# Patent

## Title
Host cell specific binding molecules capable of neutralizing viruses and uses thereof

## Abstract

Provided are human binding molecules that specifically bind to a host cell protein and have virus neutralizing activity, nucleic acid molecules encoding such human binding molecules, compositions comprising the human binding molecules, and methods of identifying or producing the human binding molecules. The human binding molecules can be used in the diagnosis, prophylaxis and/or treatment of viral infections.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application is a divisional of U.S. patent application Ser. No. 11/919,265, filed Oct. 24, 2007, now U.S. Pat. No. 8,052,974, issued Nov. 8, 2011, which is a national phase entry under 35 U.S.C. §371 of International Patent Application PCT/EP2006/062250, filed May 11, 2006, published in English as International Patent Publication WO 2006/120230 A2 on Nov. 16, 2006, which claims the benefit under Article 8 of the Patent Cooperation Treaty to International Patent Application Serial No. PCT/EP2005/054002, filed Aug. 15, 2005, to International Patent Application Serial No. PCT/EP2005/052946, filed Jun. 23, 2005, to International Patent Application Serial No. PCT/EP2005/052648, filed Jun. 8, 2005, and to International Patent Application Serial No. PCT/EP2005/052160, filed May 12, 2005, the disclosure of each of which is hereby incorporated herein by this reference in its entirety.


TECHNICAL FIELD
The invention relates to medicine. In particular, the invention relates to the diagnosis, prophylaxis and/or treatment of viral infections.
STATEMENT ACCORDING TO 37 C.F.R. §1.52(e)(5)—SEQUENCE LISTING SUBMITTED ON COMPACT DISC
Pursuant to 37 C.F.R. §1.52(e)(1)(ii), a compact disc containing an electronic version of the Sequence Listing has been submitted concomitant with this application, the contents of which are hereby incorporated by reference. A second compact disc is submitted and is an identical copy of the first compact disc. The discs are labeled “copy 1” and “copy 2,” respectively, and each disc contains one file entitled “Sequence Listing as submitted.txt” which is 260 KB and created on Aug. 24, 2011.
BACKGROUND
Several viruses assemble their core proteins and genomic material in the cytoplasm of a host cell and exit the cell by budding from the plasma membrane. Studies of these viruses, e.g., HIV-1 viruses, have shown that in addition to proteins encoded by the virus, host cell proteins can be found in viruses. While some of these proteins may be taken into the viruses simply because of their proximity to the viral assembly and budding sites, other host cell proteins are likely to be included in viruses as a result of their interaction with viral proteins during assembly and release. Additionally, some host cell proteins may be incorporated to provide a function for the virus during the infection process. Host cell proteins have been found on the surface or the interior of the viruses. Despite their detection on or in viruses, the role and function of host cell proteins in the viral assembly process is poorly understood and still highly speculative.
A variety of agents are presently used to combat viral infection. These agents include anti-viral compounds, compounds suitable for active immunization such as vaccines and compounds suitable for passive immunization such as neutralizing immunoglobulins. The latter group is often focused on neutralizing immunoglobulins that act through specific binding to viral proteins or cell surface receptors involved in viral entry. Due to their binding specificity, such immunoglobulins are however only suitable in the prophylaxis and/or treatment of specific viral diseases and are not broadly applicable in the treatment of viral diseases.
Neutralization of viruses has been described for immunoglobulins directed against virus-incorporated host cell-derived proteins. For instance, HIV-1 has been shown to incorporate the host cell derived protein Intercellular Adhesion Molecule-1 (ICAM-1) and an antibody against ICAM-1 has been shown to neutralize ICAM-1 expressing HIV-1 virions (see Rizzuto and Sodroski, 1997). Disadvantageously, ICAM-1 is also localized on the surface of uninfected host cells, so protection and/or treatment of a viral infection with the immunoglobulin against ICAM-1 may, due to interaction of the immunoglobulin with uninfected host cells, result in serious and unwanted side effects. A further disadvantage of the anti-ICAM-1 immunoglobulin is that its neutralizing activity is, similar to the viral protein-specific and cell surface receptor-specific immunoglobulins, virus (HIV-1) specific.
SUMMARY OF THE INVENTION
Provided are immunoglobulins able to bind an intracellular host cell protein that is incorporated into viruses or expressed at the cell surface. Due to the intracellular localization of the protein in uninfected host cells, fewer unwanted side effects occur upon administration of the immunoglobulins. An advantage of the immunoglobulins is that they are not dependent on specific viral identification and that they interact with a host cell protein that is commonly involved in the viral assembly and budding process and consequently are capable of neutralizing several distinct viruses.
Provided are binding molecules capable of specifically binding to a host cell protein and capable of neutralizing viruses. Also provided are nucleic acid molecules encoding at least the binding region of the binding molecules. Further provided for is the use of the binding molecules hereof in the prophylaxis and/or treatment of a subject having, or at risk of developing, a viral infection. Besides that, the binding molecules hereof may be used in the diagnosis/detection of a viral infection.
Encompassed are binding molecules capable of specifically binding to a host cell protein. Preferably, the host cell protein is an intracellular host cell protein, meaning that the host cell protein is normally (e.g., in an uninfected host cell) not exposed on the outside of the host cell such as, e.g., a cell surface receptor. In one aspect, the host cell protein is a mammalian, preferably a human, host cell protein. In normal (e.g., uninfected) host cells the intracellular host cell protein may have a cytoplasmic localization (is a cytoplasmic protein). Alternatively, it may also be localized on or inside host cell organelles including, but not limited to, mitochondria, the Golgi apparatus, nucleus, vacuoles, vesicles and/or the endoplasmic reticulum. After infection of the host cell with a virus the intracellular host cell protein may travel to the cell membrane and may become localized/incorporated in or on the cell surface of the host cell. In other words, after infection of the host cell with a virus, the intracellular host cell protein may become displayed on the surface of the host cell. The localization of the host cell protein in normal (e.g., uninfected) cells compared to infected cells may therefore differ. The binding molecules hereof may be capable of specifically binding to a host cell, once the intracellular host cell protein is exposed in or on the surface of the cell. The binding molecules hereof may also bind a fragment of a host cell protein, the fragment at least comprising an antigenic determinant recognized by the binding molecules hereof. An “antigenic determinant” as used herein is a moiety, such as a peptide or polypeptide, protein, glycoprotein, analogue or fragment thereof, that is able to bind to a binding molecule of the invention with sufficiently high affinity to form a detectable antigen-binding molecule complex.
In a certain embodiment, the intracellular host cell protein recognized by the binding molecules hereof is Fas-associated factor-1 (FAF-1; for nucleotide sequence see SEQ ID NO:1 of the incorporated SEQUENCE LISTING (nucleotides 278-2230 encode the protein) and for amino acid sequence see SEQ ID NO:2; see also GenBank No. BC067100) or NADH-dehydrogenase (ubiquinone) flavoprotein-1 (NDUFV-1; for nucleotide sequence see SEQ ID NO:3 (nucleotides 34-1428 encode the protein) and for amino acid sequence see SEQ ID NO:4; see also GenBank No. BC015645.2). In an embodiment hereof, naturally occurring truncated or secreted forms, naturally occurring variant forms (e.g., alternatively spliced forms) and naturally occurring allelic variants of FAF-1 or NDUFV-1, particularly the FAF-1 or NDUFV-1 comprising an amino acid sequence as shown in SEQ ID NO:2 and SEQ ID NO:4, respectively, are also a part hereof. A variant form of NDUFV-1 is shown under GenBank No. BC008146.1. This variant form has an amino acid sequence that is identical to SEQ ID NO:4, with the proviso that it lacks amino acids 16-24 of SEQ ID NO:4. Other variant forms of NDUFV-1 can be found under GenBank Nos. S67973.1, CR605492.1, and BC007619.1. A variant form of FAF-1 is shown under GenBank No. NM—131917. This variant form has an amino acid sequence that is identical to SEQ ID NO:2, with the proviso that as a result of the lack of an internal coding sequence it lacks amino acids 189-344 (but has the same N- and C-termini as compared to the full-length form of FAF-1). The amino acids 189-344 have been replaced by amino acids FSSR in this variant form. Binding molecules hereof may also be capable of specifically binding to the variants and alternative forms of FAF-1 or NDUFV-1, as long as the modifications in the variants and alternative forms do not abolish the binding of the binding molecules to them. FAF-1 or NDUFV-1 variants may comprise an amino acid sequence having a homology of at least 95%, preferably at least 97%, more preferably at least 98%, and even more preferably at least 99% with the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, respectively. Molecular biological techniques to make variants and forms of FAF-1 or NDUFV-1 recombinantly and/or purify them from a (natural) source are well within the reach of the person skilled in the art and are also contemplated herein.
In a further embodiment, provided is a pharmaceutical composition comprising FAF-1 and/or NDUFV-1 and a pharmaceutically acceptable excipient. Further disclosure of elements concerning pharmaceutical compositions in general is given below. The host cell proteins described herein may be used as vaccine for the prophylaxis and/or treatment of viral diseases including, but not limited to, flavivirus infections such as WNV infections.
Encompassed herein are binding molecules capable of specifically binding to a fragment of FAF-1 or NDUFV-1 or any of the variants or forms described herein. The fragment should at least comprise the antigenic determinant of FAF-1 or NDUFV-1 recognized by the respective binding molecules.
In one aspect, the intracellular host cell protein can be incorporated into a viral membrane. Incorporation can take place inside the host cell, e.g., in the cytoplasm, but may also take place inside or on the surface of one of the host cells organelles. Alternatively, incorporation can also take place near, in or on the host cell surface, e.g., when after infection of the host cell with a virus the intracellular host cell protein is displayed on the surface of the host cell. In other words, the host cell protein may be incorporated into the viral membrane of newly formed viruses after infection of a host cell with a virus. Therefore, the intracellular host cell protein may also be present in or on viruses, virus-like particles (VLP) or other material at least comprising a viral membrane or parts thereof comprising the host cell protein. Consequently, the binding molecules hereof may be capable of specifically binding to viruses, virus-like particles or the other material at least comprising a viral membrane or parts thereof comprising the host cell protein. The viruses comprising the host cell protein may be in activated or inactivated/attenuated form. Methods for inactivating/attenuating viruses are well known in the art and include, but are not limited to, heat inactivation, inactivation by UV irradiation, and inactivation by gamma irradiation. As used herein, “virus-like particle” refers to a virus particle that assembles into intact enveloped viral structures. A virus-like particle does however not contain genetic information sufficient to replicate. Virus-like particles have essentially a similar physical appearance as the wild-type virus, i.e., they are morphologically and antigenically essentially similar to authentic virions. The virus-like particles as used herein may comprise, next to the intracellular host cell protein, wild-type viral amino acid sequences. The virus-like particles may also include functional copies of certain genes. Furthermore, the virus-like particles may also include foreign nucleic acid. The virus-like particles can be naturally or unnaturally occurring viral particles. They may lack functional copies of certain genes of the wild-type virus, and this may result in the virus-like particle being incapable of some function that is characteristic of the wild-type virus, such as replication and/or cell-cell movement. The missing functional copies of the genes can be provided by the genome of a host cell or on a plasmid present in the host cell, thereby restoring the function of the wild-type virus to the virus-like particle when in the host cell. Virus-like particles may display the same cellular tropism as the wild-type virus. The virus-like particle may be non-infectious, but may be infectious. The term “infectious” as used herein means the capacity of the virus-like particle to complete the initial steps of viral cycle that lead to cell entry. In an embodiment hereof, the virus-like particle self assembles. In another embodiment, the virus-like particles are pseudoviruses. Pseudoviruses and their production are well known to the skilled person. Preferably, the pseudoviruses as used herein comprise the intracellular host cell protein on their surface, e.g., incorporated into their viral membrane. Virus-like particles can be produced in suitable host cells such as inter alia mammalian cells. They can be produced intracellularly and/or extracellularly and can be harvested, isolated and/or purified as intact virus-like particles by means known to the skilled person such as inter alia affinity chromatography, gel filtration chromatography, ion-exchange chromatography, and/or density gradient sedimentation. A virus-like particle as herein described, particularly one that comprises an intracellular host cell protein, e.g., FAF-1 and/or NDUFV-1, incorporated into its viral membrane is also part hereof.
In a particular embodiment, the virus-like particle is derived from an enveloped virus, preferably a member of the flavivirus genus such as WNV. In an embodiment, the WNV-derived virus-like particle also comprises WNV E protein. In another embodiment, this virus-like particle further comprises WNV M-protein. By a “WNV E- and M-protein” is meant an envelope and membrane protein, respectively, from any WNV strain. Preferably, the WNV E- and M-protein are derived from a same WNV strain.
In a further aspect, the binding molecules hereof have virus neutralizing activity. Preferably, the binding molecules hereof have virus neutralizing activity against viruses of more than one genus. Preferably, they neutralize viruses of at least two different genera, more preferably at least three different genera, even more preferably at least four different genera, and in particular at least five or more different genera. The viruses may be non-enveloped viruses, but preferably they are enveloped viruses including, but not limited to, Herpes viridae (e.g., Herpes Simplex Virus type 1, 2, 6, 7, 8, cytomegalovirus, varicella-zoster virus, Epstein Barr virus), poxviridae (e.g., Smallpox virus), retroviridae (e.g., HIV1, HIV2, HTLV1, HTLV2), paramyxoviridae (e.g., Mumps virus, measles virus, respiratory syncytial virus, parainfluenza virus), HepaDNAviridae (e.g., Hepatitis B virus), orthomyxoviridae (e.g., Influenza virus A or B), togaviridae (e.g., Rubella), flaviviridae (e.g., Yellow fever virus, Dengue virus, West Nile Virus, Hepatitis C virus, Japanese encephalitis virus, Venezuela encephalitis virus) rhabodiviridae (e.g., Rabies virus), arenaviridae (e.g., Lassa virus, lymphocytic choriomeningitis virus) and coronaviridae (e.g., SARS virus, metapneumoniae virus). Moreover, they may also neutralize viruses not mentioned above, including, but not limited to, Sindbis virus, poliovirus, human papilloma virus, adeno-associated virus, coxsackivirus, enterovirus, Hepatitis A virus, tick-borne encephalitis virus, astrovirus, Ebola virus, Marburg virus, La Crosse virus, California encephalitis virus, Hantaan virus, Crimean-Congo virus, Rift Valley fever, Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, Colorado tick fever, JC virus, BK virus, human adenovirus, and human parvovirus. The binding molecules hereof may have a broad virus neutralizing activity and are capable of neutralizing different, preferably all, viruses within a genus. They may neutralize different, preferably all, strains of a given virus.
In a specific aspect, the binding molecules hereof have flavivirus neutralizing activity, preferably West Nile virus neutralizing activity. The genus flavivirus is a member of the Flaviviridae family. Flaviviruses are small spherical enveloped positive-strand RNA viruses. The flavivirus genus comprises more than 80 highly related viruses including several human pathogens such as inter alia yellow fever virus, Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley encephalitis virus, tick-borne encephalitis virus, and dengue virus. Other viruses belonging to the genus flavivirus can inter alia be found in Kuno et al. (1998), which is incorporated by reference herein. Preferably, the binding molecules hereof are capable of neutralizing both WNV lineage I variants such as inter alia strain 385-99 and WNV lineage II variants such as inter alia strain H-442. In the most preferred embodiment, the binding molecules hereof are capable of neutralizing essentially all WNV variants and strains currently known. In one embodiment, the binding molecules hereof also neutralize at least one other flavivirus including, but not limited to, yellow fever virus, Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley encephalitis virus, tick-borne encephalitis virus, and dengue virus, e.g., dengue virus 1, 2, 3, 4. In addition to members of the flavivirus genera, viral members of the pestivirus en hepacivirus such as Hepatitis C virus may be neutralized. Like flavivirus, the Hepatitis C virus assembly and budding takes place in the ER.
In a further aspect, the binding molecules hereof also have rabies virus neutralizing activity. In one aspect, they are also capable of specifically binding to rabies virus. Rabies virus is member of the Lyssavirus genus. In total, the Lyssavirus genus includes eleven genotypes: rabies virus (genotype 1), Lagos bat virus (genotype 2), Mokola virus (genotype 3), Duvenhage virus (genotype 4), European bat lyssavirus 1 (genotype 5), European bat lyssavirus 2 (genotype 6), Australian bat lyssavirus (genotype 7), Aravan virus (genotype 8), Khujand virus (genotype 9), Irkut virus (genotype 10) and West Caucasian virus (genotype 11). Besides binding to rabies virus, the binding molecules hereof may also be able to bind to other genotypes of the Lyssavirus genus. The binding molecules may also be capable of neutralizing other genotypes of the Lyssavirus genus. Furthermore, the binding molecules hereof may be able to bind to and/or neutralizing viruses, other than Lyssaviruses, of the rhabdovirus family. This family includes, but is not limited to, the genera ephemerovirus, lyssavirus, rhabdovirus and vesiculovirus.
In an embodiment, the binding molecules hereof are human binding molecules. The binding molecules hereof can be intact immunoglobulin molecules such as polyclonal or monoclonal antibodies or the binding molecules can be antigen-binding fragments including, but not limited to, Fab, F(ab′), F(ab′)2, Fv, dAb, Fd, complementarity determining region (CDR) fragments, single chain antibodies (scFv), bivalent single chain antibodies, single chain phage antibodies, diabodies, triabodies, tetrabodies, and peptide or polypeptides that contain at least a fragment of an immunoglobulin that is sufficient to confer specific antigen binding to the intracellular host cell protein or fragment thereof. In one embodiment, the human binding molecules having virus neutralizing activity are administered in IgG1, IgA, or IgM format.
The binding molecules hereof can be used in non-isolated or isolated form. Furthermore, the binding molecules hereof can be used alone or in a mixture comprising at least one binding molecule (or variant or fragment thereof) hereof. In other words, the binding molecules can be used in combination, e.g., as a pharmaceutical composition comprising two or more binding molecules hereof, variants or fragments thereof. For example, binding molecules having different, but complementary activities can be combined in a single therapy to achieve a desired prophylactic, therapeutic or diagnostic effect, but alternatively, binding molecules having identical activities can also be combined in a single therapy to achieve a desired prophylactic, therapeutic or diagnostic effect. The mixture may further comprise at least one other therapeutic agent. Preferably, the therapeutic agent is useful in the prophylaxis and/or treatment of a viral infection (e.g., WNV infection).
Typically, binding molecules hereof can bind to their binding partners, i.e., the intracellular host cell proteins or fragments thereof, with an affinity constant (Kd-value) that is lower than 0.2*10−4 M, 1.0*10−5 M, 1.0*10−6 M, 1.0*10−7 M, preferably lower than 1.0*10−8 M, more preferably lower than 1.0*10−9 M, more preferably lower than 1.0*10−10 M, even more preferably lower than 1.0*10−11 M, and in particular lower than 1.0*10−12 M. The affinity constants can vary for antibody isotypes. For example, affinity binding for an IgM isotype refers to a binding affinity of at least about 1.0*10−7 M. Affinity constants can for instance be measured using surface plasmon resonance, i.e., an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIACORE system (Pharmacia Biosensor AB, Uppsala, Sweden).
The binding molecules hereof may bind to the intracellular host cell protein or fragment thereof in soluble form such as for instance in a sample or may bind to the intracellular host cell protein or a fragment thereof bound or attached to a carrier or substrate, e.g., microtiter plates, membranes and beads, etc. Carriers or substrates may be made of glass, plastic (e.g., polystyrene), polysaccharides, nylon, nitrocellulose, or Teflon, etc. The surface of such supports may be solid or porous and of any convenient shape. Furthermore, the binding molecules may bind to the intracellular host cell protein in purified/isolated or non-purified/non-isolated form. Consequently, the binding molecules may bind to the intracellular host cell protein when it is incorporated into the host cell and/or viral membrane, such as in an infected host cell, a virus or a virus-like particle.
The binding molecules hereof are capable of neutralizing virus infectivity. This may be achieved by targeting viral specific events wherein the intracellular host cell proteins are involved including, but not limited to, virus attachment to cell membranes and penetration in cells, virus uncoating, virus nucleic acid synthesis, viral protein synthesis and maturation, assembly and release of infectious particles, targeting the membrane of infected cells resulting in a signal that leads to no replication or a lower replication rate of the virus. Neutralization assays for different viruses are known in the art. WNV, yellow fever virus, Japanese encephalitis virus, St. Louis encephalitis virus, dengue virus and rabies virus neutralizing activity can for instance be measured as described herein. Alternative WNV neutralization assays are described in for instance Gollins and Porterfield (1986).
In a certain embodiment, the binding molecules hereof comprise at least a CDR3 region, preferably a heavy chain CDR3 region, comprising the amino acid sequence selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:6. In an embodiment, the binding molecules hereof may comprise two, three, four, five or even all six CDR regions of the binding molecules hereof. The heavy chain CDR1 region, heavy chain CDR2 region, light chain CDR1 region, light chain CDR2 region and light chain CDR3 region of the binding molecules hereof are shown in Table 9. CDR regions are according to Kabat et al. (1991) as described in Sequences of Proteins of Immunological Interest.
In yet another embodiment, the binding molecules hereof comprise a heavy chain comprising the variable heavy chain of the amino acid sequence selected from the group consisting of SEQ ID NO:18 and SEQ ID NO:20. In a further embodiment, the binding molecules hereof comprise a light chain comprising the variable light chain of the amino acid sequence selected from the group consisting of SEQ ID NO:22 and SEQ ID NO:24. Table 8 specifies, next to the heavy chain CDR3 region, the nucleotide and amino acid sequence of the heavy and light chains (and their variable regions) of the single chain antibodies.
In yet a further embodiment, the binding molecules hereof comprise a heavy chain comprising the amino acid sequence selected from the group consisting of SEQ ID NOS:30 and 32, and/or comprise a light chain comprising the amino acid sequence selected from the group consisting of SEQ ID NOS:34 and 36.
In another embodiment, the binding molecules hereof comprise a heavy chain CDR1 region, heavy chain CDR2 region, heavy chain CDR3 region, light chain CDR1 region, light chain CDR2 region and light chain CDR3 region as shown in SEQ ID NOS:12, 13, 6, 143, 144 and 145, respectively. In yet another embodiment, the binding molecules hereof comprise a heavy chain comprising the variable heavy chain of the amino acid sequence of SEQ ID NO:20 and/or comprise a light chain comprising the variable light chain of the amino acid sequence of SEQ ID NO:74, i.e., amino acids 1-113 of SEQ ID NO:74. In yet a further embodiment, the binding molecules comprise a heavy chain comprising the amino acid sequence of SEQ ID NO 32, and/or comprise a light chain comprising the amino acid sequence of SEQ ID NO:74.
Another aspect includes functional variants of the binding molecules as defined herein. Molecules are considered to be functional variants of a binding molecule according to the invention, if the variants are capable of competing for specifically binding to the intracellular host cell proteins or fragment thereof with the parent binding molecules. The host cell protein can be incorporated into the viral membrane. In other words, when the functional variants are still able to bind to the intracellular host cell proteins or fragment thereof. Furthermore, molecules are considered to be functional variants of a binding molecule hereof, if they have virus neutralizing activity (e.g., WNV-neutralizing activity). Functional variants include, but are not limited to, derivatives that are substantially similar in primary structural sequence, but which contain, e.g., in vitro or in vivo modifications, chemical and/or biochemical, that are not found in the parental binding molecule. Such modifications include inter alia acetylation, acylation, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, cross-linking, disulfide bond formation, glycosylation, hydroxylation, methylation, oxidation, pegylation, proteolytic processing, phosphorylation, and the like.
Alternatively, functional variants can be binding molecules as defined herein comprising an amino acid sequence containing substitutions, insertions, deletions or combinations thereof of one or more amino acids compared to the amino acid sequences of the parental binding molecules. Furthermore, functional variants can comprise truncations of the amino acid sequence at either or both the amino or carboxyl termini. Functional variants may have the same or different, either higher or lower, binding affinities compared to the parental binding molecule but are still able to bind to the intracellular host cell proteins or fragment thereof. For instance, functional variants may have increased or decreased binding affinities for the intracellular host cell proteins or fragment thereof compared to the parental binding molecules. Preferably, the amino acid sequences of the variable regions, including, but not limited to, framework regions, hypervariable regions, in particular the CDR3 regions, are modified. Generally, the light chain and the heavy chain variable regions comprise three hypervariable regions, comprising three CDRs, and more conserved regions, the so-called framework regions (FRs). The hypervariable regions comprise amino acid residues from CDRs and amino acid residues from hypervariable loops. Functional variants intended to fall within the scope of the invention have at least about 50% to about 99%, preferably at least about 60% to about 99%, more preferably at least about 70% to about 99%, even more preferably at least about 80% to about 99%, most preferably at least about 90% to about 99%, in particular at least about 95% to about 99%, and in particular at least about 97% to about 99% amino acid sequence homology with the parental human binding molecules as defined herein. Computer algorithms such as inter alia Gap or Bestfit known to a person skilled in the art can be used to optimally align amino acid sequences to be compared and to define similar or identical amino acid residues. Functional variants can be obtained by altering the parental binding molecules or parts thereof by general molecular biology methods known in the art including, but not limited to, error prone PCR, oligonucleotide-directed mutagenesis, site-directed mutagenesis and heavy or light chain shuffling. Preferably, the functional variants of the invention have virus neutralizing activity. This neutralizing activity may either be identical, or be higher or lower compared to the parental binding molecules. Furthermore, preferably the functional variants have neutralizing activity against more than one genus, preferably against viruses of at least two, three, four, five, six, etc, different genera. Henceforth, when the term (human) binding molecule is used, this also encompasses functional variants of the (human) binding molecule.
In yet a further aspect, disclosed are immunoconjugates, i.e., molecules comprising at least one binding molecule as defined herein and further comprising at least one tag, such as inter alia a detectable moiety/agent. Also contemplated herein are mixtures of immunoconjugates according to the invention or mixtures of at least one immunoconjugates according to the invention and another molecule, such as a therapeutic agent or another binding molecule or immunoconjugate. In a further embodiment, the immunoconjugates hereof may comprise more than one tag. These tags can be the same or distinct from each other and can be joined/conjugated non-covalently to the binding molecules. The tag(s) can also be joined/conjugated directly to the human binding molecules through covalent bonding. Alternatively, the tag(s) can be joined/conjugated to the binding molecules by means of one or more linking compounds. Techniques for conjugating tags to binding molecules are well known to the skilled artisan.
The tags of the immunoconjugates hereof may be therapeutic agents, but preferably they are detectable moieties/agents. The tags may also be toxins, such as botulinum toxin or functional parts thereof. Immunoconjugates comprising a detectable agent can be used diagnostically to, for example, assess if a subject has been infected with a virus or monitor the development or progression of a virus infection as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. However, they may also be used for other detection and/or analytical and/or diagnostic purposes such as detection of viruses or virus-like particles comprising the intracellular host cell protein or host cells that have been infected and as a consequence thereof display the intracellular host cell protein on their surface. Detectable moieties/agents include, but are not limited to, enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, and non-radioactive paramagnetic metal ions. The tags used to label the binding molecules for detection and/or analytical and/or diagnostic purposes depend on the specific detection/analysis/diagnosis techniques and/or methods used such as inter alia immunohistochemical staining of (tissue) samples, flow cytometric detection, scanning laser cytometric detection, fluorescent immunoassays, enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), bioassays (e.g., neutralization assays), Western blotting applications, etc. Suitable labels for the detection/analysis/diagnosis techniques and/or methods known in the art are well within the reach of the skilled artisan.
Furthermore, the human binding molecules or immunoconjugates can also be attached to solid supports, which are particularly useful for in vitro immunoassays or purification of the intracellular host cell protein or fragment thereof, host cells displaying the intracellular host cell protein on their surface, or viruses or virus-like particles having the intracellular host cell protein incorporated into the viral membrane. Such solid supports might be porous or nonporous, planar or non-planar. The binding molecules can be fused to marker sequences, such as a peptide to facilitate purification. Examples include, but are not limited to, the hexa-histidine tag, the hemagglutinin (HA) tag, the myc tag or the flag tag. Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate. In another aspect, the binding molecules hereof may be conjugated/attached to one or more antigens. Such antigens may be antigens which are recognized by the immune system of a subject to which the binding molecule-antigen conjugate is administered. The antigens may be identical, but may also differ from each other. Conjugation methods for attaching the antigens and binding molecules are well known in the art and include, but are not limited to, the use of cross-linking agents. The binding molecules hereof will bind to the intracellular host cell protein and the antigens attached to the binding molecules will initiate a powerful T-cell attack on the conjugate, which will eventually lead to the destruction of the structure to which the intracellular host cell protein is bound or wherein it is incorporated.
Next to producing immunoconjugates chemically by conjugating, directly or indirectly, via for instance a linker, the immunoconjugates can be produced as fusion proteins comprising the binding molecules hereof and a suitable tag. Fusion proteins can be produced by methods known in the art such as, e.g., recombinantly by constructing nucleic acid molecules comprising nucleotides encoding the binding molecules in frame with nucleotides encoding the suitable tag(s) and then expressing the nucleic acid molecules.
It is another aspect to provide a nucleic acid molecule encoding at least a binding molecule or immunoconjugate hereof. Such nucleic acid molecules can be used as intermediates for cloning purposes, e.g., in the process of affinity maturation as described herein. In one embodiment, the nucleic acid molecules are isolated or purified.
The skilled person will appreciate that functional variants of these nucleic acid molecules are also intended to be a part hereof. Functional variants are nucleic acid molecules that can be directly translated, using the standard genetic code, to provide an amino acid sequence identical to that translated from the parental nucleic acid molecules.
The nucleic acid molecules may encode binding molecules comprising a CDR3 region, preferably a heavy chain CDR3 region, comprising an amino acid sequence selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:6. In a further embodiment, the nucleic acid molecules encode binding molecules comprising two, three, four, five or even all six CDR regions of the binding molecules hereof.
In another embodiment, the nucleic acid molecules encode binding molecules comprising a heavy chain comprising the variable heavy chain of the amino acid sequence selected from the group consisting of SEQ ID NO:18 and SEQ ID NO:20. In another embodiment, the nucleic acid molecules encode binding molecules comprising a light chain comprising the variable light chain of the amino acid sequence selected from the group consisting of SEQ ID NO:22 and SEQ ID NO:24.
In yet a further embodiment, the nucleic acid molecules encode binding molecules comprising a heavy chain comprising the amino acid sequence selected from the group consisting of SEQ ID NOS:30 and 32, and/or comprising a light chain comprising the amino acid sequence selected from the group consisting of SEQ ID NOS:34 and 36.
In another embodiment, the nucleic acid molecules encode binding molecules comprising a heavy chain CDR1 region, heavy chain CDR2 region, heavy chain CDR3 region, light chain CDR1 region, light chain CDR2 region and light chain CDR3 region as shown in SEQ ID NOS:12, 13, 6, 143, 144 and 145, respectively. In yet another embodiment, the nucleic acid molecules encode binding molecules comprising a heavy chain comprising the variable heavy chain of the amino acid sequence of SEQ ID NO:20 and/or comprising a light chain comprising the variable light chain of the amino acid sequence of SEQ ID NO:74, i.e., amino acids 1-113 of SEQ ID NO:74. In yet a further embodiment, the nucleic acid molecules encode binding molecules comprising a heavy chain comprising the amino acid sequence of SEQ ID NO 32, and/or comprising a light chain comprising the amino acid sequence of SEQ ID NO:74.
It is another aspect to provide vectors, i.e., nucleic acid constructs, comprising one or more nucleic acid molecules of the invention. Vectors can be derived from plasmids such as inter alia F, R1, RP1, Col, pBR322, TOL, Ti, etc.; cosmids; phages such as lambda, lambdoid, M13, Mu, P1, P22, Qβ, T-even, T-odd, T2, T4, T7, etc; plant viruses. Vectors can be used for cloning and/or for expression of the binding molecules hereof and might even be used for gene therapy purposes. Vectors comprising one or more nucleic acid molecules according to the invention operably linked to one or more expression-regulating nucleic acid molecules are also covered by the invention. The choice of the vector is dependent on the recombinant procedures followed and the host used. Introduction of vectors in host cells can be effected by inter alia calcium phosphate transfection, virus infection, DEAE-dextran mediated transfection, lipofectamin transfection or electroporation. Vectors may be autonomously replicating or may replicate together with the chromosome into which they have been integrated. The vectors may contain one or more selection markers. The choice of the markers may depend on the host cells of choice, although this is not critical to the invention as is well known to persons skilled in the art. They include, but are not limited to, kanamycin, neomycin, puromycin, hygromycin, Zeocin®, thymidine kinase gene from herpes simplex virus (HSV-TK), dihydrofolate reductase gene from mouse (dhfr). Vectors comprising one or more nucleic acid molecules encoding the human binding molecules as described herein operably linked to one or more nucleic acid molecules encoding proteins or peptides that can be used to isolate the human binding molecules are also covered by the invention. These proteins or peptides include, but are not limited to, glutathione-S-transferase, maltose binding protein, metal-binding polyhistidine, green fluorescent protein, luciferase and beta-galactosidase.
Hosts containing one or more copies of the vectors mentioned above are an additional subject of the invention. Preferably, the hosts are host cells. Host cells include, but are not limited to, cells of mammalian, plant, insect, fungal or bacterial origin. Bacterial cells include, but are not limited to, cells from Gram-positive bacteria such as several species of the genera Bacillus, Streptomyces and Staphylococcus or cells of Gram-negative bacteria such as several species of the genera Escherichia, such as E. coli, and Pseudomonas. In the group of fungal cells preferably yeast cells are used. Expression in yeast can be achieved by using yeast strains such as inter alia, Pichia pastoris, Saccharomyces cerevisiae and Hansenula polymorpha. Furthermore, insect cells such as cells from Drosophila and Sf9 can be used as host cells. Besides that, the host cells can be plant cells such as inter alia cells from crop plants such as forestry plants, or cells from plants providing food and raw materials such as cereal plants, or medicinal plants, or cells from ornamentals, or cells from flower bulb crops. Transformed (transgenic) plants or plant cells are produced by known methods, for example, Agrobacterium-mediated gene transfer, transformation of leaf discs, protoplast transformation by polyethylene glycol-induced DNA transfer, electroporation, sonication, microinjection or bolistic gene transfer. Additionally, a suitable expression system can be a baculovirus system. Expression systems using mammalian cells such as Chinese Hamster Ovary (CHO) cells, COS cells, BHK cells or Bowes melanoma cells are preferred herein. Mammalian cells provide expressed proteins with posttranslational modifications that are most similar to natural molecules of mammalian origin. Since the invention deals with molecules that may have to be administered to humans, a completely human expression system would be particularly preferred. Therefore, even more preferably, the host cells are human cells. Examples of human cells are inter alia HeLa, 911, AT1080, A549, 293 and HEK293T cells. In preferred embodiments, the human producer cells comprise at least a functional part of a nucleic acid molecule encoding an adenovirus E1 region in expressible format. In even more preferred embodiments, the host cells are derived from a human retina and immortalized with nucleic acids comprising adenoviral E1 sequences, such as 911 cells or the cell line deposited at the European Collection of Cell Cultures (ECACC), CAMR, Salisbury, Wiltshire SP4 OJG, Great Britain on 29 Feb. 1996 under number 96022940 and marketed under the trademark PER.C6® (PER.C6® is a registered trademark of Crucell Holland B.V.). For the purposes of this application “PER.C6®” refers to cells deposited under number 96022940 or ancestors, passages up-stream or downstream as well as descendants from ancestors of deposited cells, as well as derivatives of any of the foregoing. Production of recombinant proteins in host cells can be performed according to methods well known in the art. The use of the cells marketed under the trademark PER.C6® as a production platform for proteins of interest has been described in WO 00/63403 the disclosure of which is incorporated herein by reference.
A method of producing a binding molecule or an immunoconjugate hereof is an additional part hereof. The method comprises the steps of a) culturing a host according to the invention under conditions conducive to the expression of the binding molecule or immunoconjugate, and b) optionally, recovering the expressed binding molecule or immunoconjugate. The expressed binding molecules or immunoconjugates can be recovered from the cell free extract, but preferably they are recovered from the culture medium. This method of producing can also be used to make functional variants of the binding molecules and immunoconjugates hereof. Methods to recover proteins, such as binding molecules, from cell free extracts or culture medium are well known to the man skilled in the art. Binding molecules or immunoconjugates as obtainable by the aforementioned method are also a part hereof.
Alternatively, next to the expression in hosts, such as host cells, the binding molecules and immunoconjugates hereof can be produced synthetically by conventional peptide synthesizers or in cell-free translation systems using RNA nucleic acid derived from DNA molecules hereof. Binding molecules and immunoconjugates as obtainable by the aforementioned synthetic production methods or cell-free translation systems are also a part hereof.
In yet another embodiment, binding molecules can also be produced in transgenic, non-human, mammals such as inter alia rabbits, goats or cows, and secreted into for instance the milk thereof.
In yet another alternative embodiment, binding molecules, preferably human binding molecules specifically binding to the intracellular host cell protein or fragment thereof, may be generated by transgenic non-human mammals, such as, e.g., transgenic mice or rabbits that express human immunoglobulin genes. Such transgenic non-human mammals may have a genome comprising a human heavy chain transgene and a human light chain transgene encoding all or a portion of the human binding molecules as described herein. The transgenic non-human mammals can be immunized with a purified or enriched preparation of the intracellular host cell protein or fragment thereof. Protocols for immunizing non-human mammals are well established in the art. See Using Antibodies: A Laboratory Manual, Edited by: E. Harlow, D. Lane (1998), Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Current Protocols in Immunology, edited by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober (2001), John Wiley & Sons Inc., New York, the disclosures of which are incorporated herein by reference. Immunization protocols often include multiple immunizations, either with or without adjuvants such as Freund's complete adjuvant and Freund's incomplete adjuvant, but may also include naked DNA immunizations. In another embodiment, the human binding molecules are produced by B-cells or plasma cells derived from the transgenic animals. In yet another embodiment, the human binding molecules are produced by hybridomas, which are prepared by fusion of B-cells obtained from the aforementioned transgenic non-human mammals to immortalized cells. B-cells, plasma cells and hybridomas as obtainable from the above described transgenic non-human mammals and human binding molecules as obtainable from the aforementioned transgenic non-human mammals, B-cells, plasma cells and hybridomas are also a part hereof.
The intracellular host cell proteins FAF-1 and/or NDUFV-1 may also be produced and purified according to the methods and with the vectors and host cells as described herein. Alternatively, they may also be produced and/or purified from a “native” host cell, e.g., a host cell normally expressing the intracellular host cell protein. Expression in a “native” host cell may be increased by substituting the natural promoter of proteins and/or by adding, substituting and/or mutating expression enhancing elements of proteins. The elements and methods to increase expression are known to the skilled artisan.
In a further aspect, provided is a method of identifying binding molecules hereof such as human binding molecules, e.g., monoclonal antibodies or fragments thereof, specifically binding to a host cell protein or nucleic acid molecules encoding such binding molecules and comprises the steps of a) contacting a collection of binding molecules on the surface of replicable genetic packages with a virus, virus-like particle or fragment thereof under conditions conducive to binding, b) selecting at least once for a replicable genetic package binding to the virus, virus-like particle or fragment thereof, and c) separating and recovering the replicable genetic package binding to the virus, virus-like particle or fragment thereof from replicable genetic packages that do not bind. The host cell protein may be an intracellular host cell protein.
A replicable genetic package as used herein can be prokaryotic or eukaryotic and includes cells, spores, yeasts, bacteria, viruses, (bacterio)phage, ribosomes and polysomes. A preferred replicable genetic package is a phage. The binding molecules, such as for instance single chain Fvs, are displayed on the replicable genetic package, i.e., they are attached to a group or molecule located at an exterior surface of the replicable genetic package. The replicable genetic package is a screenable unit comprising a binding molecule to be screened linked to a nucleic acid molecule encoding the binding molecule. The nucleic acid molecule should be replicable either in vivo (e.g., as a vector) or in vitro (e.g., by PCR, transcription and translation). In vivo replication can be autonomous (as for a cell), with the assistance of host factors (as for a virus) or with the assistance of both host and helper virus (as for a phagemid). Replicable genetic packages displaying a collection of binding molecules is formed by introducing nucleic acid molecules encoding exogenous binding molecules to be displayed into the genomes of the replicable genetic packages to form fusion proteins with endogenous proteins that are normally expressed from the outer surface of the replicable genetic packages. Expression of the fusion proteins, transport to the outer surface and assembly results in display of exogenous binding molecules from the outer surface of the replicable genetic packages.
In one embodiment, the selection step in a method according to the invention is performed in the presence of virus that is inactivated. The inactivation of the virus may be performed by viral inactivation methods well known to the skilled artisan. Methods to test if a virus is still infective or partly or completely inactivated are well known to the person skilled in the art. The virus used in the above method may be non-isolated, e.g., present in serum and/or blood of an infected individual. The virus used may also be isolated either before or after inactivation. Purification may be performed by means of, e.g., well known purification methods suitable for viruses such as for instance centrifugation through a glycerol cushion. The virus contains the (intracellular) host cell protein incorporated into its viral membrane.
Alternatively, the selection step may be performed in the presence of a fragment of the virus such as a fragment comprising the viral membrane or virus-like particles having the (intracellular) host cell protein incorporated into their membranes. The inactivated virus, virus-like particle or fragment thereof may be immobilized to a suitable material before use. In a specific embodiment, the selection can be performed on different materials comprising the (intracellular) host cell proteins. For instance, the first selection round can be performed on (inactivated) virus, while the second selection round can be performed on WNV-like particles. Alternatively, the first selection round can be performed on host cells displaying the (intracellular) host cell protein on their surface, while the second selection round can be performed on (inactivated) virus. Of course, other combinations are also suitable. For instance, the first and second selection round can be performed on identical material such as WNV-like particles. Different materials comprising the (intracellular) host cell protein can also be used during one selection/panning step.
In yet a further aspect, provided is a method of obtaining a binding molecule specifically binding to a host cell protein or a nucleic acid molecule encoding such a binding molecule specifically binding to a host cell protein, wherein the method comprises the steps of a) performing the above described method of identifying binding molecules, and b) isolating from the recovered replicable genetic package the binding molecule and/or the nucleic acid molecule encoding the binding molecule. The collection of binding molecules on the surface of replicable genetic packages can be a collection of scFvs or Fabs. Once a new scFv or Fab has been established or identified with the above mentioned method of identifying binding molecules or nucleic acid molecules encoding the binding molecules, the DNA encoding the scFv or Fab can be isolated from the bacteria or phages and combined with standard molecular biological techniques to make constructs encoding bivalent scFvs or complete human immunoglobulins of a desired specificity (e.g., IgG, IgA or IgM). These constructs can be transfected into suitable cell lines and complete human monoclonal antibodies can be produced (see Huls et al., 1999; Boel et al., 2000).
As mentioned before, the preferred replicable genetic package is a phage. Phage display methods for identifying and obtaining (human) binding molecules, e.g., monoclonal antibodies, are by now well-established methods known by the person skilled in the art. They are, e.g., described in U.S. Pat. No. 5,696,108; Burton and Barbas, 1994; de Kruif et al., 1995b; and Phage Display: A Laboratory Manual, edited by C. F. Barbas, D R Burton, J. K. Scott and G. J. Silverman (2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. All of these references are herewith incorporated herein in their entirety. For the construction of phage display libraries, collections of human monoclonal antibody heavy and light chain variable region genes are expressed on the surface of bacteriophage, preferably filamentous bacteriophage, particles, in for example single chain Fv (scFv) or in Fab format (see de Kruif et al., 1995b). Large libraries of antibody fragment expressing phages typically contain more than 1.0*109 antibody specificities and may be assembled from the immunoglobulin V-regions expressed in the B-lymphocytes of immunized or non-immunized individuals. In a specific embodiment, the phage library of binding molecules, e.g., scFv phage library, is prepared from RNA isolated from cells obtained from a subject that has been vaccinated or exposed to a virus. RNA can be isolated from inter alia bone marrow or peripheral blood, preferably peripheral blood lymphocytes. The subject can be an animal vaccinated or exposed to a virus, but is preferably a human who has been vaccinated or has been exposed to a virus. Preferably the human subject has recovered from the viral infection. In an embodiment hereof, the virus is a flavivirus such as WNV.
Alternatively, phage display libraries may be constructed from immunoglobulin variable regions that have been partially assembled in vitro to introduce additional antibody diversity in the library (semi-synthetic libraries). For example, in vitro assembled variable regions contain stretches of synthetically produced, randomized or partially randomized DNA in those regions of the molecules that are important for antibody specificity, e.g., CDR regions. Host cell protein specific phage antibodies can be selected from the library by for instance immobilizing target antigens such as viruses, virus-like particles, host cells or host cell proteins, e.g., intracellular host cell proteins, on a solid phase and subsequently exposing the target antigens to a phage library to allow binding of phages expressing antibody fragments specific for the solid phase-bound antigen(s). Unbound phages are removed by washing and bound phages eluted from the solid phase for infection of E. coli bacteria and subsequent propagation. Multiple rounds of selection and propagation are usually required to sufficiently enrich for phages binding specifically to the target antigen(s). If desired, before exposing the phage library to target antigens the phage library can first be subtracted by exposing the phage library to non-target antigens bound to a solid phase. Phages may also be selected for binding to complex antigens such as complex mixtures of host cell proteins or peptide or polypeptides, (expression) host cells expressing one or more host cell proteins or peptide or polypeptides, virus-like particles comprising host cell proteins, or whole inactivated viruses. Antigen specific phage antibodies can be selected from the library by incubating a solid phase with bound thereon virus-like particles with the phage antibody library to let for example the scFv or Fab part of the phage bind to the virus-like particle. Of course, in an alternative embodiment, selections and subtractions may also be performed in solution. After incubation and several washes to remove unbound and loosely attached phages, the phages that have bound with their scFv or Fab part to the virus-like particle are eluted and used to infect E. coli to allow amplification of the new specificity. Generally, one or more selection rounds are required to separate the phages of interest from the large excess of non-binding phages. Alternatively, the host cell proteins can be expressed in expression host cells, e.g., PER.C6® cells, and these cells can be used for selection of phage antibodies specific for the proteins or peptide or polypeptides. A phage display method using (expression) host cells can be extended and improved by subtracting irrelevant binders during screening by addition of an excess of host cells comprising no target molecules or non-target molecules that are similar, but not identical, to the target, and thereby strongly enhance the chance of finding relevant binding molecules. Of course, the subtraction may also be performed before or after the screening with material comprising the host cell protein or fragment thereof. In an embodiment, the selection can take place on host cells that have been infected with a virus or a virus-like particle. These cells display the normally intracellular located host cell protein after infection on their surface. Subtraction may be performed before, during, or after the selection and may be performed with host cells that have not been infected and thus comprise the intracellular host cell protein in its original cellular location, i.e., inside the host cell. The subtraction process is referred to as the Mabstract® process (MABSTRACT® is a registered trademark of Crucell Holland B.V., see also U.S. Pat. No. 6,265,150, which is incorporated herein by this reference).
In yet another aspect, provided is a method of obtaining a binding molecule potentially having neutralizing activity against a virus and being capable of specifically binding to a host cell protein, wherein the method comprises the steps of a) performing the method of obtaining a binding molecule specifically binding to the host cell protein or a nucleic acid molecule encoding such a binding molecule specifically binding to the host cell protein as described herein, and b) verifying if the binding molecule isolated has neutralizing activity against a virus. Preferably, the host cell protein is an intracellular host cell protein. In an embodiment, the methods described herein are performed with a flavivirus such as WNV. In one embodiment of the method, it is verified if the binding molecule isolated has neutralizing activity against viruses of at least two different genera. More preferably, it is verified if the binding molecules are broadly neutralizing binding molecules and are able to neutralize many different genera. Assays for verifying if a binding molecule has neutralizing activity for a specific virus are well known in the art.
Also described is a host cell protein specific binding molecule obtainable by the methods as described herein. Preferably, the binding molecule has neutralizing activity against at least one virus.
In yet a further aspect, provided are compositions comprising at least one binding molecule, at least one functional variant thereof, at least one immunoconjugate hereof or a combination thereof. Also included are compositions comprising at least two binding molecules, preferably human binding molecules, at least one of which may be a binding molecule hereof. Preferably, both binding molecules have virus neutralizing activity. The activity may be against the same virus, but also against different viruses. In an embodiment, the compositions comprising two or more binding molecules having virus neutralizing activity exhibit synergistic virus neutralizing activity. In an embodiment, the binding molecules acting synergistically in neutralizing one virus may also be capable of neutralizing other viruses synergistically. As used herein, the term “synergistic” means that the combined effect of the binding molecules when used in combination is greater than their additive effects when used individually. A way of calculating synergy is by means of the combination index. The concept of the combination index (CI) has been described by Chou and Talalay, 1984. In an alternative embodiment, the compositions comprise two or more binding molecules having different modes of action. In an embodiment, the binding molecules hereof have flavivirus-, e.g., WNV-, neutralizing activity. In addition to that, the compositions may comprise inter alia stabilizing molecules, such as albumin or polyethylene glycol, or salts. Preferably, the salts used are salts that retain the desired biological activity of the binding molecules and do not impart any undesired toxicological effects. If necessary, the human binding molecules hereof may be coated in or on a material to protect them from the action of acids or other natural or unnatural conditions that may inactivate the binding molecules.
In yet a further aspect, compositions are provided comprising at least one nucleic acid molecule as defined herein. The compositions may comprise aqueous solutions such as aqueous solutions containing salts (e.g., NaCl or salts as described herein), detergents (e.g., SDS), and/or other suitable components.
Furthermore, provided are pharmaceutical compositions comprising at least one binding molecule (or functional fragment or variant thereof), at least one immunoconjugate according to the invention, at least one composition according to the invention, or combinations thereof. The pharmaceutical composition hereof further typically comprises at least one pharmaceutically acceptable excipient. A pharmaceutical composition hereof can further comprise at least one other therapeutic, prophylactic and/or diagnostic agent. Preferably, the pharmaceutical composition comprises at least one other prophylactic and/or therapeutic agent. Preferably, the further therapeutic and/or prophylactic agents are agents capable of preventing and/or treating an infection and/or a condition resulting from a virus, e.g., a flavivirus such as WNV. Therapeutic and/or prophylactic agents include, but are not limited to, anti-viral agents. Such agents can be binding molecules, small molecules, organic or inorganic compounds, enzymes, polynucleotides etc. Agents capable of preventing and/or treating a viral infection and/or a condition resulting from a virus that are in the experimental phase might also be used as other therapeutic and/or prophylactic agents useful herein.
The binding molecules or pharmaceutical compositions hereof can be tested in suitable animal model systems prior to use in humans. Such animal model systems include, but are not limited to, a murine model, a hamster model, and a geese model system.
Typically, pharmaceutical compositions must be sterile and stable under the conditions of manufacture and storage. The binding molecules, immunoconjugates, nucleic acid molecules or compositions hereof can be in powder form for reconstitution in the appropriate pharmaceutically acceptable excipient before or at the time of delivery. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Alternatively, the binding molecules, immunoconjugates, nucleic acid molecules or compositions hereof can be in solution and the appropriate pharmaceutically acceptable excipient can be added and/or mixed before or at the time of delivery to provide a unit dosage injectable form. Preferably, the pharmaceutically acceptable excipient used herein is suitable to high drug concentration, can maintain proper fluidity and, if necessary, can delay absorption.
The choice of the optimal route of administration of the pharmaceutical compositions will be influenced by several factors including the physico-chemical properties of the active molecules within the compositions, the urgency of the clinical situation and the relationship of the plasma concentrations of the active molecules to the desired therapeutic effect. For instance, if necessary, the binding molecules hereof can be prepared with carriers that will protect them against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can inter alia be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Furthermore, it may be necessary to coat the binding molecules with, or co-administer the binding molecules with, a material or compound that prevents the inactivation of the human binding molecules. For example, the binding molecules may be administered to a subject in an appropriate carrier, for example, liposomes or a diluent.
The routes of administration can be divided into two main categories, oral and parenteral administration. The preferred administration route is intravenous.
Oral dosage forms can be formulated inter alia as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard capsules, soft gelatin capsules, syrups or elixirs, pills, dragees, liquids, gels, or slurries. These formulations can contain pharmaceutically excipients including, but not limited to, inert diluents, granulating and disintegrating agents, binding agents, lubricating agents, preservatives, coloring, flavoring or sweetening agents, vegetable or mineral oils, wetting agents, and thickening agents.
The pharmaceutical compositions hereof can also be formulated for parenteral administration. Formulations for parenteral administration can be inter alia in the form of aqueous or non-aqueous isotonic sterile non-toxic injection or infusion solutions or suspensions. The solutions or suspensions may comprise agents that are non-toxic to recipients at the dosages and concentrations employed such as 1,3-butanediol, Ringer's solution, Hank's solution, isotonic sodium chloride solution, oils, fatty acids, local anesthetic agents, preservatives, buffers, viscosity or solubility increasing agents, water-soluble antioxidants, oil-soluble antioxidants, and metal chelating agents.
In a further aspect, the binding molecules (functional fragments and variants thereof), immunoconjugates, compositions, or pharmaceutical compositions hereof can be used as a medicament. So, a method of treatment and/or prevention of a viral infection using the binding molecules, immunoconjugates, compositions, or pharmaceutical compositions hereof is another part hereof. The above-mentioned molecules can inter alia be used in the diagnosis, prophylaxis, treatment, or combination thereof, of one or more conditions resulting from a virus or viral infection. Preferably, they are broadly applicable to prevent, detect and/or treat several viral infections caused by viruses of different genera and/or strains, including, but not limited to, herpes viridae (e.g., Herpes Simplex Virus type 1, 2, 6, 7, 8, cytomegalovirus, varicella-zoster virus, Epstein Barr virus), Poxviridae (e.g., Smallpox virus), Retroviridae (e.g., HIV 1, HIV2, HTLV1, HTLV2), paramyxoviridae (e.g., mumps virus, measles virus, respiratory syncytial virus, parainfluenza virus), HepaDNAviridae (e.g., Hepatitis B virus), Orthomyxoviridae (e.g., Influenza virus A or B), Togaviridae (e.g., Rubella), Flaviviridae (e.g., Yellow fever virus, Dengue virus, West Nile Virus, Hepatitis C virus, Japanese encephalitis virus, Venezuela encephalitis virus) rhabodiviridae (e.g., rabies virus), Arenaviridae (e.g., Lassa virus, lymphocytic choriomeningitis virus) and Coronaviridae (e.g., SARS virus, metapneumoniae virus). Moreover, they may also be applicable to prevent, detect and/or treat several viral infections caused by viruses not mentioned above including, but not limited to, Sindbis virus, poliovirus, human papilloma virus, adeno-associated virus, coxsackivirus, enterovirus, hepatitis A virus, tick-borne encephalitis virus, astrovirus, Ebola virus, Marburg virus, La Crosse virus, California encephalitis virus, Hantaan virus, Crimean-Congo virus, Rift Valley fever, Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, Colorado tick fever, JC virus, BK virus, human adenovirus, and human parvovirus. In other words, the binding molecules hereof are broad spectrum anti-viral binding molecules. They are suitable for treatment of yet untreated patients suffering from a condition resulting from a virus and patients who have been or are treated from a condition resulting from a virus or a viral infection. They protect against further infection by a virus for approximately one month and/or will retard the onset or progress of the symptoms associated with a virus. They may also be used in post-exposure prophylaxis, when there is a chance of infection but symptoms are absent. They may also be used as prophylaxis in the transplant of infected organs or in other patient populations at high risk of exposure and progression to disease due to inter alia age or immune status. In a specific embodiment, the compounds and compositions are used to treat and/or prevent a flavivirus, e.g., WNV, infection and/or a rabies virus infection.
The above-mentioned molecules or compositions may be employed in conjunction with other molecules useful in diagnosis, prophylaxis and/or treatment. They can be used in vitro, ex vivo or in vivo. For instance, the binding molecules, immunoconjugates or pharmaceutical compositions hereof can be co-administered with a vaccine against the intracellular host cell proteins. Alternatively, the vaccine may also be administered before or after administration of the molecules of the invention. Instead of a vaccine, other anti-viral agents can also be employed in conjunction with the binding molecules of the invention. In an aspect the invention is also directed to the use of an intracellular host cell protein, preferably FAF-1 and/or NDUFV-1, as a vaccine suitable for preventing and/or treating a viral infection, e.g., a flavivirus infection such as WNV infection.
The molecules are typically formulated in the compositions and pharmaceutical compositions hereof in a therapeutically or diagnostically effective amount. Alternatively, they may be formulated and administered separately. For instance the other molecules such as the anti-viral compounds may be applied systemically, while the binding molecules hereof may be applied intrathecally, intraventricularly or intradermally.
Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic response). A suitable dosage range may for instance be 0.1-100 mg/kg body weight, preferably 0.5-15 mg/kg body weight. Furthermore, for example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. The molecules and compositions hereof are preferably sterile. Methods to render these molecules and compositions sterile are well known in the art. The other molecules useful in diagnosis, prophylaxis and/or treatment can be administered in a similar dosage regimen as proposed for the binding molecules hereof. If the other molecules are administered separately, they may be administered to a patient prior to (e.g., 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 7 days, 2 weeks, 4 weeks or 6 weeks before), concomitantly with, or subsequent to (e.g., 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 7 days, 2 weeks, 4 weeks or 6 weeks after) the administration of one or more of the human binding molecules or pharmaceutical compositions hereof. The exact dosing regimen is usually sorted out during clinical trials in human patients.
Human binding molecules and pharmaceutical compositions comprising them are particularly useful, and often preferred, for administration to human beings as in vivo therapeutic agents, since recipient immune response to the administered antibody will often be substantially less than that occasioned by administration of a monoclonal murine, chimeric or humanized binding molecule.
Also described is the use of the human binding molecules or binding molecules (functional fragments and variants thereof), immunoconjugates, nucleic acid molecules, compositions or pharmaceutical compositions described herein in the preparation of a medicament for the diagnosis, prophylaxis, treatment, or combination thereof, of a viral infection and/or a condition resulting thereof.
Next to that, kits comprising at least one binding molecule (functional fragments and variants 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 according to the invention or a combination thereof are also a part hereof. Optionally, the aforementioned components of the kits hereof are packed in suitable containers and labeled for diagnosis, prophylaxis and/or treatment of the indicated conditions. The above-mentioned components may be stored in unit or multi-dose containers as an aqueous, preferably sterile, solution or as a lyophilized, preferably sterile, formulation for reconstitution. The containers may be formed from a variety of materials such as glass or plastic and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The kit may further comprise more containers comprising a pharmaceutically acceptable buffer. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, culture medium for one or more of the suitable hosts and, possibly, even at least one other therapeutic, prophylactic or diagnostic agent. Associated with the kits can be instructions customarily included in commercial packages of therapeutic, prophylactic or diagnostic products, that contain information about for example the indications, usage, dosage, manufacture, administration, contraindications and/or warnings concerning the use of such therapeutic, prophylactic or diagnostic products.
Further described is a method of detecting an intracellular host cell protein such as FAF-1 and/or NDUFV-1 in a sample, wherein the method comprises the steps of a) contacting a sample with a diagnostically effective amount of a binding molecule (functional fragments and variants thereof) or an immunoconjugate described herein, and b) determining whether the binding molecule or immunoconjugate specifically binds to a molecule of the sample. The sample may be a biological sample including, but not limited to blood, serum, urine, tissue or other biological material from (potentially) infected subjects, or a non-biological sample such as water, drink, etc. The (potentially) infected subjects may be human subjects, but also animals that are suspected as carriers of a virus might be tested for the presence of the virus using the human binding molecules or immunoconjugates hereof. By detecting the intracellular host cell proteins a viral infection (infected host cell and/or virus particle) may be detected. The sample may first be manipulated to make it more suitable for the method of detection. Manipulation means inter alia treating the sample suspected to contain and/or containing a virus in such a way that the virus will disintegrate into antigenic components such as proteins, peptide or polypeptides or other antigenic fragments. Preferably, the human binding molecules or immunoconjugates hereof are contacted with the sample under conditions which allow the formation of an immunological complex between the human binding molecules and intracellular host cell protein or antigenic components thereof that may be present in the sample. The formation of an immunological complex, if any, indicating the presence of a virus in the sample, is then detected and measured by suitable means. Such methods include, inter alia, homogeneous and heterogeneous binding immunoassays, such as radio-immunoassays (RIA), ELISA, immunofluorescence, immunohistochemistry, FACS, BIACORE and Western blot analyses.
Preferred assay techniques, especially for large-scale clinical screening of patient sera and blood and blood-derived products are ELISA and Western blot techniques. ELISA tests are particularly preferred. For use as reagents in these assays, the binding molecules or immunoconjugates hereof are conveniently bonded to the inside surface of microtiter wells. The binding molecules or immunoconjugates hereof may be directly bonded to the microtiter well. However, maximum binding of the binding molecules or immunoconjugates hereof to the wells might be accomplished by pre-treating the wells with poly-lysine prior to the addition of the binding molecules or immunoconjugates hereof. Furthermore, the binding molecules or immunoconjugates hereof may be covalently attached by known means to the wells. Generally, the binding molecules or immunoconjugates are used between 0.01 to 100 μg/ml for coating, although higher as well as lower amounts may also be used. Samples are then added to the wells coated with the binding molecules or immunoconjugates hereof.
Furthermore, binding molecules hereof can be used to identify epitopes of the intracellular host cell proteins such as FAF-1 and/or NDUFV-1. The epitopes can be linear, but also structural and/or conformational. In one embodiment, binding of binding molecules hereof to a series of overlapping peptides, such as 15-mer peptides, of the host cell protein can be analyzed by means of PEPSCAN analysis (see inter alia WO 84/03564, WO 93/09872, Slootstra et al., 1996). The binding of the molecules to each peptide can be tested in a PEPSCAN-based enzyme-linked immunoassay (ELISA). In another embodiment, a random peptide library comprising peptides from the host cell proteins can be screened for peptides able to bind to the binding molecules hereof. In the above assays the use of neutralizing binding molecules may identify one or more neutralizing epitopes. The peptides/epitopes found can be used as vaccines and for the diagnosis of a viral infection.
In a further aspect, provided is a method of screening a binding molecule (or a functional fragment or variant thereof) for specific binding to the same epitope of the intracellular host cell protein, such as FAF-1 and/or NDUFV-1, as the epitope bound by a human binding molecule hereof, wherein the method comprises the steps of a) contacting a binding molecule to be screened, a binding molecule hereof and material comprising the intracellular host cell protein or antigenic fragment thereof, b) measure if the binding molecule to be screened is capable of competing for specifically binding to the intracellular host cell protein comprising material or antigenic fragment thereof with the binding molecule of the invention. The material comprising the intracellular host cell protein or antigenic fragment thereof may be a host cell displaying the protein on its surface, a virus having the protein incorporated into its viral membrane, a virus-like particle having the protein incorporated into the viral membrane or the protein itself in purified or non-purified form. In a further step it may be determined, if the screened binding molecules that are capable of competing for specifically binding to the intracellular host cell protein or antigenic fragment thereof have neutralizing activity. A binding molecule that is capable of competing for specifically binding to the intracellular host cell protein or an antigenic fragment thereof with the binding molecule of the invention is another part hereof. In the aforementioned screening method, “specifically binding to the same epitope” also contemplates specific binding to substantially or essentially the same epitope as the epitope bound by the a binding molecule of the invention. The capacity to block, or compete with, the binding of the binding molecules hereof to the intracellular host cell protein typically indicates that a binding molecule to be screened binds to an epitope or binding site on the intracellular host cell protein that structurally overlaps with the binding site on the intracellular host cell protein that is immunospecifically recognized by the binding molecules hereof. Alternatively, this can indicate that a binding molecule to be screened binds to an epitope or binding site that is sufficiently proximal to the binding site immunospecifically recognized by the binding molecules hereof to sterically or otherwise inhibit binding of the binding molecules hereof to the intracellular host cell protein.
In general, competitive inhibition is measured by means of an assay, wherein an antigen composition, i.e., a composition comprising the intracellular host cell protein or antigenic fragments thereof, is admixed with reference binding molecules, i.e., the binding molecules hereof, and binding molecules to be screened. Usually, the binding molecules to be screened are present in excess. Protocols based upon ELISAs and Western blotting are suitable for use in such simple competition studies. In certain embodiments, one may pre-mix the reference binding molecules with varying amounts of the binding molecules to be screened (e.g., 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90 or 1:100) for a period of time prior to applying to the antigen composition. In other embodiments, the reference binding molecules and varying amounts of binding molecules to be screened can simply be admixed during exposure to the antigen composition. In yet another embodiment, the reference binding molecules or binding molecules to be screened are contacted before the binding molecules to be screened or reference binding molecules, respectively, are contacted with the intracellular host cell protein or antigenic fragment thereof. In any event, by using species or isotype secondary antibodies one will be able to detect only the bound reference binding molecules, the binding of which will be reduced by the presence of a binding molecule to be screened that recognizes substantially the same epitope. In conducting a binding molecule competition study between a reference binding molecule and any binding molecule to be screened (irrespective of species or isotype), one may first label the reference binding molecule with a detectable label, such as, e.g., biotin, an enzymatic, a radioactive or other label to enable subsequent identification. In these cases, one would pre-mix or incubate the labeled reference binding molecules with the binding molecules to be screened at various ratios (e.g., 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90 or 1:100) and (optionally after a suitable period of time) then assay the reactivity of the labeled reference binding molecules and compare this with a control value in which no potentially competing binding molecule was included in the incubation. The assay may again be any one of a range of immunological assays based upon antibody hybridization, and the reference binding molecules would be detected by means of detecting their label, e.g., using streptavidin in the case of biotinylated reference binding molecules or by using a chromogenic substrate in connection with an enzymatic label (such as 3,3′5,5′-tetramethylbenzidine (TMB) substrate with peroxidase enzyme) or by simply detecting a radioactive label. A binding molecule to be screened that binds to the same epitope as the reference binding molecule will be able to effectively compete for binding and thus will significantly reduce reference binding molecule binding, as evidenced by a reduction in bound label. The reactivity of the (labeled) reference binding molecule in the absence of a completely irrelevant binding molecule would be the control high value. The control low value would be obtained by incubating the labeled reference binding molecule with unlabelled reference binding molecules of exactly the same type, when competition would occur and reduce binding of the labeled reference binding molecule. In a test assay, a significant reduction in labeled reference binding molecule reactivity in the presence of a binding molecule to be screened is indicative of a binding molecule that recognizes the same epitope, i.e., one that “cross-reacts” with the labeled reference binding molecule.
Binding molecules identified by these competition assays (“competitive binding molecules” or “cross-reactive 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, i.e., a binding molecule described herein, as well as antibodies, antibody fragments and other binding agents that bind to an epitope or binding site sufficiently proximal to an epitope bound by the reference binding molecule for competitive binding between the binding molecules to be screened and the reference binding molecule to occur. Preferably, competitive binding molecules hereof will, when present in excess, inhibit specific binding of a reference binding molecule to a selected target species by at least 10%, preferably by at least 25%, more preferably by at least 50%, and most preferably by at least 75%-90% or even greater. The identification of one or more competitive binding molecules that bind to about, substantially, essentially or at the same epitope as the binding molecules hereof is a straightforward technical matter. As the identification of competitive binding molecules is determined in comparison to a reference binding molecule, i.e., a binding molecule of the invention, it will be understood that actually determining the epitope to which the reference binding molecule and the competitive binding molecule bind is not required in order to identify a competitive binding molecule that binds to the same or substantially the same epitope as the reference binding molecule.
In a further aspect, disclosed are new leader peptide sequences (also called signal sequences or presequences) and nucleotides encoding the leader peptides. The leader peptides are useful in methods for producing recombinant proteins, e.g., immunoglobulin molecules, from a host cell. The entry of almost all secreted polypeptides to the secretory pathway, in both prokaryotes and eukaryotes, is directed by specific leader peptides at the N terminus of the polypeptide chain. Leader peptides direct nascent polypeptides towards the machinery of the cell that exports polypeptides from the cell into the surrounding medium or, in some cases, into the periplasmic space. The mechanisms by which leader peptides direct nascent polypeptide chains to the secretion pathway and direct the precise and efficient proteolytic cleavage to release mature proteins are incompletely understood. The leader peptide is usually, although not necessarily, located at the N terminus of the primary translation product and is generally, although not necessarily, cleaved off the desired polypeptide during the secretion process, to yield the “mature” polypeptide. The secretion of polypeptides is a dynamic and multi-step process involving several elements of the cellular secretory apparatus and specific sequence elements in the signal peptide. It further involves translation, translocation and post-translational processing, and one or more of these steps may not necessarily be completed before another is either initiated or completed. Signal sequences are indispensible for production of phage antibodies, in which antibody fragments such as scFv or Fab are fused to the phage coat proteins such as pIII or pVIII.
Signal sequences are predominantly hydrophobic in nature, a feature which may be important in directing the nascent peptide to the membrane and transfer of secretory proteins across the inner membrane of prokaryotes or the endoplasmic reticulum membranes of eukaryotes. In mammalian cells, leader peptides are recognized by the 54K protein of the signal recognition particle (SRP), which is believed to hold the nascent chain in a translocation competent conformation until it contacts the endoplasmic reticulum membrane. The SRP consists of a 7S RNA and six different polypeptides. The 7S RNA and the 54K leader peptide-binding protein (SRP54) of mammalian SRP exhibit strong sequence similarity to the 4.5S RNA and P48 protein (Ffh) of Escherichia coli, which forms the signal recognition particle in bacteria. It is likely that the various sequences found in different signal peptides interact in unique ways with the secretion apparatus. There are no eukaryotic signal sequences available that result in correct protein processing and high protein expression in bacteria or vice versa.
The antibody phage format or scFv fragment format is often not considered a suitable format in assays of functionalities other than binding, e.g., assays for testing neutralizing activity where bivalent binding is required or where functional Fc regions are required. In for instance assays testing neutralizing activity data obtained with phage antibodies or scFv fragments derived from phage antibodies are not representative of the neutralizing activity of the complete immunoglobulin molecules. Therefore, complete immunoglobulin molecules, e.g., IgG1, are needed to test particular specificities for functional activity. To increase the efficiency by which scFv molecules can be reformatted into IgG1 a new vector system making use of new leader peptides was designed. The new vectors and leader peptides were designed to enable direct shuttling of immunoglobulin heavy chain regions (VH region) and immunoglobulin light chain regions (VL region) from bacterial expression vectors, such as phage display vectors, e.g., PDV, pHEN and vectors derived thereof, into a eukaryotic expression system. This approach has a significant time and cost advantage over the standard systems wherein each VH and VL region had to be PCR amplified, cloned and checked for frequent mutations that occur through the amplification process before being shuttled into eukaryotic expression vectors. In short the procedure is as follows. VH and VL fragment repertoires are cloned into prokaryotic expression vectors such as pHEN1 and PDV-C06 using specific restriction enzymes. These sites are chosen to be outside of the V region encoding sequences because there is too much sequence diversity within the V regions for efficient PCR amplification and cloning of V gene repertoires. Typically, the enzyme recognition site at the 5′-end of the VH antibody fragments is located in the coding region of the prokaryotic leader sequence such as the SfiI site in the PelB leader sequence of pHEN1 or PDV-C06. Therefore, SfiI is one of the available sites for cloning at the 5′-end that is present in all phage display derived scFv clones. When using this site for cloning, the sequence encoded by the SfiI site and the downstream sequence are transferred to the eukaryotic vector; this means that the C-terminal part of the prokaryotic leader is transferred to the eukaryotic vector. Herein, a eukaryotic leader was designed that is functional when the region encoding the C-terminal part of the prokaryotic leader sequence (e.g., PelB) is transferred into the eukaryotic vector. VL antibody fragment repertoires are cloned using a Sail site that is located within the scFv linker sequence. When this site is used for cloning, the sequence encoded by the SalI site is transferred to the eukaryotic vector and will be part of the eukaryotic leader sequence. Therefore, leaders were designed that contain a sequence upstream of the SalI restriction site that together with the SalI restriction site encoded sequence and the sequence downstream of the restriction site after transcription and translation form a functional leader in eukaryotic cells. The VH region is cut from a phage display vector with restriction enzymes and cloned in frame between a “truncated” eukaryotic leader peptide and the hinge and constant domains of an immunoglobulin heavy chain present in the expression vector. In an identical way, the VL region is cloned in frame with a “truncated” eukaryotic leader peptide and the constant domain of the immunoglobulin light (either lambda or kappa) chain. The newly designed leader peptides have a sequence that is recognized by the cellular secretory apparatus and is proteolitically cleaved during secretion of the immunoglobulin chains from host cells, e.g., eukaryotic host cells, resulting in correctly processed mature Ig heavy and light chain proteins. The invention also provides an expression vector (pIG-CHG1) for production of a full length IgG1 heavy chain immunoglobulin molecule and two vectors encoding a complete light chain fragment. One vector (pIG-Ckappa) produces the kappa light chain sub-type and the other (pIG-Clambda) the lambda light chain variant. It is essential to determine beforehand what sub-type the VL region belongs to, so that the right constant light chain domain can be fused to it. Of course, the IgG1 constant domain in the vector can be replaced by an IgG2, IgG3 or IgG4 domain to produce heavy chains of the IgG2, IgG3 or IgG4 format. Other proteins that need a leader sequence in both prokaryotes and eukaryotes can also be made by means of the new leaders. For convenient and efficient cloning the pIG vectors were extended with large stuffer fragments such that double cut vectors are easily separated from linear single cut vectors.
Also disclosed is a eukaryotic leader peptide comprising an amino acid molecule selected from the group consisting of SEQ ID NO:150, SEQ ID NO:152 and SEQ ID NO:154. In a further aspect, provided is a recombinant polypeptide (e.g., recombinant protein) comprising a eukaryotic leader peptide described herein and a polypeptide of interest. The polypeptide of interest can be any recombinant protein, but preferably it is a eukaryotic protein such as a mammalian protein, or more preferably a human protein. In one embodiment, the polypeptide of interest is selected from the group consisting of an immunoglobulin heavy chain, an immunoglobulin light chain, an immunoglobulin heavy chain fragment and an immunoglobulin light chain fragment. Preferred fragments are immunoglobulin variable regions (VH or VL regions). Preferably, the immunoglobulin chains and fragments are human. In one aspect, the leader peptide is used to direct or even enhance the secretion of the recombinant polypeptide produced in a recombinant (i.e., transformed) host organism, e.g., a host cell. In an embodiment, the carboxy terminus of the eukaryotic leader peptide is joined/fused to the amino terminus of the polypeptide of interest. Preferably, the leader peptide comprising SEQ ID NO:150 is fused to an immunoglobulin heavy chain or fragment thereof. This leader peptide is the result of joining a “truncated” eukaryotic leader peptide encoded by the nucleotide sequence of SEQ ID NO:155 to a nucleotide sequence encoding an immunoglobulin heavy chain or fragment thereof, e.g., a heavy chain variable region from plasmid pHEN1 or PDV-C06. The leader peptides comprising SEQ ID NO:152 and SEQ ID NO:154 are joined to an immunoglobulin kappa and lambda light chain or fragment thereof, respectively. These leader peptides are the result of joining a “truncated” eukaryotic leader peptide encoded by the nucleotide sequence of SEQ ID NO:156 or SEQ ID NO:157 to a nucleotide sequence encoding an immunoglobulin kappa or lambda light chain or fragment thereof, e.g., a kappa or lambda light chain variable region from plasmid pHEN1 or PDV-C06. Thus, provided is a fusion polypeptide comprising a leader peptide sequence of the invention and a recombinant protein sequence. The fusion polypeptide may be designed such that there are additional amino acids present between the leader peptide and the recombinant protein. In these instances, cleavage of the leader peptide from the fusion polypeptide may produce a modified recombinant protein having additional amino acids at the N terminus. Alternatively, the fusion polypeptide may be designed such that the site for cleavage of the leader peptide occurs a few amino acids into the sequence of the recombinant protein. In these instances, a modified recombinant protein may be produced which has an altered N terminus.
In another aspect, provided is a nucleic acid molecule encoding a eukaryotic leader peptide described herein. The eukaryotic leader peptides according to the invention comprise nucleotides GGCCCAGCCGGCC (SEQ ID NO:158), i.e., a SfiI-site, or nucleotides TCGAC (SEQ ID NO:159), i.e., a combined XhoI/SalI-site, within the nucleic acid molecule encoding the leader peptides. The combined XhoI/SalI-site is made by ligating the XhoI-site in a prokaryotic leader into a eukaryotic vector that has been digested with the restriction enzyme SalI. The sites may be located within the carboxy terminal part of the leader peptide encoding sequences. In one embodiment, the nucleic acid molecule encoding the new leader peptides according to the invention comprise a nucleic acid molecule selected from the group consisting of SEQ ID NO:149, SEQ ID NO:151 and SEQ ID NO:153. The nucleic acid molecule may further comprise a nucleic acid molecule encoding a polypeptide of interest. Suitable polypeptides of interest are mentioned herein. The nucleic acid molecule encoding the leader peptides and the nucleic acid molecule encoding the polypeptides of interest can form a so-called fusion construct. By “fusion construct” is intended a nucleic acid comprising the coding sequence for a leader peptide and the coding sequence, with or without introns, for a recombinant protein, in which the coding sequences are adjacent and in the same reading frame such that, when the fusion construct is transcribed and translated in a host cell, a protein is produced in which the C terminus of the leader peptide is joined to the N terminus of the recombinant protein. The protein product of the fusion construct may be referred to herein as “fusion polypeptide.” Nucleic acid encoding the leader peptide can be operably joined/linked to nucleic acid containing the coding region of the recombinant protein in such manner that the leader peptide coding region is upstream of (that is, 5′ of) and in the same reading frame with the recombinant protein coding region to provide a fusion construct. Typically, the 3′-end of the nucleic acid encoding the leader peptide is joined to the 5′-end of the nucleic acid encoding the recombinant protein. The two coding regions are joined such that they are in the same reading frame. In this way, the fusion construct will encode a single protein, having the leader peptide at the N terminal end followed by the recombinant protein at the C terminal end. The leader peptide and the recombinant protein may be joined directly or there may be one or several amino acids connecting them. Certain amino acids are well known to interfere with cleavage by signal peptidases and these residues are avoided in designing the cleavage site for the fusion polypeptide. The fusion construct can be expressed in a host cell to provide a fusion polypeptide comprising the leader peptide joined, at its carboxy terminus, to the recombinant protein at its amino terminus. The fusion polypeptide can be secreted from the host cell. Typically, the leader peptide is cleaved from the fusion polypeptide during the secretion process, resulting in the accumulation of secreted recombinant protein in the external cellular environment.
Further provided is an expression vector comprising a nucleic acid molecule of the invention. Preferably, such a nucleic acid molecule comprises a nucleic acid molecule encoding a leader peptide hereof. They may further comprise a nucleic acid molecule encoding the polypeptide of interest. Expression vectors can be prepared containing the nucleic acids encoding the leader peptide or the fusion construct by methods that are well known in the art. In general, the expression vectors will contain nucleic acid encoding the leader peptide, or the fusion construct, under the control of a promoter. In some embodiments, more than one leader peptide or fusion construct may be placed under the control of a single promoter. In such embodiments, the additional fusion construct(s) will be placed downstream of the first fusion construct and separated from the upstream fusion construct by nucleotides. The promoter is chosen so that it is capable of directing transcription in a host of interest. Promoters capable of directing transcription in various host cells are well known. In an embodiment, the promoter is the CMVlong promoter, but any other suitable promoter may also be chosen. In general, a “promoter” will include all nucleotides upstream of the translational start necessary for the transcription of the leader peptide and/or fusion polypeptide coding region. The promoter may include or overlap the sequence of the ribosome binding site. Selection of promoter will often influence the selection of ribosome binding site as well. The expression vector may also contain other expression regulating nucleic acid molecules including selectable marker genes, origins of replication, polyadenylation signal sequences, etc. Other expression regulating nucleic acid molecules and further sequences, e.g., sequences of polypeptides useful for isolation, which can be included in the vectors are mentioned above. The expression vectors may contain one or more selectable marker genes, including ampicillin and/or neomycin, for selection in the host of interest and/or one or more origins of replication, including a pUc ori and/or a SV40 on and/or a fl ori, to provide autonomous replication of the vector in the host. Additionally, they may contain one or more polyadenylation signal sequences including a SV40-polyA signal and/or a BGH-polyA signal. Alternatively, or in addition, the expression vector may contain nucleotides to aid in integration of the vector into the host chromosome. An expression vector according to the invention may further comprise a XhoI-site or a NotI-site. An immunoglobulin heavy and/or light chain variable region may for instance be located between a Sfi-site and a XhoI-site (e.g., in an expression vector for the production of heavy chain (IgG1) immunoglobulin chains), and located between a XhoI-site and a NotI-site (e.g., in an expression vector for the production of light chain (kappa and lambda) immunoglobulin chains). The restriction sites may be located downstream of a nucleic acid molecule encoding the promoter, e.g., the CMVlong promoter, and downstream of the eukaryotic leader peptide of the invention. The sites may be located upstream of a nucleic acid molecule encoding an immunoglobulin constant domain.
Further provided is an expression vector a nucleic acid molecule selected from the group consisting of SEQ ID NO:155, SEQ ID NO:156 and SEQ ID NO:157, i.e., a “truncated” eukaryotic leader peptide. The expression vector comprising the nucleic acid molecule of SEQ ID NO:155 comprises a SfiI-site at its carboxy terminus. The expression vectors comprising the nucleic acid molecule of SEQ ID NO:156 or SEQ ID NO:157 comprise a XhoI-site at their carboxy termini. The vectors may further comprise downstream of the nucleic acid molecule selected from the group consisting of SEQ ID NO:155, SEQ ID NO:156 and SEQ ID NO:157 a XhoI-site (for SEQ ID NO:155) or a NotI-site (for SEQ ID NO:156 and SEQ ID NO:157). They may further comprise a stuffer sequence between the nucleic acid molecule selected from the group consisting of SEQ ID NO:155, SEQ ID NO:156 and SEQ ID NO:157 and the XhoI-site or NotI-site. The nucleic acid molecule selected from the group consisting of SEQ ID NO:155, SEQ ID NO:156 and SEQ ID NO:157, the stuffer sequence and the XhoI-site and NotI-site may be located downstream of a promoter sequence such as a CMVlong promoter sequence. They may be located upstream of an immunoglobulin heavy or light chain constant region. The expression vector may further comprise other expression regulating nucleic acid molecules or other sequences. Examples of suitable expression regulating nucleic acid molecules or other sequences are mentioned above. In an embodiment, the expression vector according to the invention comprises a nucleic acid molecule selected from the group consisting of SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:58. These vectors comprise a stuffer sequence.
A host cell comprising at least an expression vector hereof is another aspect of the disclosure. Preferably, the host cell is a human host cell. Other suitable host cells are mentioned above. Furthermore, described is the use of an expression vector hereof and/or a host cell hereof for the production of an immunoglobulin chain and/or molecule.
In yet a further aspect, provided is a method for producing expression vectors. In one embodiment, the expression vector produced is suitable for producing an immunoglobulin heavy chain. The method comprises the steps of: digesting/cutting an expression vector comprising the nucleic acid molecule of SEQ ID NO:155 and downstream thereof an XhoI-site and an immunoglobulin heavy chain constant region with the restriction enzymes SfiI and XhoI, digesting/cutting a phage display vector comprising an immunoglobulin heavy chain variable region with the restriction enzymes SfiI and XhoI, inserting the immunoglobulin heavy chain variable region into the expression vector, and isolating the expression vector. In a specific embodiment, the expression vector comprising the nucleic acid molecule of SEQ ID NO:155 and downstream thereof an XhoI-site and an immunoglobulin heavy chain constant region comprises the nucleic acid molecule of SEQ ID NO:53. In another embodiment, the expression vector produced is suitable for producing an immunoglobulin light chain. The method comprises the steps of digesting/cutting an expression vector comprising the nucleic acid molecule of SEQ ID NO:156 or SEQ ID NO:157 and downstream thereof a NotI-site and an immunoglobulin light chain constant region with the restriction enzymes XhoI and NotI, digesting/cutting a phage display vector comprising an immunoglobulin light chain variable region with the restriction enzymes SalI and NotI, inserting the immunoglobulin light chain variable region into the expression vector, and isolating the expression vector. In a specific embodiment, the expression vector comprising the nucleic acid molecule of SEQ ID NO:156 or SEQ ID NO:157 and downstream thereof an NotI-site and an immunoglobulin light chain constant region comprises the nucleic acid molecule of SEQ ID NO:54 or SEQ ID NO:58, respectively. The phage display vector can be a phagemid but also any other suitable vehicle. It is clear that the digesting/cutting steps can be performed in any order and even concomitantly. It is further to be understood that the variable heavy chain and variable light chain regions in the phage display vector, such as pHEN1 or PDV, are located between the sites SfiI and XhoI and SalI and NotI, respectively. By digesting the display vectors with the respective restriction enzymes the regions are obtained and can be used for insertion into the eukaryotic expression vectors.
Further disclosed is a method for producing an immunoglobulin heavy or light chain, the method comprising the steps of transforming at least one host cell with an expression vector as produced above, culturing the host cell under conditions conducive to the expression of an immunoglobulin chain, and optionally, purifying the immunoglobulin chain from the medium or cellular extract. In an embodiment, the host cell may be transformed with an expression vector suitable for producing a heavy chain and an expression vector suitable for producing a light chain and a complete immunoglobulin may be produced. Many commercially significant proteins are produced by recombinant gene expression in appropriate prokaryotic or eukaryotic host cells. It may be desirable to isolate the expressed protein product after secretion into the culture medium. Secreted proteins are typically soluble and can be separated readily from contaminating host proteins and other cellular components. Method for separation and/or purification are well known in the art.



DESCRIPTION OF THE FIGURES
FIG. 1 shows the titration of anti-WNV monoclonal antibody CR4354L4328 in a murine WNV challenge model. From top to bottom titration of anti-WNV monoclonal antibody CR4354L4328 using doses of 0.03, 0.01, 0.003, and 0.001 mg/kg and titration with a control antibody at a concentration of 10 mg/kg are shown. On the X-axis days are shown and on the Y-axis the survival probability (%) is represented.
FIG. 2 shows the titration of anti-WNV monoclonal antibody CR4348 in a murine WNV challenge model. From top to bottom titration of anti-WNV monoclonal antibody CR4348 using doses of 0.1, 0.03, 0.01, 0.003, and 0.001 mg/kg and titration with a control antibody at a concentration of 10 mg/kg are shown. On the X-axis days are shown and on the Y-axis the survival probability (%) is represented.
FIG. 3 shows the ELISA binding of dilutions of the optimized antibodies CR4354L4261, CR4354L4267, CR4354L4328, CR4354L4335, CR4354L4383 and the parent antibody CR4354 to WNV. On the Y-axis the absorbance (OD) at 492 nm is shown and on the X-axis the amount of antibody in μg/ml is shown.
FIG. 4 shows neutralization data of CR4348 (closed circles) and CR4354L4328 (open circles) of the flaviviruses yellow fever virus, Japanese encephalitis virus, St. Louis encephalitis virus and dengue virus 2 in a plaque reduction assay.
FIG. 5 shows an immunoblot with from left to right mycNDUFV-1 cell lysate and lysate immunoprecipitated with CR4348, CR4361, CR4374 or CR4354L4328.



DETAILED DESCRIPTION
Here below follow definitions of terms as used herein.
Amino acid sequence. The term “amino acid sequence” (or “amino acid molecule”), as used herein, refers to naturally occurring or synthetic molecules and to a peptide, oligopeptide, polypeptide, or protein sequence.
Binding molecule. As used herein, the term “binding molecule” refers to an intact immunoglobulin including monoclonal antibodies, such as chimeric, humanized or human monoclonal antibodies, or to an antigen-binding and/or variable domain-comprising fragment of an immunoglobulin that competes with the intact immunoglobulin for specific binding to the binding partner of the immunoglobulin, e.g., a host cell protein. Regardless of structure, the antigen-binding fragment binds with the same antigen that is recognized by the intact immunoglobulin. An antigen-binding fragment can comprise a peptide or polypeptide comprising an amino acid sequence of at least two contiguous amino acid residues, at least five contiguous amino acid residues, at least ten contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 30 contiguous amino acid residues, at least 35 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least contiguous 80 amino acid residues, at least contiguous 90 amino acid residues, at least contiguous 100 amino acid residues, at least contiguous 125 amino acid residues, at least 150 contiguous amino acid residues, at least contiguous 175 amino acid residues, at least 200 contiguous amino acid residues, or at least contiguous 250 amino acid residues of the amino acid sequence of the binding molecule.
The term “binding molecule,” as used herein includes all immunoglobulin classes and subclasses known in the art. Depending on the amino acid sequence of the constant domain of their heavy chains, binding molecules can be divided into the five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgA 1, IgA2, IgG 1, IgG2, IgG3 and IgG4.
Antigen-binding fragments include, inter alia, Fab, F(ab′), F(ab′)2, Fv, dAb, Fd, complementarity determining region (CDR) fragments, single chain antibodies (scFv), bivalent single chain antibodies, single chain phage antibodies, diabodies, triabodies, tetrabodies, peptide or polypeptides that contain at least a fragment of an immunoglobulin that is sufficient to confer specific antigen binding to the peptide or polypeptide, etc. The above fragments may be produced synthetically or by enzymatic or chemical cleavage of intact immunoglobulins or they may be genetically engineered by recombinant DNA techniques. The methods of production are well known in the art and are described, for example, in Antibodies: A Laboratory Manual, Edited by: E. Harlow and D, Lane (1988), Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., which is incorporated herein by reference. A binding molecule or antigen-binding fragment thereof may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or they may be different.
The binding molecule can be a naked or unconjugated binding molecule but can also be part of an immunoconjugate. A naked or unconjugated binding molecule is intended to refer to a binding molecule that is not conjugated, operatively linked or otherwise physically or functionally associated with an effector moiety or tag, such as inter alia a toxic substance, a radioactive substance, a liposome, an enzyme. It will be understood that naked or unconjugated binding molecules do not exclude binding molecules that have been stabilized, multimerized, humanized or in any other way manipulated, other than by the attachment of an effector moiety or tag. Accordingly, all post-translationally modified naked and unconjugated binding molecules are included herewith, including where the modifications are made in the natural binding molecule-producing cell environment, by a recombinant binding molecule-producing cell, and are introduced by the hand of man after initial binding molecule preparation. Of course, the term naked or unconjugated binding molecule does not exclude the ability of the binding molecule to form functional associations with effector cells and/or molecules after administration to the body, as some of such interactions are necessary in order to exert a biological effect. The lack of associated effector group or tag is therefore applied in definition to the naked or unconjugated binding molecule in vitro, not in vivo.
Biological sample. As used herein, the term “biological sample” encompasses a variety of sample types, including blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures, or cells derived thereof and the progeny thereof. The term also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as proteins or polynucleotides. The term encompasses various kinds of clinical samples obtained from any species, and also includes cells in culture, cell supernatants and cell lysates.
Complementarity determining regions (CDR). The term “complementarity determining regions” as used herein means sequences within the variable regions of binding molecules, such as immunoglobulins, that usually contribute to a large extent to the antigen binding site which is complementary in shape and charge distribution to the epitope recognized on the antigen. The CDR regions can be specific for linear epitopes, discontinuous epitopes, or conformational epitopes of proteins or protein fragments, either as present on the protein in its native conformation or, in some cases, as present on the proteins as denatured, e.g., by solubilization in SDS. Epitopes may also consist of posttranslational modifications of proteins.
Deletion. The term “deletion,” as used herein, denotes a change in either amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues, respectively, are absent as compared to the parent, often the naturally occurring, molecule.
Expression-regulating nucleic acid molecule. The term “expression-regulating nucleic acid molecule” as used herein refers to polynucleotides necessary for and/or affecting the expression of an operably linked coding sequence in a particular host organism. The expression-regulating nucleic acid molecules, such as inter alia appropriate transcription initiation, termination, promoter, enhancer sequences; repressor or activator sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic Mrna; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion, can be any nucleic acid molecule showing activity in the host organism of choice and can be derived from genes encoding proteins, which are either homologous or heterologous to the host organism.
Functional variant. The term “functional variant,” as used herein, refers to a binding molecule that comprises a nucleotide and/or amino acid sequence that is altered by one or more nucleotides and/or amino acids compared to the nucleotide and/or amino acid sequences of the parental binding molecule and that is still capable of competing for binding to the binding partner, e.g., an intracellular host cell protein, with the parental binding molecule. In other words, the modifications in the amino acid and/or nucleotide sequence of the parental binding molecule do not significantly affect or alter the binding characteristics of the binding molecule encoded by the nucleotide sequence or containing the amino acid sequence, i.e., the binding molecule is still able to recognize and bind its target. The functional variant may have conservative sequence modifications including nucleotide and amino acid substitutions, additions and deletions. These modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and random PCR-mediated mutagenesis, and may comprise natural as well as unnatural nucleotides and amino acids.
Conservative amino acid substitutions include the ones in which the amino acid residue is replaced with an amino acid residue having similar structural or chemical properties. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cystine, tryptophan), nonpolar side chains (e.g., 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, histidine). It will be clear to the skilled artisan that other classifications of amino acid residue families than the one used above can also be employed. Furthermore, a variant may have non-conservative amino acid substitutions, e.g., replacement of an amino acid with an amino acid residue having different structural or chemical properties. Similar minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing immunological activity may be found using computer programs well known in the art.
A mutation in a nucleotide sequence can be a single alteration made at a locus (a point mutation), such as transition or transversion mutations, or alternatively, multiple nucleotides may be inserted, deleted or changed at a single locus. In addition, one or more alterations may be made at any number of loci within a nucleotide sequence. The mutations may be performed by any suitable method known in the art.
Host. The term “host,” as used herein, is intended to refer to an organism or a cell into which a vector such as a cloning vector or an expression vector has been introduced. The organism or cell can be prokaryotic or eukaryotic. It should be understood that this term is intended to refer not only to the particular subject organism or cell, but to the progeny of such an organism or cell as well. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent organism or cell, but are still included within the scope of the term “host” as used herein.
Human. The term “human,” when applied to binding molecules as defined herein, refers to molecules that are either directly derived from a human or based upon a human sequence. When a binding molecule is derived from or based on a human sequence and subsequently modified, it is still to be considered human as used throughout the specification. In other words, the term human, when applied to binding molecules is intended to include binding molecules having variable and constant regions derived from human germline immunoglobulin sequences or based on variable or constant regions occurring in a human or human lymphocyte and modified in some form. Thus, the human binding molecules may include amino acid residues not encoded by human germline immunoglobulin sequences, comprise substitutions and/or deletions (e.g., mutations introduced by for instance random or site-specific mutagenesis in vitro or by somatic mutation in vivo). “Based on” as used herein refers to the situation that a nucleic acid molecule may be exactly copied from a template, or with minor mutations, such as by error-prone PCR methods, or synthetically made matching the template exactly or with minor modifications. Semi-synthetic molecules based on human sequences are also considered to be human as used herein.
Insertion. The term “insertion,” also known as the term “addition,” denotes a change in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid or nucleotide residues, respectively, as compared to the parent sequence.
Isolated. The term “isolated,” when applied to binding molecules as defined herein, refers to binding molecules that are substantially free of other proteins or polypeptides, particularly free of other binding molecules having different antigenic specificities, and are also substantially free of other cellular material and/or chemicals. For example, when the binding molecules are recombinantly produced, they are preferably substantially free of culture medium, and when the binding molecules are produced by chemical synthesis, they are preferably substantially free of chemical precursors or other chemicals, i.e., they are separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. The term “isolated” when applied to nucleic acid molecules encoding binding molecules as defined herein, is intended to refer to nucleic acid molecules in which the nucleotides encoding the binding molecules are free of other nucleotides, particularly nucleotides encoding binding molecules that bind binding partners other than host cell proteins. Furthermore, the term “isolated” refers to nucleic acid molecules that are substantially separated from other cellular components that naturally accompany the native nucleic acid molecule in its natural host, e.g., ribosomes, polymerases, or genomic sequences with which it is naturally associated. Moreover, “isolated” nucleic acid molecules, such as Cdna molecules, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
Monoclonal antibody. The term “monoclonal antibody” as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope. Accordingly, the term “human monoclonal antibody” refers to an antibody displaying a single binding specificity that has variable and constant regions derived from or based on human germline immunoglobulin sequences or derived from completely synthetic sequences. The method of preparing the monoclonal antibody is not relevant.
Naturally occurring. The term “naturally occurring” as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.
Nucleic acid molecule. The term “nucleic acid molecule” as used herein refers to a polymeric form of nucleotides and includes both sense and anti-sense strands of RNA, Cdna, genomic DNA, and synthetic forms and mixed polymers of the above. A nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide. The term also includes single- and double-stranded forms of DNA. In addition, a polynucleotide may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or unnaturally occurring nucleotide linkages. The nucleic acid molecules may be modified chemically or biochemically or may contain unnatural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analogue, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.). The above term is also intended to include any topological conformation, including single-stranded, double-stranded, partially duplexed, triplex, hair-pinned, circular and padlocked conformations. Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule. A reference to a nucleic acid molecule encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid molecule having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence. The complementary strand is also useful, e.g., for anti-sense therapy, hybridization probes and PCR primers.
Operably linked. The term “operably linked” refers to two or more nucleic acid molecule elements that are usually physically linked and are in a functional relationship with each other. For instance, a promoter is operably linked to a coding sequence, if the promoter is able to initiate or regulate the transcription or expression of a coding sequence, in which case the coding sequence should be understood as being “under the control of” the promoter.
Pharmaceutically acceptable excipient. By “pharmaceutically acceptable excipient” is meant any inert substance that is combined with an active molecule such as a drug, agent, or binding molecule for preparing an agreeable or convenient dosage form. The “pharmaceutically acceptable excipient” is an excipient that is non-toxic to recipients at the dosages and concentrations employed, and is compatible with other ingredients of the formulation comprising the drug, agent or binding molecule.
Specifically Binding. The term “specifically binding,” as used herein, in reference to the interaction of a binding molecule, e.g., an antibody, and its binding partner, e.g., an antigen, means that the interaction is dependent upon the presence of a particular structure, e.g., an antigenic determinant or epitope, on the binding partner. In other words, the antibody preferentially binds or recognizes the binding partner even when the binding partner is present in a mixture of other molecules or organisms. The binding may be mediated by covalent or non-covalent interactions or a combination of both. In yet other words, the term “specifically binding” means immunospecifically binding to an antigen or a fragment thereof and not immunospecifically binding to other antigens. A binding molecule that immunospecifically binds to an antigen may bind to other peptides or polypeptides with lower affinity as determined by, e.g., radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), BIACORE, or other assays known in the art. Binding molecules or fragments thereof that immunospecifically bind to an antigen may be cross-reactive with related antigens. Preferably, binding molecules or fragments thereof that immunospecifically bind to an antigen do not cross-react with other antigens.
Substitutions. A “substitution,” as used herein, denotes the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.
Therapeutically effective amount. The term “therapeutically effective amount” refers to an amount of the binding molecule as defined herein that is effective for preventing, ameliorating and/or treating a condition resulting from a viral infection.
Treatment. The term “treatment” refers to therapeutic treatment as well as prophylactic or preventative measures to cure or halt or at least retard disease progress. Those in need of treatment include those already inflicted with a condition resulting from a viral infection as well as those in which a viral infection is to be prevented. Subjects partially or totally recovered from a viral infection might also be in need of treatment. Prevention encompasses inhibiting or reducing the spread of a virus or inhibiting or reducing the onset, development or progression of one or more of the symptoms associated with a viral infection.
Vector. The term “vector” denotes a nucleic acid molecule into which a second nucleic acid molecule can be inserted for introduction into a host where it will be replicated, and in some cases expressed. In other words, a vector is capable of transporting a nucleic acid molecule to which it has been linked. Cloning as well as expression vectors are contemplated by the term “vector,” as used herein. Vectors include, but are not limited to, plasmids, cosmids, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC) and vectors derived from bacteriophages or plant or animal (including human) viruses. Vectors comprise an origin of replication recognized by the proposed host and in case of expression vectors, promoter and other regulatory regions recognized by the host. A vector containing a second nucleic acid molecule is introduced into a cell by transformation, transfection, or by making use of viral entry mechanisms. Certain vectors are capable of autonomous replication in a host into which they are introduced (e.g., vectors having a bacterial origin of replication can replicate in bacteria). Other vectors can be integrated into the genome of a host upon introduction into the host, and thereby are replicated along with the host genome.
EXAMPLES
To further describe the invention, the following illustrative examples are provided.
Example 1
Construction of a scFv Phage Display Library Using RNA Extracted from Peripheral Blood of WNV Convalescent Donors
From three convalescent WNV patients samples of blood were taken 1, 2 and 3 months after infection. Peripheral blood leukocytes were isolated by centrifugation and the blood serum was saved and frozen at −80° C. All donors at all time points had high titers of neutralizing antibodies to WNV as determined using a virus neutralization assay. Total RNA was prepared from the cells using organic phase separation and subsequent ethanol precipitation. The obtained RNA was dissolved in RNAse free water and the concentration was determined by OD 260 nm measurement. Thereafter, the RNA was diluted to a concentration of 100 ng/μl. Next, 1 μg of RNA was converted into cDNA as follows: To 10 μl total RNA, 13 μl DEPC-treated ultrapure water and 1 μl random hexamers (500 ng/μl) were added and the obtained mixture was heated at 65° C. for 5 minutes and quickly cooled on wet-ice. Then, 8 μl 5× First-Strand buffer, 2 μl dNTP (10 mM each), 2 μl DTT (0.1 M), 2 μl Rnase-inhibitor (40 U/μl) and 2 μl SuperscriptTM III MMLV reverse transcriptase (200 U/μl) were added to the mixture, incubated at room temperature for 5 minutes and incubated for 1 hour at 50° C. The reaction was terminated by heat inactivation, i.e., by incubating the mixture for 15 minutes at 75° C.
The obtained cDNA products were diluted to a final volume of 200 μl with DEPC-treated ultrapure water. The OD 260 nm of a 50 times diluted solution (in 10 mM Tris buffer) of the dilution of the obtained cDNA products gave a value of 0.1.
For each donor 5 to 10 μl of the diluted cDNA products were used as template for PCR amplification of the immunoglobulin gamma heavy chain family and kappa or lambda light chain sequences using specific oligonucleotide primers (see Tables 1-6). PCR reaction mixtures contained, besides the diluted cDNA products, 25 pmol sense primer and 25 pmol anti-sense primer in a final volume of 50 μl of 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl2, 250 μM dNTPs and 1.25 units Taq polymerase. In a heated-lid thermal cycler having a temperature of 96° C., the mixtures obtained were quickly melted for 2 minutes, followed by 30 cycles of: 30 seconds at 96° C., 30 seconds at 60° C. and 60 seconds at 72° C.
In a first round amplification, each of seventeen light chain variable region sense primers (eleven for the lambda light chain (see Table 1) and six for the kappa light chain (see Table 2) were combined with an anti-sense primer recognizing the C-kappa called HuCk 5′-ACACTCTCCCCTGTTGAAGCTCTT-3′ (see SEQ ID NO:37) or C-lambda constant region HuCλ2 5′-TGAACATTCTGTAGGGGCCACTG-3′ (see SEQ ID NO:38) and HuCλ7 5′-AGAGCATTCTGCAGGGGCCACTG-3′ (see SEQ ID NO:39) (the HuCλ2 and HuCλ7 anti-sense primers were mixed to equimolarity before use), yielding 4 times 17 products of about 600 base pairs. These products were purified on a 2% agarose gel and isolated from the gel using Qiagen gel-extraction columns. 1/10 of each of the isolated products was used in an identical PCR reaction as described herein using the same seventeen sense primers, whereby each lambda light chain sense primer was combined with one of the three Jlambda-region specific anti-sense primers and each kappa light chain sense primer was combined with one of the five Jkappa-region specific anti-sense primers. The primers used in the second amplification were extended with restriction sites (see Table 3) to enable directed cloning in the phage display vector PDV-C06 (see SEQ ID NO:40). This resulted in 4 times 63 products of approximately 350 base pairs that were pooled to a total of 10 fractions. This number of fractions was chosen to maintain the natural distribution of the different light chain families within the library and not to over or under represent certain families. The number of alleles within a family was used to determine the percentage of representation within a library (see Table 4). In the next step, 2.5 μg of pooled fraction and 100 μg PDV-C06 vector were digested with SalI and NotI and purified from gel. Thereafter, a ligation was performed overnight at 16° C. as follows. To 500 ng PDV-C06 vector 70 ng pooled fraction was added in a total volume of 50 μl ligation mix containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM DTT, 1 mM ATP, 25 μg/ml BSA and 2.5 μl T4 DNA Ligase (400 U/μl). This procedure was followed for each pooled fraction. The ligation mixes were purified by phenol/chloroform, followed by a chloroform extraction and ethanol precipitation, methods well known to the skilled artisan. The DNA obtained was dissolved in 50 μl ultrapure water and per ligation mix two times 2.5 μl aliquots were electroporated into 40 μl of TG1 competent E. coli bacteria according to the manufacturer's protocol (Stratagene). Transformants were grown overnight at 37° C. in a total of 30 dishes (three dishes per pooled fraction; dimension of dish: 240 mm×240 mm) containing 2TY agar supplemented with 50 μg/ml ampicillin and 4.5% glucose. A (sub)library of variable light chain regions was obtained by scraping the transformants from the agar plates. This (sub)library was directly used for plasmid DNA preparation using a QIAGENTM QIAFilter MAXI prep kit.
For each donor the heavy chain immunoglobulin sequences were amplified from the same cDNA preparations in a similar two round PCR procedure and identical reaction parameters as described herein for the light chain regions with the proviso that the primers depicted in Tables 5 and 6 were used. The first amplification was performed using a set of nine sense directed primers (see Table 5; covering all families of heavy chain variable regions) each combined with an IgG specific constant region anti-sense primer called HuCIgG 5′-GTC CAC CTT GGT GTT GCT GGG CTT-3′ (SEQ ID NO:41) yielding four times nine products of about 650 base pairs. These products were purified on a 2% agarose gel and isolated from the gel using Qiagen gel-extraction columns. 1/10 of each of the isolated products was used in an identical PCR reaction as described herein using the same nine sense primers, whereby each heavy chain sense primer was combined with one of the four JH-region specific anti-sense primers. The primers used in the second round were extended with restriction sites (see Table 6) to enable directed cloning in the light chain (sub)library vector. This resulted per donor in 36 products of approximately 350 base pairs. These products were pooled for each donor per used (VH) sense primer into nine fractions. The products obtained were purified using Qiagen PCR purification columns. Next, the fractions were digested with SfiI and XhoI and ligated in the light chain (sub)library vector, which was cut with the same restriction enzymes, using the same ligation procedure and volumes as described herein for the light chain (sub)library. Alternatively, the fractions were digested with NcoI and XhoI and ligated in the light chain vector, which was cut with the same restriction enzymes, using the same ligation procedure and volumes as described herein for the light chain (sub)library. Ligation purification and subsequent transformation of the resulting definitive library was also performed as described herein for the light chain (sub)library and at this point the ligation mixes of each donor were combined per VH pool. The transformants were grown in 27 dishes (three dishes per pooled fraction; dimension of dish: 240 mm×240 mm) containing 2TY agar supplemented with 50 μg/ml ampicillin and 4.5% glucose. All bacteria were harvested in 2TY culture medium containing 50 μg/ml ampicillin and 4.5% glucose, mixed with glycerol to 15% (v/v) and frozen in 1.5 ml aliquots at −80° C. Rescue and selection of each library were performed as described below.
Example 2
Selection of Phages Carrying Single Chain Fv Fragments Specifically Recognizing WNV Envelope (E) Protein
Antibody fragments were selected using antibody phage display libraries, general phage display technology and MABSTRACT® technology, essentially as described in U.S. Pat. No. 6,265,150 and in WO 98/15833 (both of which are incorporated by reference herein). The antibody phage libraries used were two different semi-synthetic scFv phage libraries (JK1994 and WT2000) and the immune library prepared as described in Example 1. The first semi-synthetic scFv phage library (JK1994) has been described in de Kruif et al., 1995b, the second one (WT2000) was build essentially as described in de Kruif et al., 1995b. Briefly, the library has a semi-synthetic format whereby variation was incorporated in the heavy and light chain V genes using degenerated oligonucleotides that incorporate variation within CDR regions. Only VH3 heavy chain genes were used, in combination with kappa- and lambda light chain genes. CDR1 and CDR3 of the heavy chain and CDR3 of the light chain were recreated synthetically in a PCR-based approach similar as described in de Kruif et al., 1995b. The thus created V region genes were cloned sequentially in scFv format in a phagemid vector and amplified to generate a phage library as described before. Furthermore, the methods and helper phages as described in WO 02/103012 (incorporated by reference herein) were used herein. For identifying phage antibodies recognizing WNV E protein, phage selection experiments were performed using whole WNV (called strain USA99b or strain 385-99) inactivated by gamma irradiation (50 Gy for 1 hour), recombinantly expressed WNV E protein (strain 382-99), and/or WNV-like particles expressing WNV E protein (strain 382-99) on their surface.
The recombinantly expressed E protein was produced as follows. The nucleotide sequence coding for the preM/M-protein and the full length E protein of WNV strain 382-99 (see SEQ ID NO:42 for the amino acid sequence of a fusion protein comprising both WNV polypeptides) was synthesized. Amino acids 1-93 of SEQ ID NO:42 constitute the WNV preM-protein, amino acids 94-168 of SEQ ID NO:42 constitute the WNV M-protein, amino acids 169-669 of SEQ ID NO:42 constitute the WNV E protein (the soluble WNV E protein (ectodomain) constitutes amino acids 169-574 of SEQ ID NO:42, while the WNV E protein stem and transmembrane region constitutes amino acids 575-669 of SEQ ID NO:42) The synthesized nucleotide sequence was cloned into the plasmid pAdapt and the plasmid obtained was called pAdapt.WNV.prM-E (FL).
To produce a soluble secreted form of the E protein a construct was made lacking the transmembrane spanning regions present in the final 95 amino acids at the carboxyl terminal of the full length E protein (truncated form). For that purpose the full length construct pAdapt.WNV.prM-E (FL) was PCR amplified with the primers CMV-Spe (SEQ ID NO:43) and WNV-E-95 REV (SEQ ID NO:44) and the fragment obtained was cloned into the plasmid pAdapt.myc.his to create the plasmid called pAdapt.WNV-95. Next, the region coding for the preM-protein, the truncated E protein, the Myc tag and His tag were PCR amplified with the primers clefsmaquwnv (SEQ ID NO:45) and reverse WNVmychis (SEQ ID NO:46) and cloned into the vector pSyn-C03 containing the HAVT20 leader peptide using the restriction sites EcoRI and SpeI. The expression construct obtained, pSyn-C03-WNV-E-95, was transfected into 90% confluent HEK293T cells using lipofectamine according to the manufacturer's instructions. The cells were cultured for 5 days in serum-free ultra CHO medium, then the medium was harvested and purified by passage over HisTrap chelating columns (Amersham Bioscience) pre-charged with nickel ions. The truncated E protein was eluted with 5 ml of 250 mM imidazole and further purified by passage over a G-75 gel filtration column equilibrated with phosphate buffered saline (PBS). Fractions obtained were analyzed by SDS-PAGE analysis and Western blotting using the WNV-E protein specific murine antibody 7H2 (Biorelience, see Beasley and Barrett 2002). Three 5 ml fractions containing a single band of  ̃45 kDa that was immunoreactive with antibody 7H2 were aliquoted and stored at −20° C. until further use. The protein concentration was determined by OD 280 nm.
WNV-like particles were produced as follows. The construct pSyn-C03-WNV-E-95 described herein and pcDNA3.1 (Invitrogen) were digested with the restriction endonucleases MunI and XbaI and the construct pAdapt.WNV.prM-E (FL) described herein was digested with the restriction endonucleases ClaI and XbaI. The resulting fragments were combined in a three-point ligation to produce the construct pSyn-H-preM/E FL. This construct contained the full length E protein and expressed the two structural WNV proteins, protein M and E, required for assembly of an enveloped virion. The construct was transfected into 70% confluent HEK293T cells using lipofectamine according to the manufacturer's instructions. The cells were cultured for three days in serum-free ultra CHO medium, then the medium was harvested, layered on to a 30% glycerol solution at a 2:1 ratio and pelleted by centrifugation for 2 hours at 120,000*g at 4° C. The WNV-like particles were resuspended in PBS, aliquoted and stored at −80° C. Aliquots were analyzed by SDS-PAGE analysis and Western blotting using the WNV E protein specific murine antibody 7H2 (Biorelience).
Before inactivation, whole WNV was purified by pelleting through a 30% glycerol solution as described herein for WNV-like particles. The purified WNV was resuspended in 10 mM Tris/HCl pH 7.4 containing 10 mM EDTA and 200 mM NaCl, the obtained preparation was kept on dry ice during inactivation, tested for infectivity and stored at −80° C. in small aliquots. Aliquots were analyzed by SDS-PAGE analysis and Western blotting using the WNV E protein specific murine antibody 7H2 (Biorelience).
Whole inactivated WNV, WNV-like particles or recombinantly expressed soluble E protein were diluted in PBS. 2-3 ml of the preparation was added to MaxiSorpTM Nunc-Immuno Tubes (Nunc) and incubated overnight at 4° C. on a rotating wheel. An aliquot of a phage library (500 μl, approximately 1013 cfu, amplified using CT helper phage (see WO 02/103012)) was blocked in blocking buffer (2% Protifar in PBS) for 1-2 hours at room temperature. The blocked phage library was added to the immunotubes, incubated for 2 hours at room temperature, and washed with wash buffer (0.1% v/v Tween-20 in PBS) to remove unbound phages. Bound phages were eluted from the antigen by incubation with 1 ml of 50 mM Glycine-HCl pH 2.2 for 10 minutes at room temperature. Subsequently, the eluted phages were mixed with 0.5 ml of 1 M Tris-HCl pH 7.5 to neutralize the pH. This mixture was used to infect 5 ml of an XL1-Blue E. coli culture that had been grown at 37° C. to an OD 600 nm of approximately 0.3. The phages were allowed to infect the XL1-Blue bacteria for 30 minutes at 37° C. Then, the mixture was centrifuged for 10 minutes at 3200*g at room temperature and the bacterial pellet was resuspended in 0.5 ml 2-trypton yeast extract (2TY) medium. The obtained bacterial suspension was divided over two 2TY agar plates supplemented with tetracyclin, ampicillin and glucose. After overnight incubation of the plates at 37° C., the colonies were scraped from the plates and used to prepare an enriched phage library, essentially as described by De Kruif et al. (1995a) and WO 02/103012. Briefly, scraped bacteria were used to inoculate 2TY medium containing ampicillin, tetracycline and glucose and grown at a temperature of 37° C. to an OD 600 nm of  ̃0.3. CT helper phages were added and allowed to infect the bacteria after which the medium was changed to 2TY containing ampicillin, tetracycline and kanamycin. Incubation was continued overnight at 30° C. The next day, the bacteria were removed from the 2TY medium by centrifugation after which the phages in the medium were precipitated using polyethylene glycol (PEG) 6000/NaCl. Finally, the phages were dissolved in 2 ml of PBS with 1% bovine serum albumin (BSA), filter-sterilized and used for the next round of selection.
Typically, two rounds of selections were performed before isolation of individual phage antibodies. After the second round of selection, individual E. coli colonies were used to prepare monoclonal phage antibodies. Essentially, individual colonies were grown to log-phase in 96 well plate format and infected with CT helper phages after which phage antibody production was allowed to proceed overnight. The produced phage antibodies were PEG/NaCl-precipitated and filter-sterilized and tested in ELISA for binding to WNV-like particles purified as described supra.
Example 3
Validation of the WNV Specific Single Chain Phage Antibodies
Selected single chain phage antibodies that were obtained in the screens described herein were validated in ELISA for specificity, i.e., binding to WNV E protein, whole inactivated WNV and WNV-like particles, all purified as described supra. For this purpose, whole inactivated WNV, the WNV E protein, or WNV-like particles were coated to MaxisorpTM ELISA plates. In addition, whole inactivated rabies virus was coated onto the plates as a control. After coating, the plates were blocked in PBS containing 1% Protifar for 1 hour at room temperature. The selected single chain phage antibodies were incubated for 15 minutes in an equal volume of PBS containing 1% Protifar to obtain blocked phage antibodies. The plates were emptied, and the blocked single chain phage antibodies were added to the wells. Incubation was allowed to proceed for one hour, the plates were washed in PBS containing 0.1% v/v Tween-20 and bound phage antibodies were detected (using OD 492 nm measurement) using an anti-M13 antibody conjugated to peroxidase. As a control, the procedure was performed simultaneously without single chain phage antibody, with a negative control single chain phage antibody directed against rabies virus glycoprotein (antibody called SC02-447), with a negative control single chain phage antibody directed against SARS-CoV (antibody called SC03-014) and a positive control single chain phage antibody directed against rabies virus. As shown in Table 7, the selected phage antibodies called SC04-348 and SC04-354 displayed significant binding to immobilized whole inactivated WNV and WNV-like particles. Both single chain phage antibodies were selected when WNV-like particles were used in the first and second round of selection. When the ELISA was performed with recombinantly expressed purified soluble WNV E protein prepared as described supra or rabies virus, the single chain phage antibodies SC04-348 and SC04-354 did not bind, suggesting they either bind to a region not present in the truncated soluble E protein, bind to an unrelated protein on the virion surface, do not bind to the monomeric form of the E protein or do not bind because of the phage antibody format.
Example 4
Characterization of the WNV Specific scFvs
From the selected specific single chain phage antibody (scFv) clones plasmid DNA was obtained and nucleotides were determined according to standard techniques. The nucleotides of the scFvs (including restriction sites for cloning) called SC04-348 and SC04-354 are shown in SEQ ID NO:25 and SEQ ID NO:27, respectively. The amino acid sequences of the scFvs called SC04-348 and SC04-354 are shown in SEQ ID NO:26 and SEQ ID NO:28, respectively.
The VH and VL gene identity (see I. M. Tomlinson, S. C. Williams, O. Ignatovitch, S. J. Corbett, G. Winter. V-BASE Sequence Directory. Cambridge United Kingdom: MRC Centre for Protein Engineering (1997)) and heavy chain CDR3 sequences of the scFvs specifically binding WNV are depicted in Table 8. Table 9 shows the other CDR regions of the WNV specific scFvs.
Example 5
Construction of Fully Human Immunoglobulin Molecules (Human Monoclonal Anti-WNV Antibodies) from the Selected Anti-WNV Single Chain Fvs
Heavy and light chain variable regions of the scFv called SC04-354 was PCR-amplified using oligonucleotides to append restriction sites and/or sequences for expression in the IgG expression vectors pSyn-C18-HCγ1 (see SEQ ID NO:47) and pSyn-C04-Cλ (see SEQ ID NO:48). The heavy chain variable region of the scFv called SC04-354 was cloned into the vector pSyn-C18-HCγ1; the light chain variable region of the scFv called SC04-354 was cloned into the vector pSyn-C04-Cλ. The VL lambda gene was first amplified using the following oligonucleotides set: SC04-354, 5L-C (SEQ ID NO:49) and sy3L-Cmod (SEQ ID NO:50) and the PCR product cloned into vector pSyn-C04-Cλ. Nucleotide sequence for the construct was verified according to standard techniques known to the skilled artisan. VH gene was first amplified using the following oligonucleotide set: SC04-354, 5H-A (SEQ ID NO:51) and sy3H-A (SEQ ID NO:52). Thereafter, the PCR product was cloned into vector pSyn-C18-HCγ1 and nucleotide sequence was verified according to standard techniques known to the skilled person in the art.
Heavy and light chain variable region of the scFv called SC04-348 was cloned directly by restriction digest for expression in the IgG expression vectors pIg-C911-HCgammal (see SEQ ID NO:53) and pIg-C909-Ckappa (see SEQ ID NO:54). The heavy chain variable regions of the scFv called SC04-348 was cloned into the vector pIg-C911-HCgammal by restriction digest using the enzymes SfiI and XhoI and the light chain variable region of the scFv called SC04-348 was cloned into the vector pIg-C909-Ckappa by restriction digest using the enzymes SalI and NotI. Thereafter the nucleotide sequence was verified according to standard techniques known to the person skilled in the art.
The resulting expression constructs pgG104-348C911 and pgG104-354C18 encoding the human IgG1 heavy chains and pgG104-348C909 and pgG104-354C04 encoding the human IgG1 light chains were transiently expressed in combination in 293T cells and supernatants containing human IgG1 antibodies were obtained. The nucleotides of the heavy chains of the antibodies called CR4348 and CR4354 are shown in SEQ ID NOS:29 and 31, respectively. The amino acid sequences of the heavy chains of the antibodies called CR4348 and CR4354 are shown in SEQ ID NOS:30 and 32, respectively. The nucleotides of the light chain of antibodies CR4348 and CR4354 are shown in SEQ ID NOS:33 and 35, respectively. The amino acid sequences of the light chain of antibodies CR4348 and CR4354 are shown in SEQ ID NOS:34 and 36, respectively. A person skilled in the art can determine the variable regions of the heavy and light chains of the above antibodies by following Kabat et al. (1991) as described in Sequences of Proteins of Immunological Interest. Alternatively, batches of greater than 1 mg of each antibody were produced and purified using standard procedures. The antibodies were then titrated on a fixed concentration of irradiated West Nile virus and tested in ELISA as described herein. The results are shown in Table 10. As a negative control an anti-rabies virus antibody was used. Both antibodies showed binding to the virus in a dose dependent manner. The antibodies were also able to bind WNV-like particles (data not shown).
Additionally, binding of CR4348 and CR4354L4328 (an optimized variant of antibody CR4354; see Example 8 for selection of this variant) to viral material was tested in a capture ELISA. For this purpose, CR4348, CR4283 (an anti-WNV monoclonal antibody; positive control for the inactivated WNV and WNV-like particle and negative control for rabies virus), CR4354L4328 and CR4104 (an anti-rabies virus monoclonal antibody; positive control for rabies virus and negative control for the inactivated WNV and WNV-like particle) were coated in a concentration of 5 μg/ml to MaxisorpTM ELISA plates. After coating, the plates were blocked in PBS containing 1% Protifar for 1 hour at room temperature. Next, a serial dilution of whole inactivated WNV, WNV-like particles, or rabies virus (BPL-inactivated) was performed in PBS/protifar in a volume of 100 μl until a dilution of 1/2048 was reached. The viral material was allowed to incubate for 1 hour at room temperature. The plates were emptied, and washed 3 times with 100 μl PBS containing 0.1% v/v Tween-20. Next, the mouse monoclonal antibody 7H2 (Bioreliance) was added at a concentration of 1 μg/ml (diluted in PBS/protifar) for detection of the inactivated WNV and WNV-like particles. Moreover, the mouse monoclonal antibody 1112 directed against rabies virus was added in a 1:1000 dilution (diluted in PBS/protifar) for detection of rabies virus. Bound monoclonal antibodies were detected by OD 492 nm measurement with an anti-mouse antibody conjugated to peroxidase (Jackson) in a 1:2000 dilution in PBS/protifar. CR4348, CR4283 and CR4354L4328 showed a very high binding to WNV-like particles with CR4348 binding twice as efficient compared to CR4354L4328 (data not shown). Moreover, CR4348, CR4283 and CR4354L4328 also bound to WNV (data not shown). When the capture ELISA was performed with rabies virus, binding was observed with the positive control CR4104 and antibody CR4348. No binding of antibody CR4354L4328 was observed (data not shown).
Example 6
In Vitro Neutralization of WNV by WNV Specific scFvs and IgGs (Virus Neutralization Assay)
In order to determine whether the selected scFvs are capable of blocking WNV infection, in vitro virus neutralization assays (VNA) are performed. The VNA are performed on Vero cells (ATCC CCL 81). The WNV strain 385-99 which is used in the assay is diluted to a titer of 4×103 TCID50/ml (50% tissue culture infective dose per ml), with the titer calculated according to the method of Spearman and Kaerber. The scFv preparations are serially two-fold-diluted in PBS starting from 1:2 (1:2-1:1024). 25 μl of the respective scFv dilution is mixed with 25 μl of virus suspension (100 TCID50/25 μl) and incubated for one hour at 37° C. The suspension is then pipetted twice in triplicate into 96-well plates. Next, 50 μl of a freshly trypsinized and homogenous suspension of Vero cells (1:3 split of the confluent cell monolayer of a T75-flask) resuspended in DMEM with 10% v/v fetal calf serum and antibiotics is added. The inoculated cells are cultured for three to four days at 37° C. and observed daily for the development of cytopathic effect (CPE). CPE is compared to the positive control (WNV inoculated cells) and negative controls (mock-inoculated cells or cells incubated with scFV only). The complete absence of CPE in an individual cell culture is defined as protection (=100% titer reduction). The serum dilution giving protection in 50% percent of wells (i.e., three out of six wells) is defined as the 50% neutralizing antibody titer. The murine-neutralizing antibody 7H2 (Biorelience) is used as a positive control in the assay. A 50% neutralization titer of ≦1:4 (meaning the antibody is diluted four times or more) is regarded as specific evidence of neutralizing activity of the scFv against WNV.
Alternatively, in vitro virus neutralization assays (VNA) were performed in order to determine whether the anti-WNV IgGs were capable of blocking WNV infection. The VNA were performed essentially as described for scFvs, with the proviso that the serum dilution giving protection in 66% percent of wells (i.e., two out of three wells) was defined as the 66% neutralizing antibody titer and a 66% neutralization titer of ≦1:2 was regarded as specific evidence of neutralizing activity of the IgG against WNV.
Supernatants containing the human anti-WNV antibodies called CR4348 and CR354 were expressed as described in Example 5 and subjected to the aforementioned VNA. All antibodies had a neutralizing titer≦1:2. The potency of the antibodies (in μg/ml) is given in Table 11.
Example 7
In Vivo Protection by Monoclonal Antibodies from Lethal WNV Infection in a Murine Challenge Model
A murine challenge model was adapted from the literature (see Ben-Nathan et al. 2003; Beasley et al. 2002; Wang et al. 2001). In Ben-Nathan et al. (2003) four-week-old BALB/c mice were used and the animals were inoculated intraperitoneally (i.p.) with 20-times the viral dose resulting in 50% survival (LD50) of WNV strain ISR52 (LD50 was equivalent to 5 pfu). Under this dosing mice succumbed to infection six to seven days after inoculation and reached 100% mortality after 11 days. In another study, the WNV strain USA99 (used in the experiments described here) was shown to have an LD50 of 0.5 pfu. This is ten-fold lower than the LD50 of ISR52, which may indicate a higher degree of neuro-invasiveness for this viral strain or differences associated with the mouse strain used (see Beasley et al. 2002).
To determine the i.p., LD50 of USA99 in four week BALB/c mice, animals (five per group) were injected with USA99 at TCID50 (tissue culture infectious dose) of 100, 30, 10, 3, 1, 0.3, 0.1, 0.03 the 50% in two separate experiments. The LD50 calculated from the first experiment was 5.75 TCID50 and from the second experiment 13.25 TCID50. For the calculation of the viral dose in further experiments the average of the two experiments, i.e., 9.5 TCID50, was calculated by probit regression analysis.
The protective capacity of the in vitro neutralizing antibodies CR4348 and CR4354 was tested in the in vivo model. Purified antibodies were injected i.p. into four week BALB/c mice (five animals per group) at a concentration of 15 mg/kg. After 24 hours the WNV strain USA99 was injected i.p. at a dose of 20-times the LD50 calculated. The animals were observed for signs of disease over 21 days and sacrificed when symptoms of encephalitis were evident. In the model unprotected animals generally succumbed to infection between day 8 and day 10.
Table 12 shows that the two antibodies, CR4348 and CR4354, are 100% protective in vivo at the dose of 15 mg/kg. The positive control antibody 7H2 (an anti-WNV murine monoclonal) was fully protective and the negative control antibody (binding an irrelevant antigen) showed no protection in the experiment.
To establish a dose protection relationship, the protective antibody CR4348 was titrated in the mouse model using doses of 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003 and 0.001 mg/kg. Using the same doses, an optimized variant of antibody CR4354, i.e., CR4354L4328 (see Example 8 for selection of this variant), was titrated in the mouse model. A negative control antibody binding an irrelevant antigen was included as a control at a dose 10 mg/kg.
FIG. 1 shows that the antibody CR4354L4328 is 100% protective at a dose of 0.03 mg/kg. The doses 10, 3, 1, 0.3 and 0.1 mg/kg were also 100% protective (data not shown). FIG. 1 also shows that there is a direct correlation between dose and protective capacity. The 50% protective dose calculated by probit regression analysis is 0.003 mg/kg.
FIG. 2 shows that the antibody CR4348 is 100% protective at a dose of 0.1 mg/kg. The doses 10, 3, 1 and 0.3 mg/kg were also 100% protective (data not shown). FIG. 2 further shows that there is a direct correlation between dose and protective capacity. The 50% protective dose calculated by probit regression analysis is 0.006 mg/kg.
The titration data of the antibodies were compared by probit regression analysis. Values for the Pearson Goodness-of-Fit test (Chi Square=10.38, DF=30, p=1.00) demonstrated that the model was valid and the results of the Parallelism Test (Chi Square=3.47, DF=3, p=0.324) meant that the curves could be reliably compared. The values for the 50% protective dose and 95% protective dose are summarized in Table 13.
Example 8
Selection of Optimized Variants of Neutralizing Monoclonal Antibody CR4354
Monoclonal antibody CR4354 showing to have in vitro WNV-neutralizing activity and being 100% protective in vivo was selected for improving potency and affinity. This was done based on the following hypothesis. The specificity of CR4354 (as determined by the CDR3 region on the heavy chain variable chain) is one that targets a potent neutralizing epitope of WNV, but the light chain that is randomly paired with the heavy chain (through the phage display process) does not optimally recreate the original antigen binding site. Pairing with a more optimally mutated light chain might improve the “fit” of the antibody-binding pocket for the cognate antigen. Thus, replacement of the light chain might be a way of improving the potency and affinity of the antibody.
Analysis of the heavy and light chain of antibody CR4354 showed that they belong to the VH1 1-46 (DP-7) and Vlambda 1 (1c-V1-16) gene family, respectively. Further analysis of WNV specific scFvs selected from the WNV immune library as described in Example 1 revealed 5 scFvs, i.e., SC04-261, SC04-267, SC04-328, SC04-335 and SC04-383 (these scFvs were not included in Table 8), that had light chains having the same gene family as the light chain of CR4354. None of the scFvs or their respective IgGs showed WNV-neutralizing activity. Each of these light chains contained mutations in the CDR and framework regions away from the germline indicating that they had been modified as part of the natural affinity maturation process.
In short, the construction of the antibodies went as follows. CR4354 was prepared as described in Example 5. Heavy chain variable regions of the scFvs called SC04-261, SC04-267, SC04-328 and SC04-335 were PCR-amplified using oligonucleotides to append restriction sites and/or sequences for expression in the IgG expression vector pSyn-C18-HCγ1 and cloned into this vector. Amplification was done using the following oligonucleotide sets: SC04-261, 5H-A (SEQ ID NO:51) and sy3H-A (SEQ ID NO:52); SC04-267, 5H-A (SEQ ID NO:51) and sy3H-C (SEQ ID NO:55); SC04-328, 5H-A (SEQ ID NO:51) and sy3H-A (SEQ ID NO:52); and SC04-335, 5H-C (SEQ ID NO:56) and sy3H-A (SEQ ID NO:52).
The heavy chain variable region of the scFv called SC04-383 was cloned by restriction digest using the enzymes SfiI and XhoI in the IgG expression vector pIg-C911-HCgammal.
The light chain variable region of the scFv called SC04-267 was first amplified using the oligonucleotides SC04-267, 5L-C (SEQ ID NO:49) and sy3L-Amod (SEQ ID NO:57) and the PCR product cloned into vector pSyn-C04-Clambda.
Light chain variable regions of the scFvs called SC04-261, SC04-328, SC04-335, and SC04-383 were cloned directly by restriction digest using the enzymes SalI and NotI for expression in the IgG expression vector pIg-C910-Clambda (SEQ ID NO:58).
Nucleotides for all constructs were verified according to standard techniques known to the skilled artisan.
The resulting expression constructs pgG 104-261 C18, pgG104-267C18, pgG104-328C18, pgG104-335C18 and pgG104-383C911 encoding the anti-WNV human IgG1 heavy chains and pgG104-261C910, pgG104-267C04, pgG104-328C910, pgG104-335C910 and pgG104-383C910 encoding the anti-WNV human IgG1 light chains were transiently expressed in combination in 293T cells and supernatants containing human IgG1 antibodies were obtained.
The nucleotides of the heavy chains of the antibodies called CR4261, CR4267, CR4328, CR4335, CR4354 and CR4383 are shown in SEQ ID NOS:59, 61, 63, 65, 31 and 67, respectively (the variable regions are from nucleotides 1-348; 1-381; 1-348; 1-351; 1-363; and 1-372, respectively). The amino acid sequences of the heavy chains of the antibodies called CR4261, CR4267, CR4328, CR4335, CR4354 and CR4383 are shown in SEQ ID NOS:60, 62, 64, 66, 32 and 68, respectively (the variable regions are from amino acids 1-116; 1-127; 1-116; 1-117; 1-121; and 1-124, respectively). The nucleotides of the light chain of antibodies CR4261, CR4267, CR4328, CR4335, CR4354 and CR438 are shown in SEQ ID NOS:69, 71, 73, 75, 35 and 77, respectively (the variable regions are from nucleotides 1-342; 1-330; 1-339; 1-339; 1-330; and 1-339, respectively). The amino acid sequences of the light chain of antibodies CR4261, CR4267, CR4328, CR4335, CR4354 and CR4383 are shown in SEQ ID NOS:70, 72, 74, 76, 36 and 78, respectively (the variable regions are from amino acids 1-114; 1-110; 1-113; 1-113; 1-110; and 1-113, respectively).
The expression construct encoding the heavy chain of CR4354 was combined with the constructs expressing the light chains of the respective antibodies for transfection of HEK293T cells essentially as described in Example 5. The obtained antibodies were designated CR4354L4261, CR4354L4267, CR4354L4328, CR4354L4335 and CR4354L4383. Supernatants were tested for binding by ELISA staining as described in Example 5 and for potency in the in vitro neutralization assay as described in Example 6.
The binding data showed that all shuffled variants have specificity for the pre-selected antigen (see FIG. 3).
In terms of functional activity, it was concluded that two chain shuffled variants CR4354L4328 and CR4354L4335 had a higher affinity for WNV compared to CR4354. CR4354L4261 bound the virus with a similar affinity compared to CR4354, while both CR4354L4383 and CR4354L4267 bound with a lower affinity to the virus compared to CR4354 (see FIG. 3).
Furthermore, the antibodies CR4354L4383 and CR4354L4267 did not show any WNV-neutralizing activity, which was consistent with their lower binding affinity. CR4354L4261 had a neutralization endpoint concentration similar to the original antibody CR4534, again consistent with the binding data. CR4354L4335 that bound WNV with a higher affinity compared to CR4354 had a lower neutralizing activity compared to the original antibody CR4534. In contrast, the antibody variant CR4354L4328 that had a higher affinity for WNV compared to CR4354 also had a higher neutralizing activity compared to the original antibody CR4534 (see Table 14). In 4 out of 5 cases there was a direct correlation between binding affinity and neutralization potency of the variants. It was demonstrated that substituting similar light chains can improve a functionality of interest of an antibody, e.g., affinity or neutralizing activity.
Moreover, CR4354 was converted into a fully human IgM format by removing the gamma Fc region from construct pgG104-354C18 by restriction digestion with the endonucleases NheI and XbaI. The vector pCR-IgM (SEQ ID NO:146) containing a mu Fc region was digested with the same restriction enzymes and the obtained mu Fc region was ligated into vector pgG104-354C18 and fused in frame with the variable heavy chain gene derived from SC04-354 to make vector pgM104-354C899. This construct was transiently expressed in combination together with the light chain construct pgG104-354C04 (see above) in 293T cells and supernatants containing human IgM antibodies were obtained. The nucleotide sequence of vector pgM104-354C899 is shown in SEQ ID NO:147. The amino acid sequence of the heavy chain the antibody called CRM4354 is shown in SEQ ID NO:148. The IgM antibody was purified from the supernatant by adding ammonium sulphate to a final concentration of 2.4 M and incubating the mixture overnight on ice, while stirring. The precipitated IgM was recovered by centrifugation at 10,395×g for 30 minutes. The pellet was resuspended in PBS and further purified by gel filtration. A HiLoad 26/60 Superdex 200 prep grade column (GE heathcare) equilibrated with PBS was loaded with the resuspended IgM and fractions were collected from the column, while being flushed under a constant flow rate with PBS. The first major elution peak, which contained the purified IgM, was collected. Binding activity of the antibody was confirmed by titration on West Nile virus-like particles (VLPs) (data not shown).
Example 9
In Vitro Neutralization Potency by Plaque Reduction Neutralization Test (PRNT)
To further investigate the neutralizing activity of the antibodies of the invention a PRNT was developed. Briefly, Vero-E6 cells were trypsinized and counted. 2.5×105 cells were added to each well of a 12-well plate and incubated overnight at 37° C. in a humidified CO2 incubator. Serial dilutions (ten-fold) of a titrated stock of West Nile virus USA99b were made in complete medium. Equal volume (250 μl) mixtures of virus (100 pfu) and serial dilutions of purified IgG1 antibodies were incubated in duplicate at 37° C. for 1 hour. Dilutions of both virus and antibodies were done in DMEM medium. The mixture was then added (400 μl) to the 12-well plates containing Vero cell monolayers after careful aspiration of the overnight medium. After the plates had been incubated at 37° C. for 1 hour, an 1.5 ml overlay of CMC carboxymethyl-cellulose medium with 10% FBS (v/v) (CMC:complete medium) was added per well and the plates placed in a humidified CO2 incubator for 3 days at 37° C. One day before staining the CMC:complete medium was removed from the wells and replaced with a mixture of CMC:PBS (1:1; v/v) containing 8.25 mg/ml of neutral red (2 ml neutral red at 3.3 g/l in 80 ml CMC:PBS). Plates were incubated 1 day further at 37° C. in a humidified CO2 incubator, after which the number of visible plaques was quantified.
To analyze the antibody potency data from the PRNT a binary regression model known as probit analysis was used. Probit analysis is valid, if it can be assumed that the probability of neutralizing WNV in vitro follows a normal distribution with regard to the amount of antibodies used. The assumption of normality most likely holds on a logarithmic scale, hence the neutralization of virus was modeled as a function of the logarithm of the amount of antibodies administered. Antibodies were compared directly in the regression model, with significance level alpha set at 0.05. Antibody concentrations yielding 50% and 90% neutralization were estimated from the model, together with 95% confidence intervals. A summary of the final analysis of the antibodies is given in Table 15. By converting CR4354 into IgM format (CRM4354) the in vitro potency was increased dramatically (see Table 15).
Using the assay described herein, the antibodies were tested for their neutralizing potency against other flaviviruses including yellow fever virus, Japanese encephalitis virus, St. Louis encephalitis virus and dengue virus 2. In FIG. 4 is shown that CR4348 had significant neutralizing activity against St. Louis encephalitis virus and dengue virus 2.
Example 10
Identification of the Target Antigens of CR4348 and CR4354L4328
To identify the target antigens of the CR4348 and CR4354L4328 antibodies a human fetal brain protein expression library was screened with the antibodies. The cDNA library was constructed in the bacterial expression vector pQE-30 (Qiagen) for IPTG inducible expression of (His)6-tagged fusion proteins. The library was composed of 38,016 clones with an average insert of 1500 bps and these were printed in duplicate onto the membranes. To identify target antigens CR4348, CR4354L4328 and a negative control antibody (CR4374) were subjected to protein micro-array analysis (RZPD (Heidelberg, Germany)). Two independent experiments were performed with the antibodies. The protein arrays were incubated with the primary antibody and binding was detected with a conjugated anti-human secondary antibody. Clones were considered positive, if duplicate spots appeared that were not present with the secondary antibody alone. The negative control did not show any reactivity, whereas five different clones reacted with CR4348 and four different clones with CR4354L4328. The reactive clones were sequenced and used for database search against the NCBI database using the nucleotide-nucleotide BLAST program. The identity of the clones is depicted in Table 16.
To confirm the protein-array data obtained with CR4348 and CR4354L4328 the different cDNA clones were expressed. The nine different clones and a bacterial clone expressing the E protein of WNV were streaked on an agar plate and grown overnight at 37° C. Next, 10 ml LB volumes were inoculated with the nine different clones and the control WNV E protein clone. The bacteria were grown overnight at 37° C. The next day the cultures were diluted 1:50 and grown until an OD600 nm of 0.6 was reached, subsequently the expression of the protein was induced by adding IPTG and the cells were harvested after 4 hours of induction in three portions of 1 ml. Next, each portion underwent a different extraction procedure that allowed for the extraction of soluble and insoluble proteins. The WNV E protein clone was included as a positive control in the test, since this protein is expressed as a soluble protein and is therefore present in all three extraction procedures. All methods started with a cycle of three freeze-thawings of the pellet. In the first method the pellet was extracted in a volume twice the volume of the cell pellet in a mild extraction buffer (50 mM Na2HPO4, 300 mM NaCl, 0.05% Tween-20 buffer with lysozyme 1 mg/ml) by incubation of the cell pellet for 30 minutes on ice, next cells were pelleted and the supernatant was stored for analysis (fraction 1). Subsequently, the pellet was extracted with a more stringent buffer (0.2% DOC-1% Triton X-100) and resuspended in a volume twice the volume of the cell pellet. After an incubation period of 30 minutes on ice, cells were pelleted and the supernatant was stored for analysis (fraction 2). In the second method (an adaptation of the colony blot procedure), the pellet was lysed by addition of 10% SDS (twice the volume of the cell pellet). After 10 minutes of incubation at room temperature, the suspension was denatured with an equal volume 0.5 M NaOH containing 1.5 M NaCl for 5 minutes at room temperature. By adding an equal volume of 1.5 M NaCl, 0.5 M Tris pH 7.4 the suspension was neutralized. Next, cell rests were pelleted and the supernatant was stored for analysis (fraction 3). In the third method inclusion bodies were solubilized and the pellet was extracted by addition of 8 M ureum in 100 mM Tris/HCl, 100 mM NaH2PO4 in a volume twice the volume of the cell pellet. After 30 minutes of incubation at room temperature, cells rests were pelleted and the supernatant was stored for analysis (fraction 4). Proteins extracted with each procedure were spotted on a nitrocellulose filter. Moreover, controls, i.e., purified WNV E protein (negative control) and human IgG (positive control), were spotted. Membranes were blocked overnight with 4% milk powder in TBST at 4° C. Subsequently, the blots were incubated with 5 μg/ml CR4348, CR4354L4328 or the murine anti-WNV E protein monoclonal antibody 7H2. The antibody used for WNV E protein detection recognizes a linear epitope and still reacts with recombinant protein after treatment with denaturing reagents like 8 M urea (method 3). After an incubation period of 1 hour at room temperature in a rolling incubator, the membranes were washed three times for five minutes with TBST. Binding of antibodies was detected with HRP conjugated goat anti-human (Pharmingen) or goat anti-mouse (DAKO) antibodies. Finally, the membranes were washed extensively in TBST followed by a PBS washing step. Reactive proteins were revealed by the ECL chemofluorescence detection system (Amersham).
CR4348 reacted with fractions 1, 2 and 3 of the FAF-1 expression clone (data not shown). CR4354L4328 solely reacted with fraction 3 of the NADH dehydrogenase flavoprotein 1 (NDUFV1) expression clone (data not shown). This indicates that both antibodies react with a single expression clone. It was further concluded that both antibodies recognize a conformational epitope, since they did not react with protein extracted using 8 M ureum, whereas the anti-WNV antibody that recognizes a linear epitope did react with protein extracted using this procedure (data not shown). In conclusion, CR4348 recognizes an epitope present in the protein FAF-1, while CR4354L4328 recognizes an epitope present in the protein NDUFV-1.
To confirm the identification of NDUFV-1 as the target antigen recognized by CR4354L4328, mRNA was extracted from 2*107 293T cells using the nucleotrap mRNA mini purification kit (Beckton Dickinson) according to protocols provided by the manufacturer. RT-PCR was performed on the isolated mRNA. For the PCR procedure, the following primers were designed: forward primer 5′-ATGAAGGTGACAGCGTGAGGTGAC-3′ (SEQ ID NO:160) and reverse primer 5′-ACATGGATAGACGCAGGACAGCAG-3′ (SEQ ID NO:161). PCR was performed with Pfu (Promega) in the presence of 5% DMSO and resulted in a 1500 bp product. The resulting fragment was cloned in the pCR4TOPO vector (Invitrogen) and transformed into DH5α cells. The resulting clone, TOPONDUFV1 was verified by sequence analysis and aligned with the sequence present in the database. To simplify the detection of the protein in the subsequent transfection experiments, the protein was fused with a myc tag at the 5′ prime end by means of PCR (using the construct as a template). For the 5′ myc construct the following primers were designed: forward primer 5′-AAGCTTAGCATGGAACAAAAACTTATTTCT GAAGAAGATCTGCTGGCAACACGGCGGCTGCTCGGCTG-3′(SEQ ID NO:162) and reversed primer 5′-GATATCCTTTATTGTCCAGCATTCCAC-3′ (SEQ ID NO:163). PCR was performed using Pfu polymerase and the resulting fragment of the 5′ myc tag was cloned in PCRblunt4-TOPO, and subsequently digested with HindIII-EcoRV. The resulting fragment was cloned in the corresponding sites of pcDNA3.1/zeo (Invitrogen) resulting in the mycNDUFV-1 construct. The construct was verified by sequencing. All cloning procedures were performed according to standard molecular techniques. 3*105 293T cells were seeded in T175 flasks and subjected to a transfection procedure after 72 hours. The expression construct mycNDUFV-1 and a positive control construct ATAD3Amyc were transfected with lipofectamin (Gibco) according to the manufacturer's instructions. 72 hours after transfection, cells were lysed in DOC buffer (1% Triton X-100 and 0.5% w/v desoxycholate in 0.2 M phosphate buffer containing 0.12 M NaCl, pH 7.4 and protease inhibitors (Sigma)). The insoluble material was removed by centrifugation for 30 minutes at 4° C. at 20,000*g. Next, the lysates of the transfected cells were analyzed for the quantity of myc tagged protein expressed. Hereafter, the lysate was pre-cleared with protein A beads (Amersham) for 2 hours at 4° C. In the mean time, 4 μg of CR4354L4328, control antibody CR4374 (negative control, antibody directed against WNV E protein), and control antibody CR2361 IgG1 (positive control; antibody directed against ATAD3A) were coupled to protein A beads at room temperature. Next, the pre-cleared samples were incubated with the IgGs coupled to the beads for 2 hours at 4° C. The protein A beads were washed three times for 5 minutes with 1 ml of DOC lysis buffer and bound complexes were eluted by the addition of sample loading buffer. The samples were subjected to SDS-PAGE under non-reducing conditions. After blotting on PVDF membranes, the myc tagged proteins were detected with the HRP conjugated anti-myc monoclonal antibody 9E10 (Amersham). Immunoblot developed with anti-myc antibody demonstrated that mycNDUFV-1 was only immunoprecipitated by CR4354L4328 and not by the any of the other antibodies (see FIG. 5). In addition, ATAD3Amyc protein was only immunoprecipitated by CR2361 (data not shown).
Example 11
Distribution of the Antigen Recognized by CR4348 and CR4354L4328 on 293T Cells and VLP-Transfected HEK293T Cells as Shown by FACS
The distribution of the target antigens recognized by the antibodies CR4348 and CR4354L4328 was analyzed by flow cytometry in three different ways: a) using HEK293T cells in permeabilized format, b) using HEK293T cells in non-permeablized format, and c) using non-permeabilized WNV VLP-transfected HEK293T cells. 293T cells were obtained from the ATCC CRL-11268 and were harvested with trypsin/EDTA. One part of the cells was fixed and permeabilized for intracellular staining with the DakoCytomation IntraStain (K2311) according to the manufacturer's instruction, the other part of the cells was stained extracellularly and was incubated directly after harvest with the antibodies. For each sample, 100.000 cells were incubated with 2.5 μg/ml CR4354L4328, CR4348 or the control antibodies CR2300, CR4374 and CR4104 diluted in PBS containing 1% BSA. CR2300 is a positive control antibody (recognizing CD46 which is present on nucleated cells); CR4374 recognizes WNV E protein and is the positive control antibody for VLP-transfected 293T cells and the negative antibody for non-transfected 293T cells; and CR4104 recognizes rabies glycoprotein and is included as negative control for all stainings. The antibodies were incubated with the cells for 1 hour on ice and the cells were washed three times with PBS/BSA. Binding of the antibodies to the cells was visualized after incubation for 1 hour on ice with goat anti-human phycoerythrin antibody (Pharmingen). The cells were washed twice in PBS/BSA prior to analysis. In the extracellular staining experiment dead or permeable cells were excluded from analysis by staining with 7-AAD, a dye that stains nuclear DNA. VLP-transfected 293T cells were harvested 24 hour after transfection and stained according to the aforementioned method for extracellular staining. Cells were analyzed on a FACS calibur (BD) using CellQuest software. For final analysis of the extracellular stained cells, cells were gated based on forward scatter versus low 7-AAD signal. A sample was considered positive if the mean fluorescence intensity was more than two times the signal obtained with the negative control antibody. As is shown in Table 17, CR4348 and CR4354L4328 both recognized an intracellular target antigen that is not expressed at the cell surface under normal culturing conditions. However, after transfection with the DNA construct for WNV-VLP production the target antigens of CR4348 and CR4354L4328 were detected at the cell surface. The antibodies specifically reacted with antigens expressed after WNV-VLP transfection, since they did not bind the cell surface of mock-transfected cells (data not shown). When the antibodies were applied in a serial dilution, they bound the target antigens in a dose dependent way (data not shown).
From these combined expression data it was concluded that the antigens recognized by CR4348 and CR4354L4328 are located intracellularly in healthy, normal cells. However, after mimicking a WNV-infection by introducing a WNV-VLP construct in the cells the antigens became expressed at the cellular surface.
Example 12
Intracellular Location of the Target Antigens of CR4354L4328 and CR4348 Demonstrated by Immunofluorescence
The intracellular location of the CR4354L4328 and CR4348 target antigens was analyzed by immunofluorescent staining of cells. VERO cells and 293T cells were seeded at a confluency of 20% in four-well LAB-TEK chambers (Nunc). 24 Hours after seeding of the cells one part of the VERO cells was infected with 8.1E-3 TCID50/cell of WNV strain 385-99. One part of the 293T cells was transfected with the WNV-VLP construct. The VERO and 293T cells that were not used for infection or transfection were allowed to grow for an additional 24 hours. 24 Hours after infection or transfection all VERO and 293T cells were fixed with 2% formaldehyde in PBS for 15 minutes at room temperature. Thereafter they were kept in PBS until the staining procedure. To allow the antibodies to react with the intracellular antigens in the cell, the cells were permeabilized with 0.2% Triton X-100 in PBS for 5 minutes at room temperature and rinsed for 15 minutes. Next, the cells were blocked with 2% BSA in PBS for 1 hour at room temperature. Subsequently, different stainings were performed. Dual stainings: CR4354L4328, CR4348, CR4104 and CR4283 antibodies were applied at a concentration of 5 μg/ml in 2% BSA for 1 hour at room temperature. After 3 washes of 5 minutes in PBS the antibodies were visualized by adding Alexa fluor 488 goat anti-human antibody at a concentration of 5 μg/ml in 2% BSA containing 5 units of phalloidin bodipy (Molecular probes) to stain the cytoskeletal structure. The cells were incubated for 1 hour at room temperature in the dark. Next, three washes of 5 minutes were performed with PBS and anti fading agent was added (Vectashield) to prevent bleaching of the fluorescent signal. Then, a glass cover slip was glued on top with opaque nail polish (Miss Helen, HEMA). Triple stainings were performed to analyze co-localization of CR4348 and CR4354L4328 with their target antigens FAF-1 and NDUFV1. Slides were viewed under a Leica TCS-NT confocal laser scanning microscope. Staining of uninfected VERO cells with CR4348 demonstrated a network like pattern consisting of tiny vesicles. After infection with WNV, bigger vesicles appeared that were scattered throughout the cytoplasm. Staining of uninfected VERO cells with CR4354L4328 demonstrated a bright nuclear staining. After infection of VERO cells with WNV, densely packed vesicles appeared throughout the cytoplasm and staining of the nucleus disappeared. In 293T cells the target for CR4348 was localized in vesicle-like structures close to the cellular membrane. After transfection with the VLP construct a brighter staining pattern appeared and vesicles were also localized throughout the cytoplasm. In 293T cells CR4354L4328 stains small vesicles that were localized throughout the cytoplasm in densely packed structures. After transfection of the VLP construct the appearance of the vesicles the densely packed structures disappeared and the vesicles became scattered throughout the cell and showed a bright staining pattern.
Example 12
Neutralization of Rabies Virus with CR4354L4328
To investigate the neutralizing activity of CR4354L4328 on other viral species, the antibody was tested in a RFITT experiment for the neutralization of rabies virus. The RFFIT was performed by mixing serial five-fold dilutions of CR4354L4328, CR4374 (negative control; anti-WNV E protein antibody) and CR57 (positive control; anti-rabies virus antibody) with a constant amount of rabies virus (50 FFD50/0.1 ml) in a multi-chambered slide. One unit of virus is determined as the dilution at which 50% of the observed microscopic fields contain one or more foci of infected cells, i.e., the focus forming dose, FFD50. After allowing the mixture to react in a CO2-incubator at 37° C. for 90 minutes, mouse neuroblastoma (MNA) cells in Eagle's minimum essential medium with 10% fetal bovine serum (MEM-10) were added to each monoclonal antibody-virus mixture resulting in a final concentration of 1*105 cells/ml. The monoclonal antibody-virus-cell cultures were incubated for 20 hours in a CO2-incubator at 37° C. The cultures were removed from the incubator, washed, fixed and then stained with an anti-rabies virus antibody conjugate directed against the nucleoprotein and observed under a fluorescence microscope for the presence of fluorescent cells; 20 microscopic fields (160×-200×) were read for each monoclonal antibody dilution and compared against the virus control (50 FFD50/0.1 ml), which should contain 18 to 20 fields with fluorescent cells. The 50% neutralization endpoint of a particular antibody is defined as the dilution by which 50% or more of the observed microscopic fields contain one or more infected cells and was calculated using the Reed-Muench method. Virus neutralizing antibody titers were normalized to international units (IU) using the NIH US standard human Rabies Immune Globulin reference lot R3 (SRIG). The 50% neutralization point of CR57 was obtained at a concentration of 7.3 ng/ml. The 50% neutralization point of CR4354 was obtained at a concentration of 9.2 μg/ml. The control antibody CR4374 could not neutralize rabies virus at any concentration tested. The results clearly show that CR4354L4328 has rabies virus neutralizing activity.







TABLE 1







Human lambda chain variable region primers


(sense).











Primer
Primer nucleotide




name
sequence
SEQ ID NO







HuVλ1A
5′-CAGTCTGTGCTGACT
SEQ ID NO: 79




CAGCCACC-3′




 



HuVλ1B
5′-CAGTCTGTGYTGACG
SEQ ID NO: 80




CAGCCGCC-3′




 



HuVλ1C
5′-CAGTCTGTCGTGACG
SEQ ID NO: 81




CAGCCGCC-3′




 



HuVλ2
5′-CARTCTGCCCTGACT
SEQ ID NO: 82




CAGCCT-3′




 



HuVλ3A
5′-TCCTATGWGCTGACT
SEQ ID NO: 83




CAGCCACC-3′




 



HuVλ3B
5′-TCTTCTGAGCTGACT
SEQ ID NO: 84




CAGGACCC-3′




 



HuVλ4
5′-CACGTTATACTGACT
SEQ ID NO: 85




CAACCGCC-3′




 



HuVλ5
5′-CAGGCTGTGCTGACT
SEQ ID NO: 86




CAGCCGTC-3′




 



HuVλ6
5′-AATTTTATGCTGACT
SEQ ID NO: 87




CAGCCCCA-3′




 



HuVλ7/8
5′-CAGRCTGTGGTGACY
SEQ ID NO: 88




CAGGAGCC-3′




 



HuVλ9
5′-CWGCCTGTGCTGACT
SEQ ID NO: 89




CAGCCMCC-3′

















TABLE 2







Human kappa chain variable region primers


(sense).











Primer
Primer nucleotide




name
sequence
SEQ ID NO







HuVκ1B
5′-GACATCCAGWTGACC
SEQ ID NO: 90




CAGTCTCC-3′




 



HuVκ2
5′-GATGTTGTGATGACT
SEQ ID NO: 91




CAGTCTCC-3′




 



HuVκ3
5′-GAAATTGTGWTGACR
SEQ ID NO: 92




CAGTCTCC-3′




 



HuVκ4
5′-GATATTGTGATGACC
SEQ ID NO: 93




CACACTCC-3′




 



HuVκ5
5′-GAAACGACACTCACG
SEQ ID NO: 94




CAGTCTCC-3′




 



HuVκ6
5′-GAAATTGTGCTGACT
SEQ ID NO: 95




CAGTCTCC-3′

















TABLE 3







Human kappa chain variable region primers extended with SalI restriction


sites (sense), human kappa chain J-region primers extended with NotI


restriction sites (anti-sense), human lambda chain variable region primers


extended with SalI restriction sites (sense) and human lambda chain


J-region primers extended with NotI restriction sites (anti-sense).









Primer name
Primer nucleotide sequence
SEQ ID NO





HuVκ1B-SalI
5′-TGAGCACACAGGTCGACGGACATCCAGWTGACCCAG
SEQ ID NO: 96



TCTCC-3′



 


HuVκ2-SalI
5′-TGAGCACACAGGTCGACGGATGTTGTGATGACTCAGT
SEQ ID NO: 97



CTCC-3′



 


HuVκ3B-SalI
5′-TGAGCACACAGGTCGACGGAAATTGTGWTGACRCAG
SEQ ID NO: 98



TCTCC-3′



 


HuVκ4B-SalI
5′-TGAGCACACAGGTCGACGGATATTGTGATGACCCAC
SEQ ID NO: 99



ACTCC-3′



 


HuVκ5-SalI
5′-TGAGCACACAGGTCGACGGAAACGACACTCACGCAG
SEQ ID NO: 100



TCTCC-3′



 


HuVκ6-SalI
5′-TGAGCACACAGGTCGACGGAAATTGTGCTGACTCAG
SEQ ID NO: 101



TCTCC-3′



 


HuJκ1-NotI
5′-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATTTCC
SEQ ID NO: 102



ACCTTGGTCCC-3′



 


HuJκ2-NotI
5′-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATCTCC
SEQ ID NO: 103



AGCTTGGTCCC-3′



 


HuJκ3-NotI
5′-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATATCC
SEQ ID NO: 104



ACTTTGGTCCC-3′



 


HuJκ4-NotI
5′-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATCTCC
SEQ ID NO: 105



ACCTTGGTCCC-3′



 


HuJκ5-NotI
5′-GAGTCATTCTCGACTTGCGGCCGCACGTTTAATCTCC
SEQ ID NO: 106



AGTCGTGTCCC-3′



 


HuVλ1A-SalI
5′-TGAGCACACAGGTCGACGCAGTCTGTGCTGACTCAGC
SEQ ID NO: 107



CACC-3′



 


HuVλ1B-SalI
5′-TGAGCACACAGGTCGACGCAGTCTGTGYTGACGCAG
SEQ ID NO: 108



CCGCC-3′



 


HuVλ1C-SalI
5′-TGAGCACACAGGTCGACGCAGTCTGTCGTGACGCAG
SEQ ID NO: 109



CCGCC-3′



 


HuVλ2-SalI
5′-TGAGCACACAGGTCGACGCARTCTGCCCTGACTCAGC
SEQ ID NO: 110



CT-3′



 


HuVλ3A-SalI
5′-TGAGCACACAGGTCGACGTCCTATGWGCTGACTCAG
SEQ ID NO: 111



CCACC-3′



 


HuVλ3B-SalI
5′-TGAGCACACAGGTCGACGTCTTCTGAGCTGACTCAGG
SEQ ID NO: 112



ACCC-3′



 


HuVλ4-SalI
5′-TGAGCACACAGGTCGACGCACGTTATACTGACTCAAC
SEQ ID NO: 113



CGCC-3′



 


HuVλ5-SalI
5′-TGAGCACACAGGTCGACGCAGGCTGTGCTGACTCAG
SEQ ID NO: 114



CCGTC-3′



 


HuVλ6-SalI
5′-TGAGCACACAGGTCGACGAATTTTATGCTGACTCAGC
SEQ ID NO: 115



CCCA-3′



 


HuVλ7/8-SalI
5′-TGAGCACACAGGTCGACGCAGRCTGTGGTGACYCAG
SEQ ID NO: 116



GAGCC-3′



 


HuVλ9-SalI
5′-TGAGCACACAGGTCGACGCWGCCTGTGCTGACTCAG
SEQ ID NO: 117



CCMCC-3′



 


HuJλ1-NotI
5′-GAGTCATTCTCGACTTGCGGCCGCACCTAGGACGGTG
SEQ ID NO: 118



ACCTTGGTCCC-3′



 


HuJλ2/3-NotI
5′-GAGTCATTCTCGACTTGCGGCCGCACCTAGGACGGTC
SEQ ID NO: 119



AGCTTGGTCCC-3′



 


HuJλ4/5-NotI
5′-GAGTCATTCTCGACTTGCGGCCGCACYTAAAACGGTG
SEQ ID NO: 120



AGCTGGGTCCC-3′
















TABLE 4







Distribution of the different light chain


products over the ten fractions.













Number of
Fraction
alleles/



Light chain products
alleles
number
fraction
















Vk1B/Jk1-5
19
1 and 2
9.5



Vk2/Jk1-5
9
3
9



Vk3B/Jk1-5
7
4
7



Vk4B/Jk1-5
1
5
5



Vk5/Jk1-5
1



Vk6/Jk1-5
3



Vλ1A/Jl1-3
5
6
5



Vλ1B/Jl1-3



Vλ1C/Jl1-3



Vλ2/Jl1-3
5
7
5



Vλ3A/Jl1-3
9
8
9



Vλ3B/Jl1-3



Vλ4/Jl1-3
3
9
5



Vλ5/Jl1-3
1



Vλ6/Jl1-3
1



Vλ7/8/Jl1-3
3
10
6



Vλ9/Jl1-3
3

















TABLE 5







Human IgG heavy chain variable region primers


(sense).









Primer
Primer nucleotide



name
sequence
SEQ ID NO





HuVH1B/7A
5′-CAGRTGCAGCTGGTG
SEQ ID NO: 121



CARTCTGG-3′



 


HuVH1C
5′-SAGGTCCAGCTGGTR
SEQ ID NO: 122



CAGTCTGG-3′



 


HuVH2B
5′-SAGGTGCAGCTGGTG
SEQ ID NO: 123



GAGTCTGG-3′



 


HuVH3B
5′-SAGGTGCAGCTGGTG
SEQ ID NO: 124



GAGTCTGG-3′



 


HuVH3C
5′-GAGGTGCAGCTGGTG
SEQ ID NO: 125



GAGWCYGG-3′



 


HuVH4B
5′-CAGGTGCAGCTACAG
SEQ ID NO: 126



CAGTGGGG-3′



 


HuVH4C
5′-CAGSTGCAGCTGCAG
SEQ ID NO: 127



GAGTCSGG-3′



 


HuVH5B
5′-GARGTGCAGCTGGTG
SEQ ID NO: 128



CAGTCTGG-3′



 


HuVH6A
5′-CAGGTACAGCTGCAG
SEQ ID NO: 129



CAGTCAGG-3′
















TABLE 6







Human IgG heavy chain variable region primers extended with SfiI/NcoI


restriction sites (sense) and human IgG heavy chain J-region primers


extended with XhoI/BstEII restriction sites(anti-sense).









Primer name
Primer nucleotide sequence
SEQ ID NO





HuVH1B/7A-SfiI
5′-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCC
SEQ ID NO: 130



AGRTGCAGCTGGTGCARTCTGG-3′



 


HuVH1C-SfiI
5′-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCSA
SEQ ID NO: 131



GGTCCAGCTGGTRCAG TCTGG-3′



 


HuVH2B-SfiI
5′-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCA
SEQ ID NO: 132



GRTCACCTTGAAGGAGTCTGG-3′



 


HuVH3B-SfiI
5′-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCSAG
SEQ ID NO: 133



GTGCAGCTGGTGGAGTCTGG-3′



 


HuVH3C-SfiI
5′-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGA
SEQ ID NO: 134



GGTGCAGCTGGTGGAGWCYGG-3′



 


HuVH4B-SfiI
5′-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCA
SEQ ID NO: 135



GGTGCAGCTACAGCAGTGGGG-3′



 


HuVH4C-SfiI
5′-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCC
SEQ ID NO: 136



AGSTGCAGCTGCAGGAGTCSGG-3′



 


HuVH5B-SfiI
5′-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCG
SEQ ID NO: 137



ARGTGCAGCTGGTGCAGTCTGG-3′



 


HuVH6A-SfiI
5′-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCC
SEQ ID NO: 138



AGGTACAGCTGCAGCAGTCAGG-3′



 


HuJH1/2-XhoI
5′-GAGTCATTCTCGACTCGAGACGGTGACCAGGGT
SEQ ID NO: 139



GCC-3′



 


HuJH3-XhoI
5′-GAGTCATTCTCGACTCGAGACGGTGACCATTGT
SEQ ID NO: 140



CCC-3′



 


HuJH4/5-XhoI
5′-GAGTCATTCTCGACTCGAGACGGTGACCAGGGTT
SEQ ID NO: 141



CC-3′



 


HuJH6-XhoI
5′-GAGTCATTCTCGACTCGAGACGGTGACCGTGGTC
SEQ ID NO: 142



CC-3′
















TABLE 7







Binding of single chain (scFv) phage antibodies to West


Nile virus (WNV), recombinant WNV E protein, FBS, and


rabies virus as measured by ELISA at 492 nm).












Name phage
WN
WNV E
WNV-like
FBS
Rabies


antibody
virus
protein
particle
(5%)
virus





SC04-348
1.026
0.093
0.771
ND
0.063


SC04-354
1.041
0.069
0.811
ND
0.063


SC02-447
0.094
0.057
ND
0.041
ND


SC03-014
0.061
0.060
ND
ND
0.062


Pos. control
0.067
0.056
0.062
ND
0.991





ND means not determined













TABLE 8







Data of the WNV specific single chain Fvs.













SEQ ID NO
SEQ ID NO of





Name
of nucl.
amino acid
HCDR3




scFv
sequence
sequence*
(SEQ ID NO:)
VH-locus
VL-locus





SC04-348
25
26
DKSYYYGSGTSGG
3-09 (DP-31)
Vk I




(Vh 1-124;
WFDP

(O12/O2-DPK9)




Vl 143-250)
(SEQ ID NO: 5)




 


SC04-354
27
28
DWGSNYVWGSYP
1-46 (DP-7)
Vl 1




(Vh 1-121;
KY

(1c - V1-16)




Vl 140-250)
(SEQ ID NO: 6)





*between brackets the amino acids making up the heavy chain variable region (VH) and the light chain variable region (VL) is shown













TABLE 9







Data of the CDR regions of the WNV specific single chain Fvs.













HCDR1
HCDR2
LCDR1
LCDR2
LCDR3



(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID


Name scFv
NO:)
NO:)
NO:)
NO:)
NO:)















SC04-348
7
8
9
10
11


SC04-354
12
13
14
15
16
















TABLE 10







Binding of IgG1 antibodies to WNV as measured by ELISA (OD 492 nm).









Antibody Concentration (μg/ml)


















Ab
20.000
10.000
5.000
2.500
1.250
0.630
0.310
0.160
0.078
0.039
0.000





CR4348
ND
0.370
0.343
0.300
0.251
0.192
0.143
0.122
0.104
0.085
0.003


CR4354
0.291
0.262
0.217
0.167
0.151
0.106
0.067
0.034
0.014
0.010
0.003


Neg. control
0.051
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND





ND means not determined













TABLE 11







Potency of the anti-WNV antibodies in the


66% neutralizing antibody titer assay.










Antibody name
μg/ml







CR4348
1.97



CR4354
0.48

















TABLE 12







Protection from lethal WNV challenge


in mice by monoclonal antibodies.










Antibody (15 mg/kg)
Surviving animals







CR4348
5/5



CR4354
5/5



7H2
5/5



Negative Control IgG1
0/5

















TABLE 13







Probit analysis of the protective activity of human


IgG1 in a murine lethal WNV challenge model












50% protection
95% protection



Antibody
(μg/kg)
(μg/kg)















CR4354L4328
2.74
57.4



CR4348
6.26
131

















TABLE 14







Percentage difference in 66% neutralization concentration


of IgG1 variants of CR4354 against WNV as measured by VNA.










Antibody
Potency (%)*







CR4354
100



CR4354L4261
106



CR4354L4267
Below detection



CR4354L4328
286



CR4354L4335
 60



CR4354L4383
Below detection







*Potency is represented in comparison to original antibody CR4354 (the 66% neutralizing concentration of which was set at 100%) and was calculated by dividing the 66% neutralizing concentration (in μg/ml) of CR4354 by the 66% neutralizing concentration (in μg/ml) of the chain shuffled variants and multiplying the resulting number by 100%.













TABLE 15







Neutralizing potency against West Nile virus


strain USA99b as measured by PRNT.












PRNT50 (95% CI)
PRNT90 (95% CI)



Antibody
(μg/ml)
(μg/ml)
















CR4348
3.72 (3.21-4.27)
65.2
(52.3-84.1)



CR4354L4328
21.2 (10.1-53.4)
1602
(397-20000)



CRM4354
0.17 (0.11-0.25)
4.3
(2.85-7.29)

















TABLE 16







Clones reactive with CR4348 and CR4354L4328 as recognized by protein micro-array analysis.










Antibody
Reactive clone
Blast
Target (Homo sapiens)





CR4348
MPMGp800B14568
gi|19528653|ref|NM_007051.2|
Fas(TNFRSF6) associated





factor 1 (FAF1)


CR4348
MPMGp800G07549Q170
gi|27924136|gb|BC044933.1|
clone IMAGE: 4540326,





mRNA, partial cds


CR4348
MPMGp800O05569Q170
gi|54607125|ref|NM_013364.2|
paraneoplastic antigen MA3





(PNMA3), mRNA


CR4348
MPMGp800E02577Q
gi|34364672|emb|BX640642.1
mRNA; cDNA





DKFZp686K01114


CR4348
MPMGp800M04560Q183
gi|39761682|gb|AY405708.1|
CYLN2 gene, VIRTUAL





TRANSCRIPT


CR4354L4328
MPMGp800J14549Q
gi|14198175|gb|BC008146.1|
NADH dehydrogenase





(ubiquinone) flavoprotein 1


CR4354L4328
MPMGp800K13552Q
gi|62087473|dbj|AB208947.1|
mRNA for chordin isoform a





variant protein


CR4354L4328
MPMGp800A02549Q
gi|17149812|ref|NM_057161.2|
kelch domain containing 3





(KLHDC3)


CR4354L4328
MPMGp800I05513Q
gi|4753262|gb|AC006360.2
PAC clone RP5-1140N14





from 14q24.3
















TABLE 17







Mean fluorescence measured after incubation


with 2.5 μg/ml antibody.













293T
293T
293T-VLP



Antibody
extracellular
intracellular
extracellular
















CR4348
16
225
15



CR4354L4328
16
840
11



CR4104
15
55
4



CR4374
15
57
19



CR2300
484
236
10










REFERENCES


    Beasley D. W. and A. D. Barrett (2002), Identification of neutralizing epitopes within structural domain III of the West Nile virus envelope protein. J. Virol. 76:13097-13100.
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    Ben-Nathan D., S. Lustig, G. Tam, S. Robinzon, S. Segal and B. Rager-Zisman (2003), Prophylactic and therapeutic efficacy of human intravenous immunoglobulin in treating WNV infection in mice. J. Infect. Dis. 188:5-12.
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    Wang T., J. F. Anderson, L. A. Magnarelli, S. J. Wong, R. A. Koski and E. Fikrig (2001), Immunization of mice against West Nile virus with recombinant envelope protein. J. Immunol. 167:5273-5277.

## Claims

1. A composition comprising a monoclonal antibody comprising a heavy and a light chain, wherein
the heavy chain comprises the CDR1 region of SEQ ID NO:12, the CDR2 region of SEQ ID NO:13, and the CDR3 region of SEQ ID NO:6; and
the light chain comprises:
the CDR1 region of SEQ ID NO:14, the CDR2 region of SEQ ID NO:15, and the CDR3 region of SEQ ID NO:16,
or the sequence of SEQ ID NO:74.

2. The composition of claim 1 further comprising at least one tag.

3. A nucleic acid molecule encoding a monoclonal antibody comprising a heavy and a light chain, wherein
the heavy chain comprises the CDR1 region of SEQ ID NO:12, the CDR2 region of SEQ ID NO:13, and the CDR3 region of SEQ ID NO:6; and
the light chain comprises:
the CDR1 region of SEQ ID NO:14, the CDR2 region of SEQ ID NO:15, and the CDR3 region of SEQ ID NO:16, or
the sequence of SEQ ID NO:74.

4. The composition of claim 1, further comprising at least one pharmaceutically acceptable excipient.

5. In a method of diagnosing and/or treating an infection caused by a flavivirus in a subject, the improvement comprising:
utilizing the composition of claim 1 in the diagnosis and/or treatment of the flavivirus infection in the subject, wherein said flavivirus is a West Nile Virus.