are identical to the cells used in step a, provided that they do not express the infective factor protein on their surface. The cells that are subtracted/counter-selected may be untransfected cells. Alternatively, the cell may be transfected with a protein or (extracellular) part thereof which is similar and/or highly homologous in sequence or structure to the protein of the infectious agent and/or which is derived from an infectious agent of the same family or even of the same genus.

Another aspect of the invention relates to a binding molecule having rabies virus neutralizing activity, characterized in that said human binding molecule comprises at least one heavy chain CDR3 region, which CDR3 region comprises the amino acid sequence of SEQ ID NO: 25, further characterized in that said human binding molecule has rabies virus neutralizing activity of at least 2500IU/mg protein. More preferably, the human binding molecule has rabies virus neutralizing activity of at least 2800IU/mg protein, 3000IU/mg protein, 3200IU/mg protein, 3400IU/mg protein, 3600IU/mg protein, 3800IU/mg protein, 4000IU/mg protein, 4200IU/mg protein, 4400IU/mg protein, 4600IU/mg protein, 4800IU/mg protein, 5000IU/mg protein, 5200IU/mg protein, 5400IU/mg protein. The neutralizing activity of the binding molecules was determined by in vitro neutralization assay (modified RFFIT (rapid fluorescence focus inhibition assay)). This analysis is described in detail in the examples section.

In one embodiment, the binding molecule comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 273 of a polypeptide of the formula. In another embodiment, the binding molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 123. The variable light chain of the binding molecule may comprise SEQ ID NO: 275. The light chain of the binding molecule may comprise SEQ ID NO: 125, or a pharmaceutically acceptable salt thereof.

Nucleic acid molecules encoding the above-described binding molecules are also part of the invention. Preferably, the nucleic acid molecule comprises SEQ ID NO: 122. In addition, the nucleic acid molecule may further comprise SEQ ID NO: 124. The invention also provides vectors comprising the nucleic acid molecules and host cells comprising such vectors. Preferably, the host cell is a mammalian cell such as a human cell. Examples of cells suitable for the production of human binding molecules are HeLa, 911, AT1080, a549, 293 and HEK293T cells. Preferred mammalian cells are human retinal cells such as 911 cells or the cell line deposited at 29.2.1996 with the European Collection of animal cell cultures (ECACC; CAMR, Salisbury, Wiltshire SP4OJG, Great Britain) under the trademark PER. C6, with the accession number 96022940(PER.C6 is a registered trademark of Crucell Holland B.V.). C6 "in the present application refers to the cell deposited under accession number 96022940 or an ancestor thereof, an upstream or downstream passage, and progeny of an ancestor from the deposited cell, as well as derivatives of any of the foregoing.

Examples

In order to illustrate the present 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 virus antibodies CR-57 and CR-JB

To determine whether the human monoclonal antibodies designated CR-57 and CR-JB recognized non-overlapping, non-competing epitopes, escape viruses were generated for the human monoclonal antibodies designated CR-57 and CR-JB. CR-57 and CR-JB were generated essentially as described (see Jones et al, 2003) by introducing the variable heavy and light chain coding regions of the corresponding antibody genes into a human IgG1 expression vector called pcDNA3002 (Neo). The resulting vectors pgSO57C11 and pgSOJBC11 were used for transient expression in cells from the European Collection of animal cells (ECACC; C MR, Salisbury, Wiltshire SP4OJG, Great Britain) under the trademark PER. C6 at 29 th month of 1996 with accession number 96022940The cell line of (1). The nucleotide and amino acid sequences of the heavy and light chains of these antibodies are shown in SEQ ID NOs: 122-129. Serial dilutions (0.5ml) of the rabies virus strain CVS-11 (dilution range 10)^-1-10^-8) With a constant amount (. about.4 IU/ml) of antibody CR-57 or CR-JB (0.5ml) at 37 ℃/5% CO₂Incubate for 1 hour under conditions before adding to wells containing mouse neuroblastoma (MNA cells) or BSR cells (baby hamster kidney-like cell line). After 3 days of selection in the presence of human monoclonal antibodies CR-57 or CR-JB, the medium (1ml) containing the potential escape viruses was harvested and stored at 4 ℃ until further use. Subsequently, the cells were fixed with acetone for 20 minutes at 4 ℃ and 37 ℃/5% CO₂Staining with anti-rabies virus N-FITC antibody conjugate (Centocor) was performed overnight under the conditions. The number of foci (foci) in each well was recorded by immunofluorescence and medium from wells containing 1-6 foci was selected for virus amplification. All E57 escape viruses were produced from one focus, except E57B1 (3 foci). EJB escape viruses were isolated from 1 (EJB3F), 3 (EJB2B), 4 (EJB2C), 5 (EJB2E, 2F) or 6 (EJB2D) foci, respectively. Each escape virus is first amplified on a small scale on BSR or MNA cells according to its growth characteristics. These small batches of virus are then used at the MNFurther large scale expansion on A or BSR cells. The amplified virus was then titrated on MNA cells to determine the titer of each batch of escape virus and the optimal dilution of escape virus (resulting in 80-100% infection after 24 hours) for use in the virus neutralization assay.

A modified RFFIT (Rapid fluorescence Focus inhibition assay) assay was performed to detect the cross-protection of E57(CR-57 escape virus) and EJB (CR-JB escape virus) with CR-JB and CR-57, respectively. Thus, a series of 3-fold dilutions of CR-57 or CR-JB was performed starting from a 1: 5 dilution. To each dilution a concentration of rabies virus (CVS-11 strain) was added, so that 80-100% infection was produced. The virus/IgG mixture was incubated at 37 ℃/5% CO₂Incubated under conditions for 1 hour before addition to MNA cells. 24 hours after infection (at 34 ℃/5% CO)₂Under conditions), cells were fixed with acetone for 20 min at 4 ℃ and stained with anti-rabies virus N-FITC antibody conjugate (Centocor) for a minimum of 3 hours. Wells were then analyzed under a fluorescence microscope for rabies virus infection to determine the 50% endpoint dilution. This is the dilution at which viral infection was blocked by 50% in this assay. To calculate efficacy, international Standards (Rabies ImmuneGlobulin Lot R3, reference material from Standards and Testing DMPQ/CBER/FDA laboratories) were included in each modified RFFIT. The standard 50% end point dilution corresponds to a potency of 2 IU/ml. The neutralizing potency of the individual human monoclonal antibodies CR-57 and CR-JB and the combination of these antibodies were tested.

EJB viruses were no longer neutralized by either CR-JB or CR-57 (see Table 1), suggesting that these two antibodies bind to a similar region of the rabies glycoprotein and induce amino acid changes therein. The E57 virus was no longer neutralized by CR-57, whereas 4 of 6E 57 viruses were still neutralized by CR-JB, but at a lower potency (see Table 1). A mixture of antibodies CR-57 and CR-JB (1: 1IU/mg ratio) provided similar results to the single antibody (data not shown).

To identify possible mutations in the rabies virus glycoprotein, the nucleotide sequence of the glycoprotein Open Reading Frame (ORF) of each EJB and E57 escape viruses was determined. Viral RNA from each escape virus and CVS-11 was isolated from virus-infected MNA cells and converted to cDNA by standard RT-PCR. Subsequently, the rabies virus glycoprotein ORF was nucleotide sequenced using cDNA to identify mutations.

Both E57 and EJB escape viruses showed mutations in the same region of the glycoprotein (shown in FIGS. 1 and 2; see the complete sequences depicted in FIGS. 1 and 2, SEQ ID NO: 130-151, respectively). This indicates that the two antibodies recognize overlapping epitopes. From the above it can be concluded that the combination of CR-57 and CR-JB in the mixture does not prevent the escape of the neutralizing resistant variants and is therefore not an ideal immunoglobulin preparation for prevention after rabies exposure.

Example 2

Construction of scFv phage display libraries with rabies-vaccinated donor peripheral blood lymphocytes

50ml of blood was taken from the veins of 4 rabies vaccinated human individuals one week after the last booster immunization. Peripheral Blood Lymphocytes (PBLs) were isolated from these blood samples using Ficoll cell density fractionation. Serum was stored frozen at-20 ℃. Serum was positive for the presence of anti-rabies antibodies by FACS staining on 293T cells transfected with rabies virus glycoprotein. Separation by organic phase (TRIZOL)^TM) And subsequent ethanol precipitation total RNA was prepared from PBL. The obtained RNA was dissolved in DEPC-treated ultrapure water, and the concentration was determined by OD260nm measurement. Thereafter, the RNA was diluted to a concentration of 100 ng/. mu.l. Next, 1. mu.g of RNA was converted to cDNA as follows: to 10. mu.l of total RNA were added 13. mu.l of DEPC-treated ultrapure water and 1. mu.l of random hexamer (500 ng/. mu.l), and the resulting mixture was heated at 65 ℃ for 5 minutes and rapidly cooled on wet ice. Then, 8. mu.l of 5 Xfirst strand buffer, 2. mu.l of dNTPs (each 10mM), 2. mu.l of DTT (0.1M), 2. mu.l of RNase inhibitor (40U/. mu.l), and 2. mu.l of Superscription were added to the mixture^TMIII MMLV reverse transcriptase (200U/. mu.l), incubated for 5 minutes at room temperature and 1 hour at 50 ℃. The reaction was stopped by thermal deactivation, i.e. the mixture was incubated at 75 ℃ for 15 minutes.

The cDNA product obtained was diluted with DEPC-treated ultrapure water to a final volume of 200. mu.l. The OD260nm of a 50-fold dilution (in 10mM Tris buffer) of the obtained dilution of the cDNA product was 0.1.

For each donor, 5-10. mu.l of diluted cDNA product was used as template for PCR amplification of immunoglobulin gamma heavy chain family and kappa or lambda light chain sequences using specific oligonucleotide primers (see tables 2-7). 50 μ l in final volume of 20mM Tris-HC1(pH 8.4), 50mM KCl, 2.5mM MgCl₂250 μ M dNTPs and 1.25 units Taq polymerase, the PCR reaction mixture contained 25pmol of sense primer and 25pmol of antisense primer in addition to the diluted cDNA product. The resulting mixture was rapidly melted for 2 minutes in a heated lid thermocycler at 96 ℃ followed by 30 cycles of 96 ℃ for 30 seconds, 60 ℃ for 30 seconds, and 72 ℃ for 60 seconds.

In the first round of amplification, each of the 17 light chain variable region sense primers (11 for λ light chain (see Table 2) and 6 for κ light chain) was combined with an antisense primer (HuC λ 2 and HuC λ 7 antisense primers were mixed equimolar prior to use) called HuCk5 '-ACACTCTCCCCTGTTGAAGCTCTT-3' (see SEQ ID NO: 152) that recognized C- κ or HuC λ 25 '-TGAACATTCTGTAGGGGCCACTG-3' (see SEQ ID NO: 153) and HuC λ 75 '-AGAGCATTCTGCAGGGGCCACTG-3' (see SEQ ID NO: 154) that recognized the C- λ constant region to yield 4-fold 17 products of about 600 base pairs. These products were purified on a 2% agarose gel and separated from the gel using a Qiagen gel extraction column. 1/10 each isolated product was used in an identical PCR reaction as described above using the same 17 sense primers, each lambda light chain sense primer in combination with one of 3J lambda region-specific antisense primers and each kappa light chain sense primer in combination with one of 5J kappa region-specific antisense primers. The primers used in the second amplification were extended with restriction sites (see Table 4) to allow direct cloning into the phage display vector PDV-C06 (see FIG. 3 and SEQ ID NO: 155). This produced 63 products of 4 times about 350 base pairs, which were mixed (pooled) into a total of 10 fractions. This number of fractions was chosen to maintain the natural distribution of the different light chain families within the library without over-representing certain families or certain familial generationsThe table is not sufficient. The number of alleles within the family was used to determine the percentage presented within the library (see table 5). In the next step, 2.5. mu.g of the mixed fraction and 100. mu.g of PDV-C06 vector were digested with SalI and NotI and purified from the gel. After this time, ligation was performed overnight at 16 ℃ as follows: in the presence of 50mM Tris-HCl (pH 7.5), 10mM MgCl₂To a total volume of 50. mu.l of ligation mix, 10mM DTT, 1mM ATP, 25. mu.g/ml BSA and 2.5. mu. l T4DNA ligase (400U/. mu.l) was added 70ng of the mixed fraction to 500ng of PDV-C06 vector. This procedure was performed for each mixed fraction. The ligation mixture was purified by phenol/chloroform, chloroform extraction and ethanol precipitation using methods well known to those skilled in the art. The DNA obtained was dissolved in 50. mu.l of ultrapure water and 2-fold 2.5. mu.l aliquots of each ligation mixture 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 ℃ on 30 plates of 2TY agar supplemented with 50. mu.g/ml ampicillin and 4.5% glucose (3 plates per mixed fraction, plate size 240mmX240 mm). A (sub) library of light chain variable regions was obtained by scraping transformants from agar plates. This (sub) library was used directly in Qiagen^TMPlasmid DNA preparation was performed with QIAFiltermAXI prep kit.

For each donor, heavy chain immunoglobulin sequences were amplified from the same cDNA preparations with similar two rounds of PCR procedures and the same reaction parameters as described above for the light chain regions, provided that the primers of table 6 and table 7 were used. A first amplification was performed with a set of 9 sense-oriented primers (see Table 6; covering the entire heavy chain variable region family), each combined with an IgG-specific constant region antisense primer called HuCIgG5 '-GTC CAC CTT GGT GTT GCT GGG CTT-3' (SEQ ID NO: 156), yielding 9 products of 4-fold approximately 650 base pairs. These products were purified on a 2% agarose gel and separated from the gel using a Qiagen gel extraction column. 1/10 each isolated product was used in the same PCR reaction using the same 9 sense primers as described above, each heavy chain sense primer in combination with one of the 4 JH region-specific antisense primers. The primers used in the second round were extended with restriction sites (see table 7) to allow for directional cloning in the light chain (sub) library vectors. This results in 36 products of about 350 base pairs per donor. These products were mixed into 9 fractions for each donor according to each (VH) sense primer used. The obtained product was purified using Qiagen PCR purification columns. Next, the fractions were digested with SfiI and XhoI and ligated into light chain (sub) library vectors that had been cleaved with the same restriction enzymes using the same ligation procedures and volumes described above for the light chain (sub) library. Alternatively, the fractions were digested with NcoI and XhoI and ligated into light chain vectors that had been cut with the same restriction enzymes using the same ligation procedures and volumes described above for the light chain (sub) library. Ligation purification and subsequent transformation of the resulting defined library was also performed as described above for the light chain (sub) library, at which point the ligation mix for each donor was combined per VH library. Transformants were grown on 27 plates containing 2TY agar supplemented with 50. mu.g/ml ampicillin and 4.5% glucose (3 plates per mixed fraction; plate size: 240mmX240 mm). All bacteria were collected in 2TY medium containing 50. mu.g/ml ampicillin and 4.5% glucose, mixed with glycerol to 15% (v/v) and frozen in 1.5mi aliquots at-80 ℃. Rescue and selection of each library was performed as follows.

Example 3

Selection of phage carrying a Single chain Fv fragment specifically recognizing rabies Virus glycoprotein

Antibody phage display libraries, general phage display technology and Mabstract, substantially as described in U.S. Pat. Nos. 6,265,150 and WO 98/15833 (both incorporated herein by reference)Antibody fragments are selected by the technique. The antibody phage libraries used were two different semi-synthetic scFv phage libraries (JK1994 and WT2000) as well as the immune scFv phage libraries prepared in example 2 (RAB-03-G01 and RAB-04-G01). The 1 st semi-synthetic scFv phage library (JK1994) is described in de Kruif et al, (1995b) and the 2 nd (WT2000) is essentially as de Kruif et al, (1)995b) And (4) constructing. Briefly, the library has a semi-synthetic format in which variations are incorporated into the heavy and light chain V genes using degenerate oligonucleotides that incorporate the variations into the CDR regions. Only the VH3 heavy chain gene was used, in combination with the kappa and lambda light chain genes. The CDRs 1 and 3 of the heavy chain and the CDR3 of the light chain were constructed synthetically in a PCR-based approach similar to that described by deKruif et al (1995 b). The V region genes thus constructed were sequentially cloned in a phagemid vector in scFv form and amplified to generate a phage library as described previously. In addition, the methods and helper phages described in WO 02/103012 (incorporated herein by reference) are useful in the present invention. To identify phage antibodies that recognize rabies virus glycoprotein, phage selection experiments were performed with either complete rabies virus inactivated by β -propiolactone treatment (rabies virus Pitman-Moore strain), purified rabies virus glycoprotein (rabies virus ERA strain), and/or transfected cells expressing rabies virus G protein (rabies virus ERA strain).

The G protein was purified from rabies virus ERA strain as follows. To the virus solution was added 1/10 volumes of 10% octyl- β -glucopyranoside and gently mixed. The virus samples were incubated at 4 ℃ for 30 minutes and centrifuged (36000rpm, 4 ℃) in a SW51 rotor. The supernatant was collected and dialyzed against 0.1M Tris/EDTA overnight at 4 ℃. Subsequently, the glycoproteins were collected from the dialysis chamber, aliquoted and stored at-80 ℃ until further use. Protein concentration was measured at OD280 and G protein integrity was analyzed by SDS-PAGE.

Intact inactivated rabies virus or rabies virus G protein was diluted in Phosphate Buffered Saline (PBS), 2-3ml was added to MaxiSorp Nunc-Immuno Tubes (Nunc) and incubated overnight at 4 ℃ on a rotating wheel. An aliquot of phage library (500. mu.l, about 10)¹³cfu, amplified with CT helper phage (see WO 02/103012)) were blocked in blocking buffer (2% complement (Protifar) in PBS) for 1-2 hours at room temperature. The blocked phage library was added to an immune tube (preincubated with or without CR-57scFv to block the epitope recognized by CR-57), incubated for 2 hours at room temperature, and washed with wash buffer (0.1% Tween-20(Serva) in PBS) to remove unbound phage. Bound phageThereafter, the antigen was eluted by incubation with 1ml of 50mM glycine-HCl pH 2.2 for 10 minutes at room temperature. Subsequently, the eluted phage was mixed with 0.5ml of 1M Tris-HCl pH7.5 to neutralize the pH. 5ml of a mixture which had been grown to an OD600nm of about 0.3 at 37 ℃ were infected

XL1-Blue E.coli culture. The phage were infected with XL1-Blue bacteria for 30 min at 37 ℃. The mixture was then centrifuged at 3200 Xg for 10 minutes at room temperature and the bacterial pellet resuspended in 0.5ml of 2-trypton yeast extract (2TY) medium. The resulting bacterial suspension was split between two 2TY agar plates supplemented with tetracycline, ampicillin and glucose. After incubation of the plates overnight at 37 ℃, colonies were scraped from the plates and used to prepare enriched phage libraries, essentially as described by De Kruifet al (1995a) and WO 02/103012. Briefly, 2TY medium containing ampicillin, tetracycline and glucose was inoculated with the scraped bacteria and grown at 37 ℃ to an OD600nm of about 0.3. CT helper phage was added and the bacteria were infected, after which the medium was changed to 2TY containing ampicillin, tetracycline and kanamycin. Incubation was continued overnight at 30 ℃. On day 2, bacteria were removed from the 2TY medium by centrifugation, after which the phage in the medium were precipitated with polyethylene glycol (PEG) 6000/NaCl. Finally, the phage were dissolved in 2ml PBS containing 1% Bovine Serum Albumin (BSA), filter sterilized and used for the next round of selection.

Phage selection was also performed on cells transfected with rabies virus glycoprotein. The cells used were cells from the cell line deposited at 29.2.1996 with the European Collection of Animal Cell Cultures (ECACC), CAMR, Salisbury, Wiltshire SP4OJG, Great Britain under the accession number 96022940, per.C6Is marketed. C6, hereinafter they are referred to as perA cell. Here, a closed phage library (2ml) was first added to 1X 10⁷subtractor cells (in DMEM/10% FBS) andincubate at 4 ℃ for 1 hour on a rotating wheel. C 6. the Subtractor cells express on their surface the ectodomain of the glycoprotein of Vesicular Stomatitis Virus (VSV) fused to the transmembrane and cytoplasmic domains of rabies virusA cell. C 6. c.The phage of the antigen of the cell are removed from the phage library. The phage/cell mixture was centrifuged (500 Xg for 5 min, 4 ℃) to remove cell-bound phage, and the supernatant was added to a volume of 1X 10 containing 3ml⁷subtractor cells in new tubes. This subtraction step was repeated 2 times with each supernatant. Subsequently, the subtracted phage were combined with transfected cells expressing rabies virus glycoprotein (per.c 6) on a rotating wheelCells (3X 10)⁶Cells) were incubated at 4 ℃ for 1.5 hours. Previously, transfected cells were preincubated with or without CR-57scFv to block the epitope recognized by CR-57. After incubation, cells were washed 5 times with 1ml of DMEM/10% FBS (cells were resuspended and transferred to a new tube for each wash), and phage were eluted and processed as described above.

Typically, two rounds of selection are performed prior to isolation of each phage antibody. After round 2 selection, monoclonal phage antibodies were prepared with each E.coli colony. Essentially, individual colonies were grown to log phase in 96-well plates and infected with VCSM13 helper phage, after which phage antibody production was allowed to proceed overnight. The phage antibodies generated were precipitated with PEG/NaCl, filter sterilized and tested in ELISA for binding to intact inactivated rabies virus and purified rabies virus G protein. A large panel of phage antibodies was obtained from the selection, demonstrating binding to both intact inactivated rabies virus and rabies virus G protein (see examples below). Two selection strategies were performed using the above-described immune libraries. In the first strategy, 736 phage antibodies were selected after two rounds of selection with inactivated virus or purified G protein in the first and second rounds of selection. In the second selection strategy, 736 phage antibodies were selected after two rounds of selection with cell surface expressed recombinant G protein in the first round of selection and inactivated virus or purified G protein in the second round of selection. The number of unique phage antibodies obtained with the first strategy was 97, while the second strategy produced 70 unique phage antibodies. The 97 unique phage antibodies found in the first strategy produced 18 neutralizing antibodies, and the 70 unique clones identified in the second strategy produced 33 neutralizing antibodies. This clearly indicates that the selection of cells comprising rabies virus glycoprotein transfected cells, i.e. cell surface expressed recombinant G protein as antigen, appears to produce more neutralizing antibodies than the selection using purified G protein and/or inactivated virus alone.

Example 4

Identification of rabies virus glycoprotein-specific single-chain phage antibodies

The selected single chain phage antibodies obtained in the above screen were confirmed for specificity in ELISA, i.e. binding to rabies virus G protein purified as described above. In addition, single chain phage antibodies were also tested for binding to 5% FBS. For this purpose, Maxisorp was coated with rabies G protein or 5% FBS^TMELISA plates. After coating, plates were blocked in PBS/1% complement for 1 hour at room temperature. Selected single chain phage antibodies were incubated in an equal volume of PBS/1% alexin for 15 minutes to obtain blocked phage antibodies. The plate is emptied and blocked phage antibody is added to the well. After 1 hour incubation, the plates were washed in PBS containing 0.1% Tween-20 and bound phage antibodies (measured by OD492 nm) were detected with peroxidase-conjugated anti-M13 antibody. As controls, the procedure was performed simultaneously without single-chain phage antibody, with a negative control single-chain phage antibody against CD8 (SC02-007), or with a positive control single-chain phage antibody against rabies virus glycoprotein (scFv SO 57). Shown in Table 8, referred to as SC04-001, SC04-004, SC04-008, SC04-010, SC04-018, SC04-021, SC04-026, SC04-031, Sc,Selected phage antibodies of 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 immobilized purified rabies virus G protein, but no binding to FBS was observed. The same results were obtained in an ELISA using whole inactivated rabies virus prepared as described above (data not shown).

Example 5

Identification of rabies virus-specific scFv

Plasmid DNA was obtained from selected specific single chain phage antibody (scFv) clones and the nucleotide sequence was determined according to standard techniques. The nucleotide sequences (including restriction sites for cloning) of the scFvs designated 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. the amino acid sequences of the scFvs designated 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.

the VH and VL gene identities of scFv that specifically bind rabies G Protein (see Tomlinson IM, Williams SC, Ignatovitch O, Corbett SJ, Winter G.V-BASE Sequence directory. Cambridge Unit Kingdom: MRC centre for Protein Engineering (1997)) and heavy chain CDR3 composition are shown in Table 9.

Example 6

In vitro neutralization of rabies virus by rabies virus-specific scFv (modified RFFIT)

To determine whether the selected scFv can block rabies virus infection, an in vitro neutralization assay (modified RFFIT) was performed. The scFv preparation was diluted by a series of 3-fold dilutions starting with a 1: 5 dilution. Rabies virus (strain CVS-11) was added to each dilution at a concentration that produced 80-100% infection. Virus/scFv mixtures were added to MNA cells at 37 ℃/5% CO₂The temperature is kept for 1 hour. 24 hours post infection (at 34 ℃/5% CO)₂) Cells were fixed with acetone for 20 min at 4 ℃ and stained with anti-rabies N-FITC antibody conjugate (Centocor) for a minimum of 3 hours. Cells were then analyzed under a fluorescent microscope for rabies virus infection to determine the 50% endpoint dilution. This is the dilution at which viral infection was blocked by 50% in this assay (see example 1). Several scfvs were identified that showed neutralizing activity against rabies virus (see table 10).

In addition, it was investigated whether the selected scFv could neutralize the E57 escape virus prepared in example 1 (E57a2, E57A3, E57B1, E57B2, E57B3, and E57C3) by an in vitro neutralization assay (modified RFFIT) as described above. Several scfvs were identified that showed neutralizing activity against the E57 escape virus (see tables 11A and 11B).

Example 7

Rabies virus G protein competition ELISA Using scFv

To identify antibodies that bind to non-overlapping, non-competing epitopes, a rabies glycoprotein competition ELISA was performed. Nunc-Immuno coating with purified rabies virus glycoprotein (1 mg/ml; rabies virus ERA strain) diluted 1: 1000 in PBS (50. mu.l) at 4 ℃^TMMaxisorp F96 plates (Nunc) were used overnight. Uncoated proteins were washed away, and wells were then blocked with 100. mu.l of PBS/1% alexin for 1 hour at room temperature. The blocking solution was then discarded and 50 μ l of unpurified anti-rabies virus scFv (2X diluted) in PBS/1% voxels was added. The wells were washed 5 times with 100. mu.l PBS/0.05% Tween-20. Then, 50. mu.l of biotinylated anti-rabies virus competitor IgG, CR-57bio, was added to each well, incubated at room temperature for 5 minutes, and the wells washed 5 times with 100. mu.l of PBS/0.05% Tween-20. To detect binding of CR-57bio, 50. mu.l of streptavidin-HRP antibody (Becton Dickinson) diluted 1: 2000 was added to the wells and incubated at room temperature for 1 hour. The wells were washed again as described above and the ELISA was further developed by adding 100. mu.l of OPD reagent (Sigma). By adding 50. mu.l of 1M H₂SO₄The reaction was stopped and OD was measured at 492 nm.

When co-incubated with scFv SO57, i.e., the scFv version of CR-57 (see SEQ ID NO: 205 and 206 for the nucleotide and amino acid sequences of SO57, respectively) or scFv SOJB, i.e., the scFv version of CR-JB (see SEQ ID NO: 312 and 313 for the nucleotide and amino acid sequences of SOJB, respectively), the signal obtained with CR-57bio alone can be reduced to background levels. This indicates that the scFv SO57 and SOJB compete for the interaction of CR-57bio with rabies virus glycoprotein by binding to the same epitope or overlapping epitopes, respectively, with CR-57 bio. In contrast, the unrelated scFv named SC02-007, i.e., the scFv that bound to CD8, did not compete for binding. Anti-rabies virus scvs, designated 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 not compete with CR-57bio, suggesting that these scvs bind an epitope different from that recognized by CR-57 (see fig. 4).

Similar results were obtained with the following experiments. First, rabies virus antibody CR-57 was added to wells coated with rabies virus G protein. Next, the competing scFv was added. In this setup, the anti-rabies scFv was detected with anti-VSV-HRP based on the presence of VSV-tag in the scFv amino acid sequence (see FIG. 5).

Example 8

Construction of fully human immunoglobulin molecules (human monoclonal anti-rabies antibody) from selected anti-rabies Single chain Fv' s

The variable regions of the heavy and light chains of the scFv designated SC04-001, SC04-008, SC04-018, SC04-040 and SC04-126 were PCR amplified with oligonucleotides to append restriction sites and/or sequences for expression in the IgG expression vectors pSyn-C03-HC γ 1 (see SEQ ID No: 277) and pSyn-C04-C λ (see SEQ ID No: 278), respectively. Amplification of V with the oligonucleotides shown in tables 12 and 13, respectively_HAnd V_LCloning PCR products into the vector pSyn-C03-HC gamma 1 and pSyn-C04-C lambda respectively.

The variable regions of the heavy and light chains of the scFv designated 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 with oligonucleotides to add restriction sites and/or sequences for expression in the IgG expression vectors pSyn-C03-HC γ 1 and pSyn-C05-C κ (see SEQ ID No: 279), respectively. Amplification of V with the oligonucleotides shown in tables 12 and 13, respectively_HAnd V_LCloning PCR products into the vector pSyn-C03-HC gamma 1 and pSyn-C05-Ck respectively. The oligonucleotides are designed so that they correct any deviations from the germline sequence introduced during the library construction process due to the limited number of oligonucleotide sets used to amplify the large number of antibody genes. The nucleotide sequence of all constructs was verified according to standard techniques known to those skilled in the art.

The resulting expression constructs pgG104-001C03, pgG104-008C03, pgG104-018C03, pgG104-040C03 and pgG104-126C03 encoding the heavy chain of anti-rabies human IgG1 were transiently expressed in 293T cells in combination with the related pSyn-C04-V λ constructs encoding the corresponding light chain and supernatants containing IgG1 antibody were obtained. Expression vectors 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, pgG104-104C03 and pgG104-164C03 encoding the heavy chain of anti-rabies virus human IgG1 are combined with related pSyn-C05-V kappa constructs encoding the corresponding light chain to obtain an antibody containing supernatant 1 transiently expressed in T cells.

The nucleotide and amino acid sequences of the heavy and light chains of antibodies designated 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. The recombinant human monoclonal antibody is then purified on a protein-A column, followed by buffer exchange on a desalting column, using standard purification methods commonly used for immunoglobulins (see, e.g., WO00/63403, incorporated herein by reference).

In addition, for CR04-098, a single human IgG1 expression vector designated pgG104-098C10 was generated as described 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: 334-337. Vectors pgSO57C11 (see example 1) and pgG104-098C10 were used for stable expression of CR-57 and CR04-098, respectively, in cells from a cell line deposited at the European Collection of Animal Cell Cultures (ECACC), CAMR, Salisbury, Wiltshire SP4OJG, Great Britain at 29.1996, under accession number 96022940, PER. C6Is marketed. The calculated isoelectric points for stably produced CR-57 and CR04-098 were 8.22 and 8.46, respectively. The isoelectric points of CR-57 observed in the experiment were between 8.1 and 8.3, and those of CR04-098 were between 9.0 and 9.2. Recombinant human monoclonal antibodies were purified as described above. Unless otherwise described, recombinant human monoclonal antibodies transiently expressed by the above two vector systems were used 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, and recombinant human monoclonal antibodies transiently expressed by one vector system described in example 1 were used for CR 57.

Example 9

Rabies virus G protein competition ELISA Using IgG

To determine whether the human monoclonal anti-rabies virus G protein IgG binds to non-overlapping, non-competitive epitopes, a competition experiment was performed. Wells with coated rabies G protein were incubated with increasing concentrations (0-50 μ G/ml) of unlabeled anti-rabies G protein IgG for 1 hour at room temperature. Then, 50. mu.l of different biotinylated anti-rabies virus IgG (1. mu.g/ml) were added to each well, incubated at room temperature for 5 minutes, and immediately washed 5 times with 100. mu.l of PBS/0.05% Tween-20. The wells were then incubated with 50. mu.l of 1: 2000 diluted streptavidin-HRP (Becton Dickinson) for 1 hour at room temperature, washed and developed as described above. The signal decreased with increasing unlabeled IgG concentration, indicating that the two antibodies compete with each other and recognize the same epitope or overlapping epitopes.

Alternatively, wells coated with rabies virus G protein (ERA strain) were incubated with 50 μ G/ml unlabeled anti-rabies virus G protein IgG for 1 hour at room temperature. Then 50. mu.l of biotinylated CR57 (0.5-5. mu.g/ml; not fully saturated level) was added to each well. Further steps were performed as described above. The signal obtained was compared with that obtained with biotinylated CR57 only (see FIG. 6; no competitor). From FIG. 6, it can be deduced that the signal was not decreased by using the antibody called CR02-428 as a negative control. In contrast, competition with unlabeled CR57 (positive control) or CR-JB reduced the signal to background levels. It can be further deduced from FIG. 6 that none of the anti-rabies virus G protein IgG competed significantly with CR-57, which is consistent with the scFv competition data described in example 7.

In addition, competition experiments were performed on rabies virus G protein (ERA strain) transfected per.c6 cells by flow cytometry. Transfected cells were incubated with 20. mu.l unlabeled anti-rabies G protein IgG (50. mu.g/ml) for 20 min at 4 ℃. After washing the cells with 1% BSA in PBS, 20. mu.l of biotinylated CR57 (0.5-5. mu.g/ml; at incomplete saturation level) were added to each well and incubated at 4 ℃ for 5 minutes, immediately washed 2 times with 100. mu.l of 1% BSA in PBS. Subsequently, the wells were incubated with 20. mu.l of 1: 200 diluted streptavidin-PE (Caltag) for 15 minutes at 4 ℃, washed and developed as described above. The signal obtained with biotinylated CR57 could not be significantly reduced 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 G protein iggs competed significantly with CR-57, except CR04-126, which reduced the signal to approximately 30% (see fig. 7). The latter did not compete in the ELISA (see figure 6). This may be due to the fact that in FACS experiments the way in which glycoproteins are presented to antibodies is different from ELISA experiments. Binding of CR04-126 may be more dependent on the conformation of the glycoprotein, resulting in a competitive effect observed with CR04-126 in FACS-based competition assays, but not in ELISA-based assays. In addition, CR04-008 and CR04-010 reduced the signal to about 50% in FACS-based competition assays (see FIG. 7), suggesting that they may compete with CR 57. For CR04-010, this was not confirmed by scFv competition data or by ELISA-based competition assays. For other iggs, FACS data were consistent with ELISA data for the respective scFv and IgG.

Example 10

Additive/synergistic effects of anti-rabies IgG in vitro neutralization of rabies virus (modified RFFIT)

To determine whether anti-rabies virus G protein IgG has an additive or synergistic effect in rabies virus neutralization, different combinations of IgG were tested. First, the potency (in IU/mg) of each antibody was determined in a modified RFFIT (see example 1). Antibody combinations were then prepared on an equal IU/mg basis and tested in modified RFFIT. The potency of each antibody combination can be determined and compared to expected potency. Antibodies have an additive effect if the potency of the antibody combination is equal to the sum of the potency of each antibody present in the combination. These antibodies have a synergistic effect in rabies virus neutralization if the potency of the antibody combination is higher.

Alternatively, additive or synergistic effects can be determined by the following experiments. First, the potency of the test antibodies, such as CR-57 and CR04-098, was determined in a standard RFFIT (see Laboratory techniques in rabies, Edited by: F. -X Meslin, M.M. Kaplan and H.Koproxski (1996), 4th edition, Chapter 15, world Organization, Geneva). Then, these antibodies were mixed in a ratio of 1: 1 based on IU/ml. This antibody mixture was tested in six independent RFFIT experiments with the same concentration of each antibody to determine the 50% neutralization endpoint. The Combination Index (CI) of the antibody mixture was then determined using the formula CI ═ C1/Cx1) + (C2/Cx2) + (C1C2/Cx1Cx2 described by Chou et al (1984). C1 and C2 are the amounts of monoclonal antibody 1 and monoclonal antibody 2 (expressed in μ g) that when used in combination result in 50% neutralization, Cx1 and Cx2 are the amounts of monoclonal antibody 1 and monoclonal antibody 2 (expressed in μ g) that when used alone result in 50% neutralization. CI-1 indicates additive effect of the monoclonal antibody, CI < 1 indicates synergistic effect of the monoclonal antibody, and CI > indicates antagonistic effect of the monoclonal antibody.

Example 11

Identification of epitopes recognized by recombinant human anti-rabies virus antibodies by PEPSCAN-ELISA

From the extracellular domain of the G protein of the rabies virus strain ERA (see SE for the complete amino acid sequence of glycoprotein G of rabies virus strain ERA)Q ID NO: 207, the extracellular domain consists of amino acids 20-458; protein id of glycoprotein of rabies virus strain ERA in EMBL database J02293) 15-mer linear and cyclic (lopped)/cyclic (cyclic) peptides were synthesized and described previously (sloottra et al, 1996; WO 93/09872) were screened using a mini-PEPSCAN card (455 peptide forms/card) in the form of a credit card (credit-card). All peptides were acetylated at the amino terminus. In all cyclic peptides, positions 2 and 14 were replaced by cysteines (acetyl-XCXXXXXXXXCX-minicard). If other cysteines are present in addition to the cysteines at positions 2 and 4 in the prepared peptide, the other cysteines are replaced with alanine. Cyclic peptides were synthesized using standard Fmoc chemistry and deprotected with trifluoric acid and scavenger. The deprotected peptide was then reacted on the card with a 0.5mM solution of 1, 3-bis (bromomethyl) benzene in ammonium bicarbonate (20mM, pH 7.9/acetonitrile (1: 1 (v/v)). the card was completely immersed in the solution while gently shaking the card in the solution for 30-60 minutes₂The cards were washed thoroughly with O and sonicated in 1% SDS/0.1% beta-mercaptoethanol in PBS (pH 7.2) in disruption buffer at 70 ℃ for 30 min, followed by H₂Sonicate in O for an additional 45 minutes. Human monoclonal antibodies were prepared as described above. The binding of these antibodies to each of the linear and cyclic peptides was tested in a PEPSCAN-based enzyme-linked immunoassay (ELISA). Polypropylene card in the form of 455-well credit card containing covalently linked peptide was incubated (4 ℃ C., overnight) with antibody (10. mu.g/ml; diluted in blocking solution containing 5% horse serum (v/v) and 5% ovalbumin (w/v)). After washing, the peptide was incubated (1 hour, 25 ℃) with anti-human antibody peroxidase (dilution 1/1000), and subsequently, after washing, the peroxidase substrate 2, 2' -azino-di-3-ethylbenzothiazoline sulfonate (ABTS) and 2. mu.l/ml 3% H were added₂O₂. Controls (for linear and cyclic peptides) were incubated with anti-human antibody peroxidase only. After 1 hour, color formation was measured. Color generation by ELISA was quantified using a CCD camera and image processing system. The device comprises a CCD camera and a 55mm lens (Sony CCD Video Camera XC-77RR, Nikon micro-nikkor 55mm f/2.8 lens), a camera adapter (Sony Camera adapter)DC-77RR) and the image processing software package Optimas, version 6.5(Media Cybernetics, Silver Spring, MD 20910, u.s.a.). Optimas runs on a Pentium II computer system.

Human anti-rabies G protein monoclonal antibodies were tested for binding to the 15-mer linear and cyclic/cyclic peptides synthesized as described above. A peptide is considered to be relatedly bound to an antibody when the OD value is equal to or higher than twice the average OD value (per antibody) of all peptides. Table 14 lists the results of binding of human monoclonal antibodies designated CR57, CRJB and CR04-010 to the linear peptide of the extracellular domain of glycoprotein G of rabies virus strain ERA. Areas showing significant binding to each antibody are highlighted in grey (see table 14).

Antibody CR57 was bound to a linear peptide 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 OD values of the peptides having the amino acid sequences GACKLKLCGVLGLRL (SEQ ID NO: 317), ACKLKLCGVLGLRLM (SEQ ID NO: 318) were lower than twice the average value. These peptides are still claimed because they are in the vicinity of the region of the antigenic peptide recognized by antibody CR 57. Binding to the peptide having amino acid sequence KLCGVLGLRLMDGTW (SEQ ID NO: 322) was most pronounced.

Antibody CR04-010 binds to a linear peptide 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 OD values of the peptides having amino acid sequences AYTIFNKTLMEADAH (SEQ ID NO: 327), YTIFNKTLMEADAHY (SEQ ID NO: 328) were lower than twice the average value. However, these peptides are still claimed because they are in the vicinity of the region of the antigenic peptide recognized by the antibody CR 04-010. Binding to peptides having amino acid sequences TIFNKTLMEADAHYK (SEQ ID NO: 329), IFNKTLMEADAHYKS (SEQ ID NO: 330) and FNKTLMEADAHYKSV (SEQ ID NO: 331) was most pronounced.

The CRJB and antibodies designated CR04-040, CR04-098 and CR04-103 (data not shown) did not recognize regions of linear antigenic peptides.

Any of the above peptides or parts thereof represent good candidates for neutralizing epitopes of rabies virus and may form the basis of a vaccine or the basis for the production of neutralizing antibodies for the treatment and/or prevention of rabies virus infection.

Based on their high reactivity in PEPSACN, SLKGACKLKLCGVLGLRLMDGTW (SEQ ID NO: 332) and GFGKAYTIFNKTLMEADAHYKSV (SEQ ID NO: 333) are regions of particular interest in glycoproteins.

From the above PEPSCAN data, it can be further concluded that human monoclonal antibodies designated CR57 and CR04-010 bind to different regions of the rabies G protein, indicating that they recognize non-competing epitopes.

Example 12

Neutralization efficacy of anti-rabies G protein IgG was determined by in vitro neutralization assay (modified RFFIT)

The neutralizing potency of each of the generated human monoclonal antibodies was determined in a modified RFFIT as described in example 1. The potency of the 16 IgG neutralizing rabies strain CVS-11 was higher than 1000IU/mg, while the potency of only two IgGs was lower than 2IU/mg (see Table 15). 8 of the 16 antibodies were more potent than transiently produced CR-57, suggesting a higher efficiency in post-exposure prophylaxis of rabies virus than CR-57. Transiently produced CR-57 exhibited a potency of approximately 3800IU/mg protein (see tables 1 and 15), while stably produced CR-57 exhibited a potency of 5400IU/mg protein (data not shown). Interestingly, most of the neutralizing human monoclonal antibodies identified contained the variable heavy chain 3-30 germline gene (see table 9).

Based on the affinity of the antibody for rabies virus (data not shown) and the 100% endpoint dilution of the antibody in the modified RFFIT assay (data not shown), a set of 6 unique iggs, namely CR04-010, CR04-040, CR04-098, CR04-103, CR04-104 and CR04-144, were selected for further development. Of this group, the antibody CR04-098 was of particular interest because it showed the highest potency, i.e., about 7300IU/mg protein (see Table 15). Similar efficacy was found for stably produced CR04-098 (data not shown).

Example 13

In vitro neutralization of E57 escape virus with anti-rabies virus IgG

To further identify new human monoclonal anti-rabies antibodies, these IgG were tested for neutralizing activity against the E57 escape virus in the modified RFFIT as described above. Most anti-rabies IgG had good neutralizing activity against all 6E 57 escape viruses (see table 16). In contrast, CR04-008, CR04-018, and CR04-126 did not neutralize 6/6, 2/6, and 3/6E 57 escape viruses, respectively. By not neutralized is meant that the 50% end point (endpoint) is not reached at 1: 100 dilution of the antibody. CR04-021, CR04-108, CR04-120, CR04-125 and CR04-164 showed a marked reduction in neutralizing activity against some escaping viruses. This suggests that the epitopes of these antibodies have been affected directly or indirectly in the E57 escape viral glycoproteins. Based on the above, some anti-rabies IgG might be compatible with CR-57 in anti-rabies mixtures for post-exposure prophylaxis treatment. In particular, the set of 6 unique iggs identified above, namely antibodies CR04-010, CR04-040, CR04-098, CR04-103, CR04-104 and CR04-144, demonstrated good neutralizing efficacy against the E57 escape virus, suggesting that the epitopes recognized by these antibodies were not affected by CR-57 induced amino acid mutations. The antibody CR04-098 appears to be the most promising, as it has a potency of more than 3000IU/mg for each escaping virus.

Example 14

Epitope recognition of anti-rabies antibodies CR-57 and CR04-098

To confirm that the human monoclonal antibodies designated CR-57 and CR04-098 recognize non-overlapping, non-competing epitopes, escape viruses were generated from the human monoclonal antibodies designated CR04-098 essentially as described for the escape virus against CR57 (see example 1). Briefly, virus amplification is performed by recording the number of foci per well by immunofluorescence, and selecting a culture medium containing wells preferably containing 1 foci. All E98 escape viruses except E98-2(2 foci) and E98-4(4 foci) were produced from 1 single foci. If the neutralization index < 2.5log, then the virus is defined as an escape variant. The neutralization index is determined as follows: the number of infectious particles/ml produced in BSR cell cultures infected with virus plus monoclonal antibody (about 4IU/ml) was subtracted from the number of infectious viral particles/ml produced in BSR or MNA cell cultures infected with virus alone ([ (log foci forming units in the absence of monoclonal antibody/ml virus) (log ffu/ml virus in the presence of monoclonal antibody) ]). Indices below 2.5log are considered evidence of escape.

To further investigate the binding of CR04-098 to a different non-overlapping non-competitive epitope than CR-57, CR-57 was tested against the E98 escape virus in a modified RFFIT assay as described above. As shown in Table 17, CR-57 had good neutralizing activity against all 5E 98 escape viruses. In addition, the neutralizing activity of antibodies CR04-010 and CR04-144 against E98 escape viruses was tested. Neither antibody neutralized the E98 escape virus (data not shown), suggesting that the epitope recognized by both antibodies was directly or indirectly affected by the amino acid mutations induced by antibody CR 04-098. Neutralizing activity of antibodies CR04-018 and CR04-126 was tested against one of the E98 escape viruses, E98-4. CR04-018 was able to neutralize the escape virus, while CR04-126 had only weak neutralizing efficacy against the escape virus. This suggests that the epitope recognized by CR04-018 is not affected by the mutation induced by antibody CR 04-098. In addition, antibodies CR04-010, CR04-038, CR04-040, CR04-073, CR04-103, CR04-104, CR04-108, CR04-120, CR04-125, and CR04-164 did not neutralize E98-4, suggesting that they recognized the same epitope as CR04-098 (data not shown).

To identify possible mutations in the rabies glycoprotein of each of the E98 escape viruses, the nucleotide sequence of the Open Reading Frame (ORF) of this glycoprotein was determined as previously described for E57 and EJB escape viruses. All E98 escape viruses showed a mutation from N to D at amino acid 336 of rabies glycoprotein (see fig. 8). This glycoprotein region has been defined as antigenic site III, which comprises amino acids 330-338 (not counting the signal peptide). In contrast, CR-57 recognizes an epitope located at amino acids 226-231 (excluding the numbering of the signal peptide), which overlaps with antigenic site I. In addition to the N336D mutation, the E98 escape virus designated E98-5 showed a mutation of H to Q at amino acid 354 of rabies glycoprotein (codon change CAT to CAG) (data not shown).

In addition, pepscan analysis of CR57 binding to peptides carrying the mutated CR57 epitope (as observed in E57 escape viruses) did show that the interaction of CR57 was disrupted (data not shown). It is noted that CR04-098 was still able to match PER. C6 as measured by flow cytometryMutant glycoproteins expressed on the cells (including the N336D mutation) bound (data not shown), although viruses containing this mutation were no longer neutralized.

In addition, BIAcore3000 was used^TMThe analysis system was subjected to epitope mapping studies and affinity ranking studies by surface plasmon resonance analysis. Purified rabies glycoprotein (ERA strain) was immobilized as a ligand on a research grade CM54 flow channel (Fc) sensor chip (Biacore AB, Sweden) using amine coupling techniques. Sequencing was performed at 25 ℃ using HBS-EP (Biacore AB, Sweden) as flow buffer. 50 μ l of each antibody was injected at a constant flow rate of 20 μ l/min. Then, flow buffer was added for 750 seconds, followed by regeneration of the CM5 chip with 5. mu.l of 2M NaOH, 5. mu.l of 45mM HC1, and 5. mu.l of 2mM NaOH. The resonance signal in Resonance Units (RU) was plotted as a function of time, and the increase and decrease in RU as a measure of association and dissociation, respectively, was determined and used as an ordering for the antibodies. By surface plasmon resonanceThe actual KD values measured for CR57 and CR04-098 were 2.4nM and 4.5nM, respectively. Epitope mapping studies further confirmed that CR57 and CR04-098 bind to different epitopes on rabies glycoprotein. Injection of CR57 produced a response of 58RU (data not shown). After injection of CR04-098, an additional increase in the level of response was obtained (24RU), suggesting that the binding site for CR04-098 was not occupied (data not shown). When the reverse order was used, similar results were observed, indicating that each antibody reached similar RU levels regardless of the injection order (data not shown). These results further demonstrate that CR57 and CR04-098 can simultaneously bind to and recognize different epitopes on rabies virus glycoprotein.

Taken together, the above data further demonstrate that antibodies CR-57 and CR04-098 recognize different non-overlapping epitopes, i.e., epitopes in antigenic sites I and III, respectively. These data are in good agreement with ELISA/FACS competition data, which indicate that CR-57 and CR04-098 do not compete for binding to ERA G and that the antibody CR04-098 has good neutralizing activity against all E57 escape viruses. Based on 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 4IU/ml of each antibody) did not produce escape virus (data not shown), the following conclusions were made: antibodies CR-57 and CR04-098 recognize non-overlapping, non-competing epitopes and can be advantageously used in anti-rabies virus antibody mixtures for post-exposure prophylactic treatment.

Example 15

Assessment of conservation of the epitopes recognized by CR57 and CR04-098

The minimal binding region of CR-57 (amino acid KLCGVL within SEQ ID NO: 332, the rabies virus glycoprotein region recognized by CR57 as determined by PEPSAN and alanine scanning techniques) was aligned with the nucleotide sequence of 229 genotype 1 rabies virus isolates to assess the conservation of this epitope (see Table 18). The sample set contains human isolates, bat isolates and isolates from canines or from livestock most susceptible to challenge with rabies-bearing canines. Frequency analysis of the amino acids at each position within the minimal binding region revealed that the key residues that make up the epitope are highly conserved. The lysine at position 1 was conserved in 99.6% of isolates, while the conserved K > R mutation was observed only in the 1/229 isolate. Positions 2 and 3 (L and C) are fully conserved. The central cysteine residue is believed to be structurally involved in glycoprotein folding and is conserved in all rabies viruses (see Badrane and Tordo, 2001). The glycine at position 4 was conserved in 98.7% of isolates, while a mutation towards charged amino acids was observed in the 3/229 isolate (G > R in 1/229; G > E in 2/229). Position 5 is also conserved, with the exception of one isolate, in which conservative V > I mutations were observed. Significant heterogeneity was observed in street virus isolates at position 6, identified as a non-critical residue by alanine substitution scanning: l in 70.7% strain, P in 26.7% strain and S in 2.6% strain. Together, it is expected that about 99% of the rabies virus that can be encountered is recognized by the CR-57 antibody.

123 of these 229 virus isolates were analyzed for the presence of mutations in the CR-57 and CR04-098 epitopes. None of these 123 street virus isolates contained mutations in both epitopes. The N > D mutation observed in the E98 escape virus was only present in 5 virus isolates. These viruses were geographically independent and isolated from african animals (see the phylogenetic tree of fig. 9; these 5 virus isolates, AF325483, AF325482, AF325481, AF325480 and AF325485, shown in bold). Phylogenetic analysis of the glycoprotein sequences revealed that rabies virus with the mutated CR57 epitope was only distantly associated with rabies virus carrying the mutated CR04-098 epitope. Thus, the possibility of encountering rabies virus resistant to neutralization by a mixture of CR-57 and CR04-098 is essentially absent.

Table 1: neutralizing potency of CR-57 and CR-JB against wild type and escape viruses

Table 2: human lambda chain variable region primer (sense)

Table 3: human kappa chain variable region primer (sense)

Table 4: human k chain variable region primer extended with SalI restriction site (sense), human k chain J-region primer extended with NotI restriction site (antisense), human lambda chain variable region primer extended with SalI restriction site (sense) and human lambda chain J-region primer extended with NotI restriction site (antisense)

Table 5: distribution of different light chain products in 10 fractions

Table 6: human IgG heavy chain variable region primer (sense)

Table 7: human IgG heavy chain variable region primer extended with SfiI/NcoI restriction site (sense) and human IgG heavy chain J-region primer extended with XhoI/BstEII restriction site (antisense)

Table 8: ELISA-determined binding of Single chain (scFv) phage antibodies to rabies Virus G protein (ERA Strain) and FBS

Table 9: data for Single chain Fv's that bind to the rabies G protein

Table 10: data for scFv rabies virus neutralization Activity assay

Table 11A: analytical data for measuring neutralizing activity of scFv against E57 escape viruses E57A2, E57A3 and E57B1

1^*Is a 50% end point dilution

2^*Is a 50% endpoint dilution WHO standard (2IU/ml)

3^*Is efficacy (IU/ml)

Table 11B: analytical data for measuring neutralizing activity of scFv against E57 escape viruses E57B2, E57B3 and E57C3

1^*Is a 50% end point dilution

2^*Is a 50% endpoint dilution WHO standard (2IU/ml)

3^*Is efficacy (IU/ml)

Table 12: for PCR amplification of V_HOligonucleotides of genes

Table 13: oligonucleotides for PCR amplification of VL genes

Table 14: binding of human monoclonal antibodies CR57, CRJB and CR04-010(10 μ G/ml) to the linear peptide of the extracellular domain of glycoprotein G of the rabies strain ERA

Table 15: neutralizing potency of anti-rabies virus G protein IgG

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 is not determined

Table 16: neutralizing potency against E57 escape virus, anti-rabies virus G protein IgG

^*0 means no 50% endpoint at 1: 100 dilution of antibody

Table 17: neutralizing potency against E98 escape virus CR57

^*0 means no 50% endpoint at 1: 1000 dilution of antibody

Table 18: occurrence of amino acid residues in the minimal binding region of CR57 in type I genotype rabies virus

^*Percentage of each amino acid present is shown in 229 rabies virus isolates

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Claims (8)

1. A binding molecule having rabies virus neutralizing activity, characterized in that said binding molecule comprises a variable heavy chain and a variable light chain, said variable heavy chain consisting of the amino acid sequence shown in SEQ ID NO. 47 and said variable light chain consisting of the amino acid sequence shown in SEQ ID NO. 71.

2. An immunoconjugate comprising the binding molecule of claim 1, said immunoconjugate further comprising at least one label.

3. A nucleic acid molecule encoding the binding molecule of claim 1.

4. A vector comprising the nucleic acid molecule of claim 3.

5. A host comprising the vector of claim 4.

6. The host of claim 5, wherein said host is a cell derived from a human cell.

7. A method of producing the binding molecule of claim 1, wherein the method comprises the steps of:

a) culturing the host of claim 5 or 6 under conditions conducive to expression of the binding molecule, and optionally

b) Recovering the expressed binding molecule.

8. A pharmaceutical composition comprising the binding molecule of claim 1, said pharmaceutical composition further comprising at least one pharmaceutically acceptable excipient.