Patent Application
20200255499
This application incorporates by reference the Sequence Listing contained in the following ASCII text file being submitted concurrently herewith:
THIS INVENTION relates to Epstein-Barr virus (EBV). More particularly, this invention relates to an antibody that at least partly blocks gp350-mediated entry of EBV into human cells.
Epstein-Barr virus (EBV) or Human Herpesvirus 4 (HHV-4) is a double stranded DNA virus that belongs to the y-herpesvirus subfamily. It is a common human pathogen that predominantly infects hosts through epithelial cells and B cells, where it can then establish long-term latency in the human host. Primary infection of EBV causes over 90% of cases of infectious mononucleosis (IM) worldwide, infecting mainly children and young adults through the dramatic expansion of EBV infected B cells (Coghill and Hildesheim 2014). EBV has been associated with several cancers, including Burkitt and Hodgkins lymphomas, gastric and nasopharyngeal carcinomas, lymphomas in HIV-infected individuals and post-transplant lymphoproliferative disorder (PTLD). Recently, EBV has also been found to be implicated in autoimmune diseases, particularly multiple sclerosis (MS) (Coghill and Hildesheim 2014).
Epstein-Barr virus encoded gp350 is one of the major targets for anti-viral humoral immunity as it mediates attachment to B cells via complement receptor 2. Gp350 is the primary glycoprotein that elicits anti-viral neutralising antibody responses after natural EBV infection. Currently, there is no commercially available prophylactic or therapeutic treatment that can prevent or cure acute EBV infection. Clinical trials utilising vaccinia-vectored gp350 demonstrated potential immunogenicity and efficacy in young children (Gu, Huang et al. 1995). Subsequent studies by GlaxoSmithKline Biologicals based on a recombinant gp350/aluminium hydroxide and 3-O-desacyl-4′-monophosphoryl lipid A (AS04) candidate vaccine showed demonstrable efficacy (mean efficacy rate, 78.0% [95% confidence interval, 1.0%-96.0%]) in preventing the development of infectious mononucleosis induced by EBV infection, but it had no efficacy in preventing asymptomatic EBV infection (Moutschen, Leonard et al. 2007). Furthermore, Haque and colleagues have also assessed potential clinical application of a murine monoclonal antibody against gp350 (72A1) both in vivo and in vitro (Hague, Johannessen et al. 2006).
The present invention is broadly directed to an anti-gp350 antibody that at least partly prevents or inhibits the binding of EBV gp350 to human cells. A particular form of the invention provides a human or humanized, recombinant anti-gp350 antibody.
In a first aspect the invention provides a recombinant, human or humanized antibody or antibody fragment that is capable of at least partly preventing or inhibiting Epstein Barr Virus (EBV) gp350 binding to a human cell.
In a particular embodiment, the human or humanized antibody or antibody fragment comprises, consists essentially of or consists of an amino acid sequence set forth in any one of SEQ ID NOS:1-156,
In a particular embodiment, the human or humanized antibody or antibody fragment comprises, consists essentially of or consists of heavy chain and/or light chain complementarity-determining region (CDR) regions that respectively comprise an amino acid sequence set forth in any one of SEQ ID NOS:1-156,
Preferred CDR amino acid sequences are set forth in SEQ ID NOS:1-144 and Tables 1-12, or an amino acid sequence at least 70% identical thereto.
In a particular embodiment, the human or humanized antibody or antibody fragment comprises, consists essentially of or consists of an amino acid sequence set forth in any one of SEQ ID NOS:145-156 and/or
In a second aspect, the invention provides an antibody or antibody fragment that comprises, consists essentially of or consists of at least one complementarity-determining region (CDR) amino acid sequence according to any one of SEQ ID NOS:1-156,
In a particular embodiment, the antibody or antibody fragment comprises, consists essentially of or consists of heavy chain and/or light chain CDR regions that respectively comprise an amino acid sequence set forth in any one of SEQ ID NOS:1-156,
Preferred CDR amino acid sequences are set forth in SEQ ID NOS:1-144 and Tables 1-12, or an amino acid sequence at least 70% identical thereto.
In a particular embodiment, the antibody or antibody fragment is a humanized antibody or antibody fragment that comprises, consists essentially of or consists of an amino acid sequence set forth in any one of SEQ ID NOS:145-156 and/or
Suitably, the antibody or antibody fragment of the second aspect is capable of at least partly preventing or inhibiting Epstein Barr virus (EBV) gp350 binding to a human cell.
Preferably, the antibody or antibody fragment of the first and second aspects is a neutralizing antibody.
In one embodiment, the antibody or antibody fragment of the first and/or second aspects is produced by phage display, wherein the phage comprise one or more nucleotide sequences of human or non-human origin encoding one or more amino acid sequences of the antibody or antibody fragment. The one or more amino acid sequences may be VH, VL, and/or CDR amino acid sequences of human origin such as set forth in SEQ ID NOS:1-156,
In a third aspect, the invention provides an isolated nucleic acid encoding the antibody or antibody fragment of the first or second aspects.
This aspect also includes genetic constructs comprising the isolated nucleic acid and/or host cells comprising the isolated nucleic acid and/or genetic construct.
In a fourth aspect, the invention provides a composition comprising the antibody or antibody fragment of the first aspect, the second aspect and/or the isolated nucleic acid of the third aspect and a pharmaceutically acceptable carrier, diluent or excipient
In a fifth aspect, the invention provides a method of treating or preventing an EBV infection in a human, said method including the step of administering the antibody of the first aspect, the second aspect and/or the composition of the fourth aspect to the human to thereby treat or prevent an EBV infection in the human.
In a sixth aspect, the invention provides a method of passively immunizing a human against an EBV infection, said method including the step of administering the antibody of the first aspect, the second aspect and/or the composition of the fourth aspect to the human to thereby passively immunize the human against an EBV infection.
In a seventh aspect, the invention provides a method of at least partly inhibiting or preventing EBV gp350 binding to a human cell, said method including the step of administering the antibody of the first aspect, the second aspect and/or the composition of the fourth aspect to the human to thereby at least partly inhibit or prevent EBV gp350 binding to a human cell.
In an eighth aspect, the invention provides a method or system for determining the efficacy of an anti-EBV antibody or antibody fragment, said method or system comprising infecting a mouse with EBV, the mouse comprising human B lymphocytes, and determining the efficacy of a candidate anti-EBV antibody or antibody fragment in the mouse.
Suitably, efficacy relates to or includes the ability of the candidate anti-EBV antibody or antibody fragment to prevent EBV gp350 binding to one or more of the B lymphocytes.
Suitably, the human B lymphocytes have been derived from human hematopoietic stem cells administered to the mouse. Preferably, the human hematopoietic stem cells are CD34+.
In a ninth aspect, the invention provides a method of detecting EBVgp350 or a cell expressing EBVgp350, said method including the step of forming a complex between the antibody or antibody fragment of the first and/or second aspects and EBV gp350 to thereby detect EBVgp350 or the cell expressing EBVgp350.
Throughout this specification, unless the context requires otherwise, the words “comprise”, “comprises” and “comprising” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
By “consists essentially of” in the context of an amino acid sequence means that the recited amino acid sequences includes an additional 1, 2, 3, 4, or 5 amino acids at an N- and/or C-terminus thereof.
As used herein, the indefinite articles ‘a’ and ‘an’ are used here to refer to or encompass singular or plural elements or features and should not be taken as meaning or defining “one” or a “single” element or feature.
The present invention is at least partly based on the creation of a humanized, recombinant antibodies directed to EBV anti-gp350 that at least partly prevent or inhibit the binding of EBV gp350 to thereby block EBV entry into human cells. This humanized, recombinant antibody may be particularly suitable for administration to humans to passively immunize against EBV infection.
In a particular aspect, the invention provides a recombinant, human or humanized antibody or antibody fragment that is capable of at least partly preventing or inhibiting Epstein Barr Virus (EBV) gp350 binding to a human cell, to thereby block EBV entry into the human cell.
In another particular aspect, the invention provides an antibody or antibody fragment comprising at least one CDR amino acid sequence according to any one of SEQ ID NOS:1-144 or present in any one of SEQ ID NOS:145-156, that is preferably capable of at least partly preventing or inhibiting Epstein Barr Virus (EBV) gp350 binding to a human cell, to thereby block EBV entry into the human cell.
Preferably, the antibody or antibody fragment of these particular aspects is a neutralizing antibody.
In one embodiment, the antibody or antibody fragment of these aspects is produced by phage display.
As used herein, by “isolated” is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in recombinant, chemical synthetic, enriched, purified or partially purified form.
As used herein a “protein” is an amino acid polymer, wherein the amino acids may include D-amino acids, L-amino acids, natural and/or non-natural amino acids. As typically used herein, a “peptide” is a protein comprising no more than sixty (60) contiguous amino acids. As typically used herein, a “polypeptide” is a protein comprising more than sixty (60) contiguous amino acids. The term “protein” should also be understood to encompass protein-containing molecules such as glycoproteins and lipoproteins, although without limitation thereto.
As used herein, an “antibody” is or comprises an immunoglobulin protein. The term “immunoglobulin” includes any antigen-binding protein product of a mammalian immunoglobulin gene complex, including immunoglobulin isotypes IgA, IgD, IgM, IgG and IgE and antigen-binding fragments thereof. Included in the term “immunoglobulin” are immunoglobulins that are recombinant, chimeric or humanized or otherwise comprise altered or variant amino acid residues, sequences and/or glycosylation, whether naturally occurring or produced by human intervention (e.g. by recombinant DNA technology).
Generally, antibodies and antibody fragments may be polyclonal or monoclonal. It will also be appreciated that antibodies may be produced as recombinant synthetic antibodies or antibody fragments by expressing a nucleic acid encoding the antibody or antibody fragment in an appropriate host cell. Non-limiting examples of recombinant antibody expression and selection techniques, inclusive of phage display methods, are provided in Chapter 17 of Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY and Zuberbuhler et al., 2009, Protein Engineering, Design & Selection 22 169. A non-limiting example of a phage display system for selecting anti-gp350 antibodies is provided hereinafter in the Examples.
Typically, an antibody comprises: respective light chain (VL) and heavy chain (VH) variable regions that each comprise complementarity determining region (CDR) 1, 2 and 3 amino acid sequences; and respective light chain (CL) and heavy chain (CH1, CH2, CH3) constant regions. CDR identification and numbering may be according to any known CDR numbering system inclusive of Kabat, Chothia, AbM and Contact. Non-limiting examples of CDR amino acid sequences are set forth in SEQ ID NOS:1-144 and shown in Tables 1-12. CDR identification and numbering was performed using abYsis version 2.7.3 and IMGT/V-QUEST. SEQ ID NOS:1-36 use Chothia numbering; SEQ ID NOS:37-72 use AbM numbering; SEQ ID NOS:73-108 use Kabat numbering; and SEQ ID NOS:109-144 use Contact numbering. Antibodies according to the invention may comprise 1, 2 or 3 VL CDR amino acid sequences (e.g CDR1, CDR2 and/or CDR3) and/or 1, 2, or 3 VH CDR amino acid sequences (e.g CDR1, CDR2 and/or CDR3), such as preferably set forth in SEQ ID NOS:1-144.
Antibody fragments include Fab and Fab′2 fragments, diabodies, triabodies, bi-specific antibodies and single chain antibody fragments (e.g. ScFvs), although without limitation thereto. In some embodiments, an antibody fragment may comprise at least a portion of a CDR1, 2 and/or 3 amino acid sequence, such as set forth in SEQ ID NOS:1-36. A preferred antibody fragment comprises at least one entire light chain variable region CDR and/or at least one entire heavy chain variable region CDR.
As broadly used herein, “humanized” antibodies may include antibodies entirely or at least partly of human origin, inclusive of modified antibodies or antibody fragments obtained from a non-human “foreign” species. In some embodiments, antibodies and antibody fragments may be modified so as to be administrable to one species having being produced in, or originating from, the same or another “foreign” species without eliciting a deleterious immune response to the “foreign” antibody. Human or non-human antibody fragments such as comprising complementarity determining regions (CDRs) or variable regions (i.e VH and VL domains) may be “grafted” onto a human antibody scaffold or backbone to produce a “humanized” antibody or antibody fragment. In a particular embodiment, human or non-human CDRs or VL and VL domains are recombinantly grafted with a human antibody constant region, preferably a human IgG1 constant region.
As disclosed herein in more detail in the Examples, human antibody VH and VL fragment-encoding nucleotide sequences were isolated by phage display and recombinantly grafted onto a hman IgG1 constant region “backbone”.
A non-limiting example of a human IgG1 constant region comprises the amino acid sequence TVSSASTKGPSVFP (SEQ ID NO:157), as originally described in Jones et al., 2010, J. Immunol. Methods 354 85.
Preferably, the antibody or antibody fragment is a neutralizing antibody or antibody fragment. By this is meant an antibody or antibody fragment that at least partly blocks, inhibits or reduces one or more infective or pathogenic properties of EBV. In a particular embodiment, the antibody or antibody fragment at least partly blocks, inhibits or reduces gp350-mediated entry of EBV into a human cell.
Suitably, the antibody or antibody fragment binds an epitope of an EBV gp350 protein. As generally used herein, an “epitope” is an antigenic protein fragment that comprises a continuous or discontinuous sequence of amino acids of a protein, wherein the epitope can be recognized or bound by an element of the immune system, such as an antibody or other antigen receptor.
The invention also includes variants of the the antibody or antibody fragment disclosed herein, such as a CDR variant.
Suitably, an antibody or antibody fragment comprising at least one variant is capable of preventing Epstein Barr Virus (EBV) gp350 binding to a human cell.
In particular embodiments, a variant has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence set forth in any one of SEQ ID NOS:1-156. The protein “variant” disclosed herein may have one or more amino acids deleted or substituted by different amino acids. It is well understood in the art that some amino acids may be substituted or deleted without changing biological activity of the peptide (conservative substitutions).
In one embodiment, the variant is an antibody or antibody fragment comprising an amino acid sequence at least 70% identical to any one of SEQ ID NOS:1-144, referred to herein as a CDR “variant”. By way of example, CDR amino acid sequences may be altered to improve recognition and/or binding to EBV gp350.
In some embodiments, variants may be produced by recombinant mutagenesis techniques.
Terms used generally herein to describe sequence relationships between respective proteins and nucleic acids include “comparison window”, “sequence identity”,“percentage of sequence identity” and “substantial identity”. Because respective nucleic acids/proteins may each comprise (1) only one or more portions of a complete nucleic acid/protein sequence that are shared by the nucleic acids/proteins, and (2) one or more portions which are divergent between the nucleic acids/proteins, sequence comparisons are typically performed by comparing sequences over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” refers to a conceptual segment of typically 6, 9 or 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence for optimal alignment of the respective sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (Geneworks program by Intelligenetics; GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA, incorporated herein by reference) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids Res. 25 3389, which is incorporated herein by reference. A detailed discussion of sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons Inc NY, 1995-2015).
The term “sequence identity” is used herein in its broadest sense to include the number of exact nucleotide or amino acid matches having regard to an appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a window of comparison. Thus, a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For example, “sequence identity” may be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, Calif., USA).
Derivatives of the antibody, antibody fragments or variants thereof disclosed herein are also provided.
As used herein, “derivative” antibodies, antibody fragments or variants thereof have been altered, for example by conjugation or complexing with other chemical moieties, by post-translational modification (e.g. phosphorylation, ubiquitination, glycosylation), chemical modification (e.g. cross-linking, acetylation, biotinylation, oxidation or reduction and the like), conjugation with labels (e.g. fluorophores, enzymes, radioactive isotopes) and/or inclusion of additional amino acid sequences as would be understood in the art.
In this regard, the skilled person is referred to Chapter 15 of CURRENT PROTOCOLS IN PROTEIN SCIENCE, Eds. Coligan et al. (John Wiley & Sons NY 1995-2015) for more extensive methodology relating to chemical modification of proteins.
Additional amino acid sequences may include fusion partner amino acid sequences which create a fusion protein. By way of example, fusion partner amino acid sequences may assist in detection and/or purification of the isolated fusion protein. Non-limiting examples include metal-binding (e.g. polyhistidine) fusion partners, maltose binding protein (MBP), Protein A, glutathione S-transferase (GST), fluorescent protein sequences (e.g. GFP), epitope tags such as myc, FLAG and haemagglutinin tags.
Another aspect of the invention provides an isolated nucleic acid encoding an antibody, antibody fragment or variant thereof disclosed herein.
As generally used herein a “nucleic acid” designates single- or double-stranded DNA and RNA. DNA includes genomic DNA and cDNA. RNA includes mRNA, RNA, RNAi, siRNA, cRNA and autocatalytic RNA. Nucleic acids may also be DNA-RNA hybrids. A nucleic acid comprises a nucleotide sequence which typically includes nucleotides that comprise an A, G, C, T or U base. However, nucleotide sequences may include other bases such as inosine, methylycytosine, methylinosine, methyladenosine and/or thiouridine, although without limitation thereto.
In a preferred form, the nucleotide sequence may be codon-optimized, by which is meant that the nucleotide sequence is a synthetic or engineered sequence, rather than the original “source” nucleotide sequence, modified for optimal expression in a particular host cell type by taking advantage of codon sequence variations or redundancies that occur across different cell types and/or species.
In some embodiments, the nucleic acid may be in a genetic construct that facilitates delivery and expression of the nucleic acid. Broadly, the genetic construct may be in the form of, or comprise genetic components of, a plasmid, a transposon, a bacteriophage, a virus, a cosmid, a yeast or bacterial artificial chromosome as are well understood in the art. Suitably, the nucleic acid is operably linked or connected to one or more elements of the construct that facilitate propagation, manipulation and/or expression of the genetic construct and/or nucleic acid. The one or more elements may include promoters, enhancers, polyadenylation sequences, splice donor/acceptor sites, multiple cloning sites, bacterial origins of replication, selection markers for bacterial, mammalian or other host cells, translation initiation and stop sequences, as are well known in the art. Promoters may be constitutive or inducible/repressible promoters as are well known in the art.
The choice of said one or more elements may be at least partly dependent on the host cell type used for expression, particularly according to the origin of the host cell (e.g. mammalian or other vertebrates, plant, bacterial, insect or yeast cells such as E. coli, CHO, COS, Vero, HeLa, HEK-293, Sf9 and Pichia pastoris host cells, although without limitation thereto).
It will be appreciated from the foregoing that an aspect of the invention provides a method of treating or preventing an EBV infection in a human, said method including the step of administering the antibody of the first aspect, the second aspect and/or the composition of the fourth aspect to the human to thereby treat or prevent an EBV infection in the human.
Another aspect of the invention provides a method of passively immunizing a human against an EBV infection, said method including the step of administering the antibody of the first aspect, the second aspect and/or the composition of the fourth aspect to the human to thereby passively immunizing the human against an EBV infection.
As generally used herein the terms “immunize”, “vaccinate” and “vaccine” refer to antibodies, antibody fragments, methods and/or compositions that elicit or provide a protective immune response against EBV. Administration of said antibody or antibody fragment to human may be referred to as “passive” immunization which provides the human with at least temporary antibody-mediated immunity to an existing or potential EBV infection.
In certain embodiments the immune response may be suitable for preventing, treating or passively immunizing the human against EBV.
As used herein, “treating”, “treat” or “treatment” refers to a therapeutic intervention that at least partly ameliorates, eliminates or reduces a symptom or pathological sign of an EBV infection after it has begun to develop. Treatment need not be absolute to be beneficial to the subject.
As used herein, “preventing”, “prevent” or “prevention” refers to a course of action initiated prior to infection by, or exposure to, EBV or molecular components thereof and/or before the onset of a symptom or pathological sign of an EBV infection, so as to at least partly prevent infection and/or reduce the symptom or pathological sign. It is to be understood that such prevention need not be absolute or complete to be beneficial to a subject.
EBV is a common human pathogen and may cause, or be associated with, one or more diseases, disorders or conditions in humans. Thus, certain embodiments of the aforementioned methods relate to passively immunization, preventing and/or treating one or more diseases, disorders or conditions caused by, or associated with, an EBV infection in humans. EBV predominantly infects human hosts through epithelial cells and B lymphocytes where it can then establish long-term latency in the human host. Primary infection of EBV causes over 90% of cases of infectious mononucleosis (IM) worldwide, infecting mainly children and young adults through the expansion of EBV infected B cells. EBV has been associated with several cancers, including Burkitt and Hodgkin's lymphomas, gastric and nasopharyngeal carcinomas, lymphomas in HIV-infected individuals and post-transplant lymphoproliferative disorder (PTLD). EBV has also been found to be implicated in autoimmune diseases, particularly multiple sclerosis.
In this context, another aspect, the invention provides at least partly inhibiting or preventing EBV gp350 binding to a human cell, said method including the step of administering the antibody of the first aspect, the second aspect and/or the composition of the fourth aspect to the human to thereby at least partly inhibit or prevent EBV gp350 binding to a human cell. Suitably the human cell is a B lymphocyte.
In some embodiments, the isolated antibodies, fragments and/or variants, or combinations of these, may be administered to a mammal in the form of a composition comprising a pharmaceutically acceptable carrier, diluent or excipient.
It will be appreciated that pharmaceutically acceptable carriers, diluents and/or excipients may include solid, semi-solid, gel or liquid fillers, diluents or encapsulating substances that may be safely used in systemic administration. Depending upon the particular route of administration, carriers, diluents and/or excipients may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, isotonic saline, pyrogen-free water, wetting or emulsifying agents, bulking agents, glidants, coatings (e.g. enteric coatings), emollients, binders, fillers, disintegrants, lubricants, pH buffering agents (e.g. phosphate buffers) and/or flavouring agents, although without limitation thereto. The composition may be administered to a human in any one or more dosage forms that include tablets, dispersions, suspensions, injectable solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches and the like.
Administration of the antibody, antibody fragment or variant thereof, or an encoding nucleic acid, or a composition comprising same may be by any known parenteral, topical or enteral route inclusive of intravenous, intramuscular, intraperitoneal, intracranial, transdermal, oral, intranasal, anal and intra-ocular, although without limitation thereto.
The composition may further include one or more additional agents inclusive of adjuvants or other immunostimulants.
By “adjuvant” is meant an agent which assists, augments or otherwise facilitates the elicitation of an immune response. Non-limiting examples of adjuvants include Freund's adjuvant, aluminium hydroxide (alum), aluminium phosphate, squalene, IL-12, CpG-oligonucleotide, Montanide ISA720, imiquimod, SBAS2, SBAS4, MF59, MPL, Quil A, QS21 and ISCOMs.
For the purposes of methods of immunization, prevention and/or treatment of EBV infections, the recipient human may be referred to as a “subject” or “patient”, which terms are used interchangeably.
As described herein, a preferred method of producing anti-gp350 antibodies or antibody fragments is by phage display, although other methods of antibody production may be utilized, as are well known in the art.
In one embodiment, “candidate” anti-gp350 antibodies or antibody fragments may subseqeuntly be tested and selected according to their ability to bind gp350 and preferably prevent EBV gp350 binding to one or more of the B lymphocytes and thereby block entry of EBV into the human cell.
Accordingly, a further aspect of the invention provides method or system for determining the efficacy of an anti-EBV gp350 antibody or antibody fragment, said method or system comprising infecting a mouse with EBV, the mouse comprising human B lymphocytes, and determining the efficacy of a candidate anti-EBV agent in the mouse.
Suitably, the efficacy relates to, or includes, the ability of the candidate anti-EBV antibody or antibody fragment to prevent EBV gp350 binding to one or more of the B lymphocytes and thereby block entry of EBV into the human cell.
Suitably, the human B lymphocytes have been derived from human hematopoietic stem cells administered to the mouse. Preferably, the human hematopoietic stem cells are CD34+. Typically, the mice are immunocompromised or immunodeficient mice. A non-limiting example is irradiated adult NOD scid gamma (NSG) mice.
Thus, it will be appreciated that the method or system provides a means whereby a mouse which has been engineered to adopt certain characteristics of the human immune system can thereby a model for testing the potential efficacy of an anti-EBV gp350 antibody to block EBV infection in humans.
In a particular embodiment, the method or system includes:
The antibodies or antibody fragments may be polyclonal, monoclonal, native or recombinant as hereinbefore described.
In another aspect, the invention provides a method of detecting EBVgp350 or a cell expressing EBVgp350, said method including the step of forming a complex between the antibody or antibody fragment of the first and/or second aspects and EBV gp350 to thereby detect EBVgp350 or the cell expressing EBVgp350.
This aspect relates to detection of gp350, such as by an EBV-infected cell.
It will therefore be understood that an antibody or antibody fragment disclosed herein may be used to assist medical diagnosis of EBV infection. Suitably, the method includes detecting gp350, such as when expressed by EBV-infected cells present in, or obtained from, a biological sample. In certain embodiments, the biological sample may be a pathology sample that comprises one or more fluids, cells, tissues, organs or organ samples obtained from a human. Non-limiting examples include blood, plasma, saliva, serum, lymphocytes, urine, faeces, amniotic fluid, cervical samples, cerebrospinal fluid, tissue biopsies, bone marrow and skin, although without limitation thereto.
In some embodiments, the antibody or antibody fragment is labeled.
The label may be selected from a group including biotin, avidin, digoxigenin, an enzyme (e.g alkaline phosphatase or horseradish peroxidase), a fluorophore (e.g. FITC, Texas Red, Coumarin), a radioisotope (e.g. 125I, 131I, 67Ga, 111In) and/or a direct visual label (e.g. a gold particle), although without limitation thereto.
Suitably, detection of gp350 includes the step of forming a detectable complex between an antibody or antibody fragment and gp350 or a cell expressing gp350. The complex so formed may be detected by any technique, assay or means known in the art including immunoblotting, immunohistochemistry, immunocytochemistry, immunoprecipitation, ELISA, flow cytometry, magnetic bead separation, biosensor-based detection systems such as surface plasmon resonance and imaging such as PET imaging, although without limitation thereto.
To facilitate detection the antibody may be directly labeled as hereinbefore described or a labeled secondary antibody may be used. The labels may be as hereinbefore described.
In some embodiments, a detection kit may be provided which comprises an antibody or antibody fragment disclosed herein together with one or more detection reagents such as enzymes, enzyme substrates (e.g Luminol, AMPPD, NBT), secondary antibodies and/or magnetic beads although without limitation thereto.
So that preferred embodiments may be described in detail and put into practical effect, reference is made to the following non-limiting Examples.
The Generation of EBV-Specific gp350 Neutralising Monoclonal Antibodies
To explore the ability to produce neutralising anti-gp350 antibodies and determine their effect against gp350, the aim was to first generate a panel of EBV-specific gp350 monoclonal antibodies. Monoclonal antibodies were generated using two different approaches. The first approach generated gp350-specific mouse hybridomas. The second approach generated recombinant anti-gp350 antibodies through a phage display system.
(i) Murine Monoclonal Antibodies via Hybridomas
To generate gp350-specific mouse hybridomas, mice were immunized with a combination of gp350 antigen and an immune adjuvant (Sigma-Aldrich cat# S6322) and methylated CpG. Serum samples were collected from the immunized mice and reactivity to the antigen was tested by ELISA at a dilution of 1:250 and 1:1250 and compared to a pre-immunization sample. Animals with the highest anti-gp350 antibody titre were selected for the generation of hybridomas.
To generate hybridoma cells the mouse spleen was excised, dissociated into a single cell suspension and fused to SP2/0-Ag14 myeloma cells using polyethylene glycol. ELISA analysis was used to screen nine hybridoma supernatants for their reactivity to gp350. Of the nine that were screened, two clones with the highest absorbance reading F1 (OD 450 nm: 21) and G1 (OD 450 nm: 15.1) were selected for further subcloning rounds to generate murine monoclonal antibodies.
After subsequent subcloning rounds, an ELISA which tested the supernatants of each subclone at 2-fold dilutions revealed four subclones with the highest reactivity against gp350. These subclones were selected and expanded into larger volumes for purification in order to generate monoclonal antibodies.
To generate purified monoclonal antibodies, the isotypes of the four subclones were determined by performing a mouse antibody isotype ELISA where each subclone was tested against six mouse immunoglobulin isotype-specific monoclonal antibodies. Analysis shows that all subclones were positively reactive to IgG2a (
A protein G column (Sigma-Aldrich cat# ge17-0405-01) was used for the purification, in which 200 mL of supernatant from each subclone was passed through the column and eluted to generate purified gp350-specific murine monoclonal antibodies. ELISA analysis revealed that subclone RG1.E11.D11b generated 521 μg/mL of purified monoclonal antibody, RG1.E11.C11 eluted 111 μg/mL, RF1.H3b.E3.B8 generated 479 μg/mL and RF1.H3b.E3.D7b contained 374 μg/mL. We then assessed whether both approaches post production of murine and humanized monoclonal antibodies are capable of neutralising EBV infection. A preliminary test of the gp350-specific monoclonal antibodies for their EBV neutralizing ability was performed. Monoclonal antibodies and serum obtained from a sero-positive donor were tested at 2-fold dilutions with PBMCs (Peripheral Blood Mononuclear Cells) at 2×105 cells per well, from a sera-negative donor against B95-8 (infectious mononucleosis-derived isolate of EBV) in a 96-well plate format. The neutralization assay was performed in quadruplicate, and the plate was left to incubate for a duration of 6 weeks. Neutralization was determined by analyzing whether each antibody neutralized B cell transformation by EBV infection. It was found that none of the murine monoclonal antibodies were able to neutralize EBV and thus failed to block transformation of B cells (
(ii) Generation of Human gp350-Specific Antibodies using Antibody Phage Display (ADP) Platform Technology
This second approach was aimed to generate fully functional recombinant human monoclonal antibodies through antibody phage display which is based on genetic engineering of bacteriophages and repeated rounds of antigen-guided selection and phage propagation. In this procedure, a library of purified phage having DNA inserts from peripheral blood B lymphocytes obtained from healthy human donors was exposed to gp350 in order to isolate a pool of phage particles that bind specifically to glycoprotein gp350. Phage particles remaining after washing away non-binders are then harvested and used to infect E. coli bacteria for amplification (
As the objective was to obtain monoclonal antibodies, individual clones from the fourth round of bio-panning was isolated from cell glycerol stocks. This procedure involved growing single colonies from the cell glycerol stocks onto 4×150 mm 2YT-Ampicillin Glucose (2%) plates overnight, after which single cell colonies were individually plated onto a 96-well plate. Helper phage was added to each well of the 96-well plate to induce the production of phage particles. Phage particles from each clone were then tested via monoclonal phage ELISA for specific binding to gp350. Of the 96 clones, it was found that 44 were positive (absorbance >1) to gp350. The CDR sequences of all 44 positive clones were analyzed and six (6) clones were found to be uniquely positive to gp350.
PCR amplification products encoding VH and VL fragments were produced from the original six (6) phage clones. The translated CDR sequences shown in Tables 1-12 (SEQ ID NOS:1-144) were identified and numbered using abYsis version 2.7.3 and IMGT/V-QUEST. These unique six (6) clones were used to generate fully functional human monoclonal antibodies by antibody reformatting with a human IgG1 backbone (
These recombinant human monoclonal antibodies were then expressed using a mammalian expression system. Target DNA sequence encoding each of full-length antibody sequences were optimized, synthesized and sub-cloned into pcDNA3.4 vector. The recombinant plasmids encoding target antibody were transiently co-transfected into suspension Expi293F cell cultures. The cell culture supernatants collected on day 6 were used for purification. Cell culture supernatants were centrifuged and followed by filtration. These filtered cell culture supernatants were loaded onto affinity purification column at an appropriate flowrate. After washing and elution with appropriate buffer, the eluted fractions were pooled and buffer exchanged to final formulation buffer. The purified protein was analyzed by SDS-PAGE, Western blotting and SEC-HPLC analysis for molecular weight and purity measurements.
Representative data for Clone B8 and Clone B7 are presented
Biophysical Analysis of Humanized Monoclonal Antibodies Bound to gp350 via Surface Plasmon Resonance
Surface Plasmon Resonance (SPR) analysis of gp350 glycoprotein and humanized monoclonal antibodies was performed using the Biacore T100 system (GE Healthcare). CM5 sensor chips were used with 1× PBS as the running buffer, at a flow rate of 30 μL/min. Glycoprotein gp350 was immobilised onto the flow cells of the sensor chip with a capture level of 1243RD with a flow rate of 10 μL/min, a flow cell of the sensor chip was left blank for referencing of the sensograms. Single cycle kinetics was then used to determine the dissociation constant (Kd) for each humanized monoclonal antibody against gp350, with concentrations ranging from 25 ug/mL to 1.2 mg/mL with 1:2 dilution series. A flow rate of 40 μL/min was used for the duration of the experiment, with contact time of 120 seconds and 600 seconds for the dissociation time per antibody for each of their concentrations. The regeneration of the surface was achieved using 10 mM glycine buffer pH 2.1 with a 30 second injection at 30 μL/min.
Single cycle kinetic binding curves (
Referring to the schematic shown in
Virus loads for each treatment group was monitored using quantitative PCR, which shows B7 (0.052 copies/mL) and B8 (0.047 copies/mL) with significantly decreased viral loads compared to PBS (97.196 copies/mL) treated mice group on day 28, p<0.0001 (
Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. It will therefore be appreciated by those of skill in the art that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention.
All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2017903197 | Aug 2017 | AU | national |
This application is the U.S. National Stage of International Application No. PCT/AU2018/050851, filed on Aug. 10, 2018, which designates the U.S., published in English, and claims priority under 35 U.S.C. § 119 or 365(c) to Australian Application No. 2017903197, filed on Aug. 10, 2017. The entire teachings of the above applications are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/AU2018/050851 | 8/10/2018 | WO | 00 |
