Patent Grant
9809643
This disclosure concerns methods of identifying lyssavirus-specific antibodies and the use of such antibodies in the treatment and prophylaxis of rabies.
Rabies is an inevitably fatal but preventable disease. In developing countries rabies remains a significant endemic disease burden. World-wide approximately 55,000 people die of rabies each year (WHO Expert Consultation on Rabies, 2004). Rabies is preventable with proper early post-exposure treatment. Currently, post-exposure prophylaxis includes thorough wound-washing with soap and water followed by administration of vaccine and anti-rabies virus immunoglobulin (RIG) of human or equine origin. RIG administered shortly after exposure at the wound site provides passive immunity which neutralizes rabies virus and prevents its spread until the patient's immune response following vaccination is elicited. Deaths due to post-exposure prophylaxis failure are most commonly attributed to deviations from the recommended regimen such as late initiation of post-exposure prophylaxis or no administration of RIG (Wilde, Vaccine, 25:7605-7609, 2007). In developing countries, availability of RIG is extremely low with only 1-2% of post-exposure prophylaxis being performed using RIG (Sudarshan et al., Int J Infect Dis, 11:29-35, 2007; WHO Consultation on a Monoclonal Antibody Cocktail for Rabies Post Exposure Treatment, 2002). In the United States, only human derived RIG is administered due to the risk of anaphylactic shock from exposure to equine immunoglobulins, but the concern of blood-born pathogen transmission in human RIG remains.
Human monoclonal antibodies (mAbs) that neutralize rabies virus have long been recognized as an alternative to overcome the limitations of RIG (Dietzschold et al., J Virol, 64:3087-3090, 1990). Adequate supplies of cell-cultured human mAbs could be produced in a cost-effective manner (Prosniak et al., J Infect Dis, 188:53-56, 2003). In addition, the use of human mAbs reduces the likelihood of an adverse immune response (Weiner, J Immunother, 29:1-9, 2006) and has been shown to be as effective as RIG in preventing rabies in animals (de Kruif et al., Annu Rev Med, 58:359-368, 2007). When a cocktail of two rabies-neutralizing, human mAbs was given with a rabies vaccine to animals experimentally infected with rabies, dose-dependent survival was observed, and all animals receiving the highest dose survived (Goudsmit et al., J Infect Dis, 193:796-801, 2006). The same cocktail was recently shown to be safe to administer to healthy humans in two phase-one clinical trials (Bakker et al., Vaccine, 26:5922-5927, 2008). However, selection of human monoclonal antibodies to include in such a cocktail has some limitations. The diversity of mAbs produced depends significantly on the diversity of viral antigens used to immunize human donors.
Rabies virus is a member (genotype 1) of the genus Lyssavirus. This genus also includes rabies-like viruses (genotypes 2-7) which can cause rabies disease in humans (Bourhy et al., Virology, 194:70-81, 1993; Gould et al., Virus Res, 54:165-187 1998). These viruses have a non-segmented, negative-sense, single-stranded RNA genome that encodes five proteins (Sokol et al., Virology, 38:651-665, 1969; Sokol et al., J Virol, 7:241-249, 1971). In the mature virion, RNA-dependent RNA polymerase, phosphoprotein, and nucleocapsid protein are associated with the genomic RNA while matrix protein and glycoprotein (G protein) surround it (Wiktor et al., J Immunol, 110:269-276, 1973). Trimeric G protein “spikes” coat the surface of the virion and as the only surface exposed protein, is responsible for attachment and entry into host cells. This also makes G protein the primary antigen for induction of virus-neutralizing antibodies, and G protein-specific mAbs are included in the cocktails currently being developed for post-exposure prophylaxis (Dietzschold et al., J Virol, 64:3087-3090, 1990; Kramer et al., Eur J Immunol, 35:2131-2145, 2005; Wunner et al., “Rabies Virus” in Rabies (Second Edition),” pp. 23-68, Academic Press, Oxford, 2007).
Disclosed herein is a method of identifying an antibody (or antigen-binding fragment thereof) that specifically binds a plurality of lyssaviruses. Antibodies identified by the methods provided herein can be used, for example, for post-exposure rabies prophylaxis or in the treatment of clinical rabies.
Provided herein is a method of identifying a monoclonal antibody or antigen-binding fragment thereof that specifically binds at least two different lyssaviruses. In some embodiments, the method includes screening a naïve antibody phage display library with at least two different lyssavirus glycoproteins, such as by panning the library against a recombinant virus expressing at least two different lyssavirus glycoproteins, panning the library against recombinant glycoprotein from at least two different lyssaviruses, panning the library against at least two different cell lines expressing different lyssavirus glycoproteins, or any combination thereof.
In particular embodiments, the method includes screening a naïve antibody phage display library with (1) a recombinant rabies virus expressing glycoprotein from rabies virus (RABV), Mokola virus (MOKV) and West Caucasian bat virus (WCBV); (2) recombinant glycoprotein from at least two of RABV, MOKV, WCBV, Lagos bat virus (LBV) and Duvenhage virus (DUVV); and/or (3) at least two different cell lines expressing different lyssavirus glycoproteins selected from RABV, MOKV, WCBV, LBV and DUVV glycoprotein. The method further includes selecting a phage display clone that specifically binds to at least two different lyssaviruses, at least two different lyssavirus glycoproteins, or both.
In some embodiments, the phage display library is a naïve human VH domain library.
Also provided are isolated monoclonal antibodies (or antigen-binding fragments thereof) identified according to the methods disclosed herein and their use in the treatment or prophylaxis of rabies.
Further provided are isolated monoclonal antibodies (or antigen-binding fragments thereof) that specifically bind at least two different lyssaviruses or that specifically bind recombinant glycoprotein from at least two different lyssaviruses. In some embodiments, the VH domain of the antibody is encoded by a nucleotide sequence at least 85% identical to any one of SEQ ID NOs: 1-110. Also provided are methods of treating rabies in a subject by administering to the subject a monoclonal antibody disclosed herein.
Expression vectors, such as an Fc IgG expression vector, comprising the nucleotide sequence of any one of SEQ ID NOs: 1-110 are also provided by the present disclosure. In some embodiments, the expression vector further comprises the nucleotide sequence of a variable light (VL) domain from a lyssavirus-specific (such as rabies virus-specific) monoclonal antibody. A cell comprising an expression vector disclosed herein is further provided. Also provided are monoclonal antibodies encoded by the expression vectors. A method of treating rabies in a subject by administering to the subject a monoclonal antibody expressed by an expressed vector disclosed herein is also provided.
The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.
The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The Sequence Listing is submitted as an ASCII text file, created on Jul. 24, 2015, 102 KB, which is incorporated by reference herein. In the accompanying sequence listing:
SEQ ID NOs: 1-43 are the nucleotide sequences of human variable heavy (VH) domain phage display clones selected using a recombinant ERA RABV expressing glycoprotein (G) from RABV, MOKV and WCBV.
SEQ ID NOs: 44-110 are the nucleotide sequences of human variable heavy (VH) domain phage display clones identified by sequential selection on cells expressing LBV glycoprotein, cells expressing MOKV glycoprotein, cells expressing WCBV glycoprotein and cells expressing DUVV glycoprotein.
SEQ ID NOs: 111 and 112 are the nucleotide sequences of RT-PCR primers for amplification of the MOKV G gene.
SEQ ID NOs: 113 and 114 are the nucleotide sequences of RT-PCR primers for amplification of the WCBV G gene.
SEQ ID NO: 115 is the nucleotide sequence of MOKV G.
SEQ ID NO: 116 is the nucleotide sequence of WCBV G.
SEQ ID NO: 117 is the nucleotide sequence of a transcription unit for incorporating heterologous ORFs.
SEQ ID NO: 118 is the nucleotide sequence of LBV G.
SEQ ID NO: 119 is the nucleotide sequence of DUVV G.
ABLV Australian bat lyssavirus
ARAV Aravan virus
BBLV Bokeloh bat lyssavirus
CDR complementarity determining region
dAb domain antibody
DUVV Duvenhage virus
EBLV-1 European bat lyssavirus-1
EBLV-2 European bat lyssavirus-2
ELISA enzyme-linked immunosorbent assay
ERA Evelyn-Rokitnicki-Abelseth
G glycoprotein
IRKV Irkut virus
KHUV Khujand virus
L RNA-dependent RNA polymerase
LBV Lagos bat virus
M matrix protein
mAb monoclonal antibody
MOKV Mokola virus
N nucleoprotein
P phosphoprotein
RABV rabies virus
RIG anti-rabies virus immunoglobulin
RNP ribonucleoprotein
RABV rabies virus
RFFIT rapid fluorescent focus inhibition test
SHIBV Shimoni bat virus
VH variable heavy
VL variable light
WCBV West Caucasian bat virus
Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).
In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:
Administer: As used herein, administering a composition, such as an antibody, to a subject means to give, apply or bring the composition into contact with the subject. Administration can be accomplished by any of a number of routes, such as, for example, topical, oral, subcutaneous, intramuscular, intraperitoneal, intravenous, intrathecal and intramuscular.
Animal: Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and non-human mammals. The term “animal” includes both human and veterinary subjects, for example, humans, non-human primates, dogs, cats, horses, raccoons, bats, rats, mice, foxes, squirrels, opossum, coyotes, wolves and cows.
Antibody: A protein (or protein complex) that includes one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
The basic immunoglobulin (antibody) structural unit is generally a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” (about 50-70 kDa) chain. The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms “variable light chain” (VL) and “variable heavy chain” (VH) refer, respectively, to these light and heavy chains.
As used herein, the term “antibody” includes intact immunoglobulins as well as a number of well-characterized fragments. For instance, Fabs, Fvs, and single-chain Fvs (scFvs) that bind to target protein (or epitope within a protein or fusion protein) would also be specific binding agents for that protein (or epitope). These antibody fragments are as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab′, the fragment of an antibody molecule obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab′ fragments are obtained per antibody molecule; (3) (Fab′)2, the fragment of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; (4) F(ab′)2, a dimer of two Fab′ fragments held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (6) single chain antibody, a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Methods of making these fragments are routine (see, for example, Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New York, 1999).
Each heavy and light chain contains a constant region and a variable region (the regions are also known as “domains”). In combination, the heavy and the light chain variable regions specifically bind the antigen. Light and heavy chain variable regions contain a “framework” region interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs.” The extent of the framework region and CDRs has been defined (see, Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991). The Kabat database is now maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species, such as humans. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.
The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. An antibody that binds a specific antigen will have a specific VH region and the VL region sequence, and thus specific CDR sequences. Antibodies with different specificities (i.e. different combining sites for different antigens) have different CDRs. Although it is the CDRs that vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs).
References to “VH” or “VH” refer to the variable region of an immunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab. References to “VL” or “VL” refer to the variable region of an immunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.
A “monoclonal antibody” is an antibody produced by a single clone of B lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies. As used herein “monoclonal antibodies” further includes antigen-binding fragments, such as Fv, scFv, dsFv or Fab fragments.
A “chimeric antibody” has framework residues from one species, such as human, and CDRs (which generally confer antigen binding) from another species, such as a murine antibody.
A “humanized” immunoglobulin is an immunoglobulin including a human framework region and one or more CDRs from a non-human (for example a mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is termed a “donor,” and the human immunoglobulin providing the framework is termed an “acceptor.” In one embodiment, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A “humanized antibody” is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. Humanized immunoglobulins can be constructed by means of genetic engineering (see for example, U.S. Pat. No. 5,585,089).
A “human” antibody (also called a “fully human” antibody) is an antibody that includes human framework regions and all of the CDRs from a human immunoglobulin. In one example, the framework and the CDRs are from the same originating human heavy and/or light chain amino acid sequence. However, frameworks from one human antibody can be engineered to include CDRs from a different human antibody. All parts of a human immunoglobulin are substantially identical to corresponding parts of natural human immunoglobulin sequences.
Antibody binding affinity: The strength of binding between a single antibody binding site and a ligand (e.g., an antigen or epitope). The affinity of an antibody binding site X for a ligand Y is represented by the dissociation constant (Kd), which is the concentration of Y that is required to occupy half of the binding sites of X present in a solution. A smaller Kd indicates a stronger or higher-affinity interaction between X and Y and a lower concentration of ligand is needed to occupy the sites. In general, antibody binding affinity can be affected by the alteration, modification and/or substitution of one or more amino acids in the epitope recognized by the antibody paratope. Binding affinity can be measured using any technique known in the art, such as end-point titration in an Ag-ELISA assay.
Antigen: A compound, composition, or substance that can stimulate the production of antibodies or a T-cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens.
Complementarity determining region (CDR): Amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native Ig binding site. The light and heavy chains of an Ig each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively.
Effector molecule (EM): The portion of a chimeric molecule that is intended to have a desired effect on a cell or system or substance to which the chimeric molecule is targeted. The term effector molecule is interchangeable with effector moiety, therapeutic agent, diagnostic agent, and similar terms.
Therapeutic agents include such compounds as nucleic acids, proteins (including monoclonal antibodies and antigen-binding fragments of monoclonal antibodies), peptides, amino acids or derivatives, glycoproteins, radioisotopes, lipids, carbohydrates, recombinant viruses or toxins. Nucleic acid therapeutic and diagnostic moieties include antisense nucleic acids, derivatized oligonucleotides for covalent cross-linking with single or duplex DNA, and triplex forming oligonucleotides. Diagnostic agents or moieties include radioisotopes and other detectable labels. Detectable labels useful for such purposes are also well known in the art, and include radioactive isotopes such as 32P, 125I, and 131I, fluorophores, chemiluminescent agents, and enzymes.
Evelyn-Rokitnicki-Abelseth (ERA): The ERA strain of rabies virus was derived from the Street-Alabama-Dufferin (SAD) strain, first isolated from a rabid dog in Alabama (USA) in 1935. The ERA strain was derived after multiple passages of SAD rabies virus in mouse brains, baby hamster kidney (BHK) cells, and chicken embryos.
Framework region: Amino acid sequences interposed between CDRs (or hypervariable regions). Framework regions include variable light and variable heavy framework regions. Each variable domain comprises four framework regions, often referred to as FR1, FR2, FR3 and FR4. The framework regions serve to hold the CDRs in an appropriate orientation for antigen binding. Framework regions typically form β-sheet structures.
Fusion partner: Refers to any molecule that is fused (such as covalently linked) to another molecule. In the context of the present disclosure, an immunoconjugate includes an antibody linked to a fusion partner. In some examples, the fusion partner is an effector molecule, a label (such as a detectable label), a heterologous polypeptide or a drug.
Fusion protein: A protein generated by expression of a nucleic acid sequence engineered from nucleic acid sequences encoding at least a portion of two different (heterologous) proteins. To create a fusion protein, the nucleic acid sequences must be in the same reading frame and contain no internal stop codons in that frame.
Immunoconjugate: A covalent linkage of a fusion partner, such as an effector molecule, label, heterologous polypeptide or other moiety, to an antibody or antigen binding fragment thereof. The linkage can be by chemical or recombinant means, for instance. In some cases, the linkage is chemical, wherein a reaction between the antibody moiety and the fusion partner has produced a covalent bond formed between the two molecules to form one molecule. A peptide linker (short peptide sequence) can optionally be included between the antibody and the effector molecule.
Inhibiting or treating a disease: Inhibiting the full development of a disease or condition, for example, in a subject who is at risk for a disease. One specific example of a disease is rabies. “Treatment” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. As used herein, the term “ameliorating,” with reference to a disease, pathological condition or symptom, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the number of relapses of the disease, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to the particular disease.
Isolated: An “isolated” or “purified” biological component (such as a nucleic acid, peptide, protein, protein complex, antibody or particle) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, that is, other chromosomal and extra-chromosomal DNA and RNA, and proteins. Nucleic acids, peptides and proteins that have been “isolated” or “purified” thus include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids or proteins. The term “isolated” or “purified” does not require absolute purity; rather, it is intended as a relative term. Thus, for example, an isolated biological component is one in which the biological component is more enriched than the biological component is in its natural environment within a cell, or other production vessel. Preferably, a preparation is purified such that the biological component represents at least 50%, such as at least 70%, at least 90%, at least 95%, or greater, of the total biological component content of the preparation.
Label: A detectable compound or composition that is conjugated directly or indirectly to another molecule to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes.
Lyssavirus: A genus of viruses that is part of the Rhabdoviridae family within the order Mononegavirales (viruses with a single-stranded, negative sense genome). Lyssaviruses are the etiological agents of rabies encephalitis in warm-blooded animals and humans. Lyssavirus species include rabies virus (RABV; genotype 1), Lagos bat virus (LBV; genotype 2), Mokola virus (MOKV; genotype 3), Duvenhage virus (DUVV; genotype 4), European bat lyssavirus-1 (EBLV-1; genotype 5), European bat lyssavirus-2 (EBLV-2; genotype 6) Australian bat lyssavirus (ABLV; genotype 7) and six additional species isolated from bats: four in central Asia and Russia (Aravan virus—ARAV; Khujand virus—KHUV; Irkut virus—IRKV; and West Caucasian bat virus—WCBV), one in Africa (Shimoni bat virus—SHIBV) and one in Europe (Bokeloh bat lyssavirus—BBLV) (Kuzmin et al., Emerg. Infect. Dis. 14(12):1887-1889, 2008; Weyer et al., Epidemiol. Infect. 136:670-678, 2007; Kuzmin et al., Virus Res. 149(2):197-210, 2010; Freuling et al., Emerg. Infect. Dis. 17(8):1519-22, 2011; Kuzmin and Rupprecht, “Bat rabies” In Rabies, 2nd Edition, New York, Academic Press, 2007, pages 259-307, Jackson and Wunner, eds.).
Neutralizing antibody: An antibody that is capable of protecting a subject (or cells) against infection.
ORF (open reading frame): A series of nucleotide triplets (codons) coding for amino acids without any termination codons. These sequences are usually translatable into a peptide.
Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame. If introns are present, the operably linked DNA sequences may not be contiguous.
Phage display: A method for the study of protein-protein, protein-peptide, and protein-DNA interactions that uses bacteriophages to connect proteins with the genetic information that encodes them. Antibody phage display libraries, and methods of generating such libraries, are well known in the art (see, for example, Famm et al., J. Mol. Biol. 376:926-931, 2008; Carmen and Jermutus, Brief Funct Genomic Proteomic 1(2):189-203, 2002; and U.S. Pat. Nos. 6,828,422 and 7,195,866). In the context of the present disclosure, an antibody phage display library is a library of any type of antigen-binding antibody fragment displayed on phage. In particular examples, the antibody phage display library is a VH domain phage display library, or a scFv phage display library. As used herein, a “naïve” antibody (or antibody domain) phage display library refers to a library constructed using subjects that have not been exposed to lyssavirus.
Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers useful in this disclosure are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds or molecules, proteins or antibodies that bind these proteins, viruses or vectors, and additional pharmaceutical agents.
In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
Polypeptide: A polymer in which the monomers are amino acid residues joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used, the L-isomers being preferred for many biological uses. The terms “polypeptide” or “protein” as used herein are intended to encompass any amino acid molecule and include modified amino acid molecules. The term “polypeptide” is specifically intended to cover naturally occurring proteins, as well as those which are recombinantly or synthetically produced.
Conservative amino acid substitutions are those substitutions that, when made, least interfere with the properties of the original protein, that is, the structure and especially the function of the protein is conserved and not significantly changed by such substitutions. Examples of conservative substitutions are shown below.
Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
Amino acids are typically classified in one or more categories, including polar, hydrophobic, acidic, basic and aromatic, according to their side chains. Examples of polar amino acids include those having side chain functional groups such as hydroxyl, sulfhydryl, and amide, as well as the acidic and basic amino acids. Polar amino acids include, without limitation, asparagine, cysteine, glutamine, histidine, selenocysteine, serine, threonine, tryptophan and tyrosine. Examples of hydrophobic or non-polar amino acids include those residues having nonpolar aliphatic side chains, such as, without limitation, leucine, isoleucine, valine, glycine, alanine, proline, methionine and phenylalanine. Examples of basic amino acid residues include those having a basic side chain, such as an amino or guanidino group. Basic amino acid residues include, without limitation, arginine, homolysine and lysine. Examples of acidic amino acid residues include those having an acidic side chain functional group, such as a carboxy group. Acidic amino acid residues include, without limitation aspartic acid and glutamic acid. Aromatic amino acids include those having an aromatic side chain group. Examples of aromatic amino acids include, without limitation, biphenylalanine, histidine, 2-napthylalananine, pentafluorophenylalanine, phenylalanine, tryptophan and tyrosine. It is noted that some amino acids are classified in more than one group, for example, histidine, tryptophan, and tyrosine are classified as both polar and aromatic amino acids. Additional amino acids that are classified in each of the above groups are known to those of ordinary skill in the art.
Substitutions which in general are expected to produce the greatest changes in protein properties will be non-conservative, for instance changes in which (a) a hydrophilic residue, for example, seryl or threonyl, is substituted for (or by) a hydrophobic residue, for example, leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, for example, lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, for example, glutamyl or aspartyl; or (d) a residue having a bulky side chain, for example, phenylalanine, is substituted for (or by) one not having a side chain, for example, glycine.
Purified: The term “purified” does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide, protein, virus, or other active compound is one that is isolated in whole or in part from naturally associated proteins and other contaminants. In certain embodiments, the term “substantially purified” refers to a peptide, protein, virus or other active compound that has been isolated from a cell, cell culture medium, or other crude preparation and subjected to fractionation to remove various components of the initial preparation, such as proteins, cellular debris, and other components.
Rabies: A viral disease that causes acute encephalitis (inflammation of the brain) in warm-blooded animals. Rabies is zoonotic (transmitted by animals), most commonly by a bite from an infected animal but occasionally by other forms of contact. Rabies is almost frequently fatal if post-exposure prophylaxis is not administered prior to the onset of severe symptoms. Rabies is caused by viruses of the Lyssavirus genus.
Rabies virus (RABV or RV): A member of the Rhabdoviridae family having a non-segmented RNA genome with negative sense polarity. Rabies virus is the prototype of the Lyssavirus genus. The rabies virus Evelyn-Rokitnicki-Abelseth (ERA) strain is a strain derived from the Street-Alabama-Dufferin (SAD) strain, first isolated from a rabid dog in Alabama (USA) in 1935. The ERA strain was derived after multiple passages of SAD RABV in mouse brains, baby hamster kidney (BHK) cells, and chicken embryos. The complete genomic sequence of the ERA strain is disclosed in PCT Publication No. WO 2007/047459.
Recombinant: A recombinant nucleic acid, protein or virus is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. In some embodiments, recombinant rabies virus is generated using reverse genetics, such as the reverse genetics system described in PCT Publication No. WO 2007/047459. In some examples, the recombinant rabies viruses comprise one or more mutations in a viral virulence factors, such as glycoprotein. In other examples, the recombinant rabies viruses comprise a heterologous gene, such as a sequence encoding a glycoprotein from another lyssavirus (such as Mokola virus, West Caucasian bat virus or Lagos bat virus).
Sequence identity: The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are.
Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman (Adv. Appl. Math., 2:482, 1981); Needleman and Wunsch (J. Mol. Biol., 48:443, 1970); Pearson and Lipman (Proc. Natl. Acad. Sci., 85:2444, 1988); Higgins and Sharp (Gene, 73:237-44, 1988); Higgins and Sharp (CABIOS, 5:151-53, 1989); Corpet et al. (Nuc. Acids Res., 16:10881-90, 1988); Huang et al. (Comp. Appls. Biosci., 8:155-65, 1992); and Pearson et al. (Meth. Mol. Biol., 24:307-31, 1994). Altschul et al. (Nature Genet., 6:119-29, 1994) presents a detailed consideration of sequence alignment methods and homology calculations.
The alignment tools ALIGN (Myers and Miller, CABIOS 4:11-17, 1989) or LFASTA (Pearson and Lipman, 1988) may be used to perform sequence comparisons (Internet Program © 1996, W. R. Pearson and the University of Virginia, “fasta20u63” version 2.0u63, release date December 1996). ALIGN compares entire sequences against one another, while LFASTA compares regions of local similarity. These alignment tools and their respective tutorials are available on the Internet at the NCSA website. Alternatively, for comparisons of amino acid sequences of greater than about 30 amino acids, the “Blast 2 sequences” function can be employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the “Blast 2 sequences” function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). The BLAST sequence comparison system is available, for instance, from the NCBI web site; see also Altschul et al., J. Mol. Biol., 215:403-10, 1990; Gish and States, Nature Genet., 3:266-72, 1993; Madden et al., Meth. Enzymol., 266:131-41, 1996; Altschul et al., Nucleic Acids Res., 25:3389-402, 1997; and Zhang and Madden, Genome Res., 7:649-56, 1997.
Orthologs (equivalent to proteins of other species) of proteins are in some instances characterized by possession of greater than 75% sequence identity counted over the full-length alignment with the amino acid sequence of specific protein using ALIGN set to default parameters. Proteins with even greater similarity to a reference sequence will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, or at least 98% sequence identity. In addition, sequence identity can be compared over the full length of one or both binding domains of the disclosed fusion proteins.
When significantly less than the entire sequence is being compared for sequence identity, homologous sequences will typically possess at least 80% sequence identity over short windows of 10-20, and may possess sequence identities of at least 85%, at least 90%, at least 95%, or at least 99% depending on their similarity to the reference sequence. Sequence identity over such short windows can be determined using LFASTA; methods are described at the NCSA website. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided. Similar homology concepts apply for nucleic acids as are described for protein. An alternative indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions.
Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences, due to the degeneracy of the genetic code. It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that each encode substantially the same protein.
Subject: Living multi-cellular vertebrate organisms, a category that includes both human and non-human mammals.
Therapeutically effective amount: A quantity of a specified agent sufficient to achieve a desired effect in a subject being treated with that agent. For example, this may be the amount of a lyssavirus-specific monoclonal antibody useful for treating rabies. The effective amount of a lyssavirus-specific monoclonal antibody useful for treating rabies in a subject will be dependent on, for example, the subject being treated, the manner of administration of the composition, and other factors.
Vector: A nucleic acid molecule that can be introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication (DNA sequences that participate in initiating DNA synthesis). A vector may also include one or more selectable marker genes and other genetic elements known in the art. In some embodiments herein, the vector is an Fc IgG expression vector.
Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Hence “comprising A or B” means including A, or B, or A and B. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Currently available anti-rabies immunoglobulins do not neutralize lyssaviruses of other genotypes, such as Lagos bat virus (LBV), Mokola virus (MOKV), and West Caucasian bat virus (WCBV). The use of immunized humans for immune library construction biases libraries towards neutralization of genotype 1 rabies viruses with lower cross-reactivity towards other genotypes. To circumvent this limitation, disclosed herein is the use of a naïve human phage display library to identify and characterize novel monoclonal antibodies (mAbs) that broadly neutralize lyssaviruses. As described in Examples 2 and 3 below, this library displays variable heavy (VH) domain antibodies (dAbs) with diversity added to the complementarity determining regions (CDR). Antibodies identified by the methods provided herein can be used, for example, for post-exposure rabies prophylaxis or in the treatment of clinical rabies.
Provided herein is a method of identifying a monoclonal antibody or antigen-binding fragment thereof that specifically binds at least two different lyssaviruses. In some embodiments, the method includes screening a naïve antibody phage display library with at least two different lyssavirus glycoproteins, such as by screening the library against a recombinant virus expressing at least two different lyssavirus glycoproteins, screening the library against recombinant glycoprotein from at least two different lyssaviruses, screening the library against cells expressing at least two different lyssavirus glycoproteins, or any combination thereof. In some embodiments, the method further includes selecting a phage display clone that specifically binds to at least two different lyssaviruses, at least two different lyssavirus glycoproteins, or both.
The at least two different lyssaviruses can be selected from any known lyssavirus, such as, for example, rabies virus (RABV), Mokola virus (MOKV), West Caucasian bat virus (WCBV), Lagos bat virus (LBV), Duvenhage virus (DUVV), European bat lyssavirus-1 (EBLV-1), European bat lyssavirus-2 (EBLV-2), Australian bat lyssavirus (ABLY), Aravan virus (ARAV), Khujand virus (KHUV) and Irkut virus (IRKV). In some embodiments, the antibody or antigen-binding fragment specifically binds at least three, at least four or at least five different lyssaviruses, or at least three, at least four or at least five different lyssavirus glycoproteins.
In particular embodiments, the method includes screening a naïve antibody phage display library with (1) a recombinant rabies virus expressing glycoprotein from RABV, MOKV and WCBV; (2) at least two different recombinant lyssavirus glycoproteins selected from the RABV glycoprotein, the MOKV glycoprotein, the WCBV glycoprotein, the LBV glycoprotein and the DUVV glycoprotein; or (3) at least two different cell lines expressing a lyssavirus glycoprotein selected from the RABV glycoprotein, the MOKV glycoprotein, the WCBV glycoprotein, the LBV glycoprotein and the DUVV glycoprotein; and selecting a phage display clone that specifically binds to at least two different lyssaviruses, at least two different lyssavirus glycoproteins, or both. In some examples, the at least two different lyssaviruses are selected from RABV, MOKV, WCBV, LBV and DUVV. In particular non-limiting examples, the antibody or antigen-binding fragment specifically binds whole virus and/or glycoprotein of RABV, MOKV and WCBV; RABV, MOKV, WCBV and LBV; RABV, MOKV, WCBV, LBV and DUVV; or MOKV, WCBV, LBV and DUVV.
In some embodiments, the phage display library is a naïve human VH domain library. In other embodiments, the phage display library is a naïve human scFv library or a naïve human Fab library. However, other naïve human antibody libraries can be used and an appropriate library can be selected by one of skill in the art.
In some embodiments, the antigen-binding fragment comprises a VH domain. In other embodiments, the antigen-binding fragment comprises a scFV. In yet other embodiments, the antigen-binding fragment comprises an Fab.
In some embodiments, the method further includes cloning the antigen-binding fragment (for example, a fragment comprising a VH domain, a scFV or a Fab) into an Fc IgG expression vector to generate an immunoglobulin molecule containing the fragment (such as an Fc IgG1 expression vector, for example pNUT-Cγ1 (Boel, et al., J. Immunol. Methods. 239:153-166, 2000)). In some examples, the Fc IgG expression vector further includes nucleic acid sequence encoding a variable light (VL) domain from a rabies virus-specific antibody.
Selecting a phage display clone that specifically binds to a lyssavirus (for example, whole virus) or lyssavirus glycoprotein can be performed using any assay known in the art for evaluating antigen binding. In some embodiments, the selecting step includes an ELISA to detect specific binding to lyssavirus glycoprotein, whole virus, or both.
In some embodiments, the method further includes screening the phage display clone for lyssavirus neutralization. Assays for evaluating virus neutralization are well known in the art and include, for example, fluorescent focus assays, including the rapid fluorescent focus inhibition test (RFFIT).
Also provided are isolated monoclonal antibodies, and antigen-binding fragments thereof, identified according to the methods disclosed herein. In some embodiments, the antibody neutralizes infectivity of the lyssaviruses.
In some embodiments, the VH domain of the antibody is encoded by a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NOs: 1-110. In some examples, the VH domain of the antibody is encoded by a nucleotide sequence comprising any one of SEQ ID NOs: 1-110.
In some embodiments, any in-frame “TAG” (stop codons) in the VH domain sequence are changed to “GAG” (glutamate codons) to allow expression in eukaryotic cells.
SEQ ID NOs: 1-43 show the nucleotide sequences of 43 different clones of a naïve human VH domain library that were selected using a recombinant rabies virus expressing glycoprotein from RABV, MOKV and WCBV. These clones bind whole virus and/or bind glycoprotein from RABV, MOKV and WCBV. Each clone includes nucleic acid sequence encoding the VH complementarity determining regions (CDRs). Identifying CDR sequences of an antibody or antibody fragment, given the nucleotide or amino acid sequence of the antibody or antibody fragment, is within the capabilities of one of skill in the art, such as by using the Kabat method (see, Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991; the Kabat database is now maintained online) or the International ImMunoGeneTics Information System™ (IMGT, available online). Thus, also provided herein is a monoclonal antibody, or antigen-binding fragment thereof, comprising at least one CDR sequence, such as two CDR sequences or all three CDR sequences, encoded by a portion of any one of SEQ ID NOs: 1-43.
SEQ ID NOs: 44-110 represent the nucleotide sequences of 67 different clones of a naïve human VH domain library that were selected by sequential panning on cell lines expressing glycoprotein from LBV, MOKV, WCBV and DUVV. CDR1, CDR2 and CDR3 sequences of each VH domain clone are provided in Table 2. Thus, provided herein is a monoclonal antibody, or antigen-binding fragment thereof, comprising at least one, at least two or all three CDR sequences from any one of SEQ ID NOs: 44-110.
Also provided is an isolated monoclonal antibody, or antigen-binding fragment thereof, that specifically binds at least two different lyssaviruses; or that specifically binds recombinant glycoprotein from at least two different lyssaviruses. In some embodiments, the VH domain of the antibody is encoded by a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 1-110. In some embodiments, the VH domain of the antibody is encoded by a nucleotide sequence comprising any one of SEQ ID NOs: 1-110. In some embodiments, the at least two lyssaviruses are selected from RABV, MOKV, WCBV, LBV and DUVV. In some examples, the isolated monoclonal antibody specifically binds RABV, MOKV and WCBV; or specifically binds recombinant glycoprotein from RABV, MOKV and WCBV. In other examples, the isolated monoclonal antibody specifically binds RABV, MOKV, WCBV and LBV; or specifically binds recombinant glycoprotein from RABV, MOKV, WCBV and LBV. In other examples, the isolated monoclonal antibody specifically binds RABV, MOKV, WCBV, LBV and DUVV; or specifically binds recombinant glycoprotein from RABV, MOKV, WCBV, LBV and DUVV. In yet other examples, the isolated monoclonal antibody specifically binds MOKV, WCBV, LBV and DUVV; or specifically binds recombinant glycoprotein from MOKV, WCBV, LBV and DUVV. In particular examples, the antibody neutralizes infectivity of the lyssaviruses.
In some embodiments, the monoclonal antibody comprises a VL domain from a rabies virus-specific antibody.
In some embodiments disclosed herein, the antibody is an IgG. In other embodiments, the antibody is an IgM. In some embodiments, the antibody is a human antibody or a humanized antibody. In some embodiments, the antibody is a Fab fragment, a Fab′ fragment, a F(ab)′2 fragment, a single chain Fv protein (scFv), or a disulfide stabilized Fv protein (dsFv).
Further provided herein is an isolated immunoconjugate comprising a monoclonal antibody disclosed herein and a fusion partner. In some embodiments, the fusion partner is an effector molecule, a label or a heterologous polypeptide.
Also provided are compositions comprising the monoclonal antibodies or immunoconjugates disclosed herein and a pharmaceutically acceptable carrier.
Compositions comprising more than one type of anti-rabies antibody are also provided herein. In some embodiments, the composition includes (1) a monoclonal antibody that specifically binds at least two lyssaviruses as disclosed herein, and (2) a monoclonal antibody specific for RABV or RABV glycoprotein. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.
Further provided is an isolated nucleic acid molecule encoding any one of the monoclonal antibodies disclosed herein.
Also provided is a method of treating rabies in a subject, comprising administering to the subject a monoclonal antibody or antigen-binding fragment thereof, an immunoconjugate or a composition disclosed herein.
Also provided herein is an expression vector comprising a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 1-110. In some embodiments, the expression vector comprises the nucleotide sequence of any one of SEQ ID NOs: 1-110. In some embodiments, the expression vector further comprises the nucleotide sequence of a VL domain from a lyssavirus-specific monoclonal antibody. In particular examples, the VL domain is from a RABV-specific monoclonal antibody. In some embodiments, the expression vector is an Fc IgG expression vector. A cell comprising an expression vectors disclosed herein is also provided. Further provided is a monoclonal antibody encoded by the Fc IgG expression vector. A monoclonal antibody expressed from such an Fc IgG expression vector can be used for rabies prophylaxis and clinical rabies treatment.
The genus Lyssavirus is a member of the Rhabdoviridae family within the order Mononegavirales (viruses with a single-stranded, negative sense genome). Lyssaviruses are the etiological agents of rabies encephalitis in warm-blooded animals and humans (Tordo et al., “Lyssaviruses” In Fauquet et al. eds. Virus taxonomy: the classification and nomenclature of viruses. The 8th Report of the International Committee on Taxonomy of Viruses. San Diego: Oxford Academic, 2006, pages 623-629; World Health Organization Expert Consultation on Rabies, 5-8 Oct. 2004, first report, World Health Organization Technical report series 931, Geneva: World Health Organization, 2005, pages 15-19). Lyssavirus species include rabies virus (RABV; genotype 1), Lagos bat virus (LBV; genotype 2), Mokola virus (MOKV; genotype 3), Duvenhage virus (DUVV; genotype 4), European bat lyssavirus-1 (EBLV-1; genotype 5), European bat lyssavirus-2 (EBLV-2; genotype 6), Australian bat lyssavirus (ABLV; genotype 7) six additional species isolated from bats: four in central Asia and Russia (Aravan virus—ARAV; Khujand virus—KHUV; Irkut virus—IRKV; and West Caucasian bat virus—WCBV), one in Africa (Shimoni bat virus—SHIBV) and one in Europe (Bokeloh bat lyssavirus—BBLV) (Kuzmin et al., Emerg. Infect. Dis. 14(12):1887-1889, 2008; Weyer et al., Epidemiol. Infect. 136:670-678, 2007; Kuzmin et al., Virus Res. 149(2):197-210, 2010; Freuling et al., Emerg. Infect. Dis. 17(8):1519-22, 2011; Kuzmin and Rupprecht, “Bat rabies” In Rabies, 2nd Edition, New York, Academic Press, 2007, pages 259-307, Jackson and Wunner, eds.).
Based on phylogeny, immunogenicity and virulence of lyssavirus isolates, two lyssavirus phylogroups have been proposed (Badrane et al., J. Virol. 75:3268-3276, 2001). The division into phylogroups generally correlates with the pattern of vaccine cross-protection observed for lyssaviruses (Badrane et al., J. Virol. 75:3268-3276, 2001; Hanlon et al., Virus Res. 111:44-54, 2005; Nel et al., Expert Rev. Vaccines 4:553-540, 2005). Phylogroup1 includes genotypes 1, 4, 5, 6 and 7, as well as ARAV, KHUV IRKV and BBLV (Kuzmin et al., Virus Res. 97:65-79, 2003; Kuzmin et al., Virus Res. 111:28-43, 2005; Hanlon et al., Virus Res. 111:44-54, 2005; Freuling et al., Emerg. Infect. Dis. 17(8):1519-22, 2011). Currently available commercial vaccines and biologicals are considered to be effective against infections of viruses from this phylogroup (Nel et al., Expert Rev. Vaccines 4:553-540, 2005). However, these vaccines and biologics for rabies do not offer full protection against infection with viruses outside of lyssavirus phylogroup 1 (i.e., genotypes 2, 3 and SHIBV). In addition, WCBV is recognized as the most divergent lyssavirus and exhibits limited relatedness to genotype 2 and 3 viruses. Previous studies have demonstrated little or no cross-neutralization of anti-RABV sera with WCBV (Botvinkin et al., Emerg. Infect. Dis. 9:1623-1625, 2003; Hanlon et al., Virus Res. 111:44-54, 2005).
Lyssaviruses are composed of two major structural components, a nucleocapsid or ribonucleoprotein (RNP), and an envelope in the form of a bilayer membrane surrounding the RNP core. The infectious component of all rhabdoviruses is the RNP core, which consists of the negative strand RNA genome encapsidated by nucleoprotein (N) in combination with RNA-dependent RNA-polymerase (L) and phosphoprotein (P). The membrane surrounding the RNP contains two proteins, the trans-membrane glycoprotein (G) and the matrix (M) protein, located at the inner side of the membrane. Thus, the viral genome codes for these five proteins: the three proteins in the RNP (N, L and P), the matrix protein (M), and the glycoprotein (G).
The molecular determinants of pathogenicity of various rabies virus strains have not been fully elucidated. RABV pathogenicity was attributed to multigenic events (Yamada et al., Microbiol. Immunol. 50:25-32, 2006). For example, some positions in the RABV genome if mutated, affect viral transcription or replication, reducing virulence. Mutations at serine residue 389 of the phosphorylation site in the N gene (Wu et al., J. Virol. 76:4153-4161, 2002) or GDN core sequence of the highly conserved C motif in the L gene (Schnell and Conzelmann, Virol. 214:522-530, 1995) dramatically reduced RABV transcription and replication.
The G protein, also referred to as spike protein, is involved in cell attachment and membrane fusion of RABV. The amino acid region at position 330 to 340 (referred to as antigenic site III) of the G protein has been identified as important for virulence of certain strains of RABV. Several studies support the concept that the pathogenicity of fixed RABV strains is determined by the presence of arginine or lysine at amino acid residue 333 of the glycoprotein (Dietzschold et al., Proc. Natl. Acad. Sci. USA 80: 70-74, 1983; Tuffereau et al., Virology 172: 206-212, 1989).
This phenomenon seems to apply at least to fixed rabies viruses such as CVS, ERA, PV, SAD-B19 and HEP-Flury strains (Anilionis et al., Nature 294:275-278, 1981; Morimoto et al., Virology 173:465-477, 1989). For example, rabies vaccine viruses possessing an amino acid differing from Arg at position 333 of the glycoprotein are described, for instance, in PCT Publication No. WO 00/32755 (describing RABV mutants in which all three nucleotides in the G protein Arg333 codon are altered compared to the parent virus, such that the Arg at position 333 is substituted with another amino acid); European Patent No. 350398 (describing an avirulent RABV mutant SAG1 derived from the Bern SAD strain of RABV, in which the Arg at position 333 of the glycoprotein has been substituted to Ser); and European patent application 583998 (describing an attenuated RABV mutant, SAG2, in which the Arg at position 333 in the G protein has been substituted by Glu).
Other strains, such as the RC-HL strain, possess an arginine residue at position 333 of the G, but do not cause lethal infection in adult mice (Ito et al., Microl. Immunol. 38:479-482, 1994; Ito et al., J. Virol. 75:9121-9128, 2001). As such, the entire G may contribute to the virulence of RABV, although the determinants or regions have not been fully elucidated.
The G gene encodes the only protein that induces viral neutralizing antibody. At least three states of RABV glycoprotein are known: the native state (N) being responsible for receptor binding; an active hydrophobic state (A) necessary in the initial step in membrane fusion process (Gaudin, J. Cell Biol. 150:601-612, 2000), and a fusion inactive conformation (I). Correct folding and maturation of the G protein play important roles for immune recognition. The three potential glycosylated positions in ERA G extracellular domain occur at Asn37, Asn247 and Asn319 residues (Wojczyk et al., Glycobiology. 8: 121-130, 1998). Nonglycosylation of G not only affects conformation, but also inhibits presentation of the protein at the cell surface.
Standard treatment for post-exposure rabies prophylaxis includes thorough wound-washing with soap and water followed by administration of vaccine and anti-rabies virus immunoglobulin (RIG) of human or equine origin. RIG administered shortly after exposure at the wound site provides passive immunity which neutralizes rabies virus and prevents its spread until the patient's immune response following vaccination is elicited. Deaths due to post-exposure prophylaxis failure are most commonly attributed to deviations from the recommended regimen such as late initiation of post-exposure prophylaxis or no administration of RIG (Wilde, Vaccine, 25:7605-7609, 2007). Provided herein are human monoclonal antibodies that specifically bind at least two different lyssaviruses. Thus, the disclosed antibodies are useful for the treatment of clinical rabies and/or for post-exposure rabies prophylaxis.
Compositions are provided that include a monoclonal antibody, or antigen-binding fragment thereof, that specifically binds at least two different lyssaviruses (such as whole virus or lyssavirus glycoprotein, or both). Compositions comprising immunoconjugates or immunotoxins are also provided. The compositions can be prepared in unit dosage forms for administration to a subject. The amount and timing of administration are at the discretion of the treating physician to achieve the desired purposes. For example, the compositions can be administered to a subject exhibiting clinical signs of rabies, or can be administered to a subject that has been exposed to or bitten by a rabid animal (or an animal suspected of being rabid). The antibody can be formulated for systemic or local (such as at a wound site) administration.
The compositions for administration can include a solution of the antibody (or antigen-binding fragment thereof) that specifically binds lyssavirus dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, for example, buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of antibody in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the subject's needs.
Doses of lyssavirus mAb generally range from about 1 to about 50 IU/kg, such as about 5 to about 40 IU/kg, or about 5 to about 20 IU/kg. In particular examples, the dose is about 5 IU/kg, about 10 IU/kg or about 20 IU/kg. However, the dose of lyssavirus mAb will vary depending upon the antibody (or antibody cocktail) selected and the particular subject to be treated. An appropriate does can be determined by a medical practitioner.
Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, Pa. (1995).
Antibodies may be provided in lyophilized form and rehydrated with sterile water before administration, although they are also provided in sterile solutions of known concentration. Antibodies can be administered by slow infusion, or by intravenous push or bolus. Antibody compositions can also be administered topically.
The antibody that specifically binds lyssavirus can be administered to prevent the development of rabies disease and/or slow the spread of lyssavirus from a wound site to distant sites in the body. In these applications, a therapeutically effective amount of an antibody is administered to a subject in an amount sufficient to inhibit virus replication or spread, or to inhibit a sign or a symptom of rabies. Suitable subjects may include those diagnosed with rabies, or subjects recently exposed to (such as bitten by) animal infected with a lyssavirus, or suspected of being infected with a lyssavirus.
A therapeutically effective amount of a lyssavirus-specific antibody will depend upon the severity of the disease and the general state of the patient's health. A therapeutically effective amount of the antibody is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer. These compositions can be administered in conjunction with other therapeutic agents (such as a rabies vaccine), either simultaneously or sequentially.
Single or multiple administrations of the compositions are administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of at least one of the antibodies disclosed herein to effectively treat the patient. The dosage can be administered once but may be applied periodically until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy. Generally, the dose is sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the patient.
The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described.
This example describes the generation and characterization of a recombinant ERA strain rabies virus encoding three different glycoprotein genes. The recombinant virus, referred to as ERA-3G, comprises rabies virus (RABV) glycoprotein, Mokola virus (MOKV) glycoprotein and West Caucasian bat virus (WCBV) glycoprotein. The cloning strategy for ERA-3G is shown in
The G genes from MOKV and WCBV were cloned into the ERA backbone (see SEQ ID NO: 7 of PCT Publication No. WO 2007/047459) by RT-PCR using viral genomic RNA from virus-infected cells as template. The following primers were used for amplification of the glycoprotein genes:
The amplified G genes for MOKV (SEQ ID NO: 115) and WCBV (SEQ ID NO: 116) were confirmed by sequencing. Two extra transcription units were synthesized (each with the sequence of SEQ ID NO: 117) and introduced into the gene junctions between the phosphoprotein (P) and the matrix protein (M), and the G and the RNA dependent RNA polymerase (L) (
Recombinant virus was recovered by transfection of the above described construct into BSR cells using the method described in PCT Publication No. WO 2007/047459.
Current anti-rabies immune globulins do not neutralize all lyssaviruses. The use of immunized humans for immune library construction biases libraries towards neutralization of RABV genotype 1 with lower cross-reactivity towards other Lyssavirus genotypes. One method to circumvent this limitation is selecting monoclonal antibodies (mAbs) from naïve immune libraries, which theoretically contain binders to any antigen.
This example describes the use of a naïve, human-heavy domain, phage display library (Famm et al., J. Mol. Biol. 376:926-931, 2008) to identify and characterize novel anti-Lyssavirus mAbs. The phage display library (3×109 clones) is based on human VH framework with diversity introduced into CDR1, CDR2 and CDR3 by PCR mutagenesis. The library was panned using a recombinant ERA RABV expressing three different G proteins (see Example 1)—the G proteins from rabies virus (RABV), Mokola virus (MOKV) and West Caucasian Bat virus (WCBV)—following established methods (Kramer et al., Eur. J. Immunol. 35:2131-2145, 2005; Lee et al., Nat. Protoc. 2:3001-3008, 2007).
Library Panning
For the first two times the library was panned, either purified G protein or whole virus was used to select binders for three rounds and then switched antigens for three more rounds so that binders selected for G protein were then panned against whole virus and vice versa. After the last round of selection, 552 individual clones were picked (276 from each panning scheme) and potential high affinity domain antibodies (dAb) were identified by ELISA.
ELISA
The ELISA procedure was standardized using the soluble dAb fragments from these clones and the same antigens used to pan the library. Approximately 5-10% of selected clones bind to the original antigen and 2-3% bind to the secondary antigen (Table 1).
Relative binding (as assessed by ELISA) of each of the clones to whole virus and glycoprotein is shown in
Sequencing of Clones
Sixty-nine percent of the clones selected for binding to whole virus and 37% of the clones selected for binding to glycoprotein were successfully sequenced. The sequences of each clone are provided in the Sequence Listing as SEQ ID NOs: 1-43 as follows:
This example describes the identification of VH domain antibodies specific for lyssavirus glycoprotein by sequential panning of a naïve, human VH domain phage display library (Famm et al., J. Mol. Biol. 376:926-931, 2008) on cell lines expressing glycoprotein (G) from several different lyssaviruses.
Flp-In-BHK cells (Invitrogen) were transfected with a pEF5/FRT/V5 plasmid (Invitrogen) encoding glycoprotein from LBV (SEQ ID NO: 118), MOKV (SEQ ID NO: 115), WCBV (SEQ ID NO: 116), or DUVV (SEQ ID NO: 119). The library was panned sequentially on cells that express LBV G protein, followed by MOKV G protein, followed by WCBV G protein and finally DUVV G protein. Selection on cells was carried out using the method described by Lee et al. (Nature Protocols 2(11):3001-3008, 2007).
Using this method, 67 unique nucleotide sequences were identified (Table 2). Each VH sequence has at least one change in amino acid sequence when translated. In-frame “TAG” (stop codons) will be changed to “GAG” (glutamate codons) to allow expression in eukaryotic cell line. VH sequences will be cloned into the pEF5/FRT/V5 plasmid to transfect Flp-In-BHK cells for expression of antibodies.
1Each CDR sequence is identified by the nucleotide positions of the provided SEQ ID NO.
A subject diagnosed with rabies or at risk of developing rabies (such as a subject bitten by a potentially rabid animal) is subjected to rabies post-exposure prophylaxis, which includes administration of a rabies vaccine, such as Imovax™ or RabAvert™, according to the recommended dosing schedule. Subjects who have not previously been vaccinated against rabies will further be treated with one or more of the lyssavirus mAbs disclosed herein.
Lyssavirus mAb is administered intramuscularly to the subject at a site distant to the site of vaccine administration. If the subject diagnosed with rabies, or at risk of developing rabies, has a bite wound, lyssavirus mAb can optionally be administered directly into the wound and to the area directly adjacent to the wound.
Doses of lyssavirus mAb generally range from 1 to 50 IU/kg. In particular examples, the dose is about 5 or about 20 IU/kg. However, the dose of lyssavirus mAb will vary depending upon the antibody (or antibody cocktail) selected and the particular subject to be treated. An appropriate does can be determined by a medical practitioner.
In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.
This is a divisional of U.S. application Ser. No. 13/879,782, filed Apr. 16, 2013, issued as U.S. Pat. No. 9,115,187 on Aug. 25, 2015, which is the U.S. National Stage of International Application No. PCT/US2011/056738, filed Oct. 18, 2011, published in English under PCT Article 21(2), which claims the benefit of U.S. Provisional Application No. 61/394,651, filed Oct. 19, 2010. All of the above-listed application are incorporated herein by reference in their entirety.
| Number | Date | Country |
|---|---|---|
| 0402 029 | Dec 1990 | EP |
| WO 2006084006 | Aug 2006 | WO |
| Entry |
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| Number | Date | Country | |
|---|---|---|---|
| 20160024185 A1 | Jan 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61394651 | Oct 2010 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13879782 | US | |
| Child | 14813427 | US |
