WO2015199618A1 - Anticorps spécifique de lmp2 du virus d'epstein-barr et ses utilisations - Google Patents

Anticorps spécifique de lmp2 du virus d'epstein-barr et ses utilisations Download PDF

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WO2015199618A1
WO2015199618A1 PCT/SG2015/050181 SG2015050181W WO2015199618A1 WO 2015199618 A1 WO2015199618 A1 WO 2015199618A1 SG 2015050181 W SG2015050181 W SG 2015050181W WO 2015199618 A1 WO2015199618 A1 WO 2015199618A1
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antibody
fragment
hla
seq
peptide
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Inventor
Paul Anthony Macary
Brendon John Hanson
Chien Tei Too
Adrian Chong Nyi SIM
Junyun LAI
Conrad En Zuo CHAN
Angeline Pei Chiew Lim
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National University of Singapore
DSO National Laboratories
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National University of Singapore
DSO National Laboratories
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—DNA viruses
    • C07K16/085—Orthoherpesviridae (F), e.g. pseudorabies virus or Epstein-Barr virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2833—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules

Definitions

  • the disclosure relates to monoclonal antibodies that target Epstein-Barr virus (EBV) associated tumor cells by binding to HLA/LMP2 peptide complexes.
  • EBV Epstein-Barr virus
  • Epstein-Barr Virus is a ubiquitous human herpesvirus, which is found as a predominantly asymptomatic infection in all human communities. EBV has been linked to numerous human tumours of diverse tissue origin. These include nasopharyngeal carcinoma (NPC), Burkitt's lymphoma (BL), Hodgkin's lymphoma (HL), gastric carcinoma, T-cell lymphomas, leiomyosarcoma, and breast cancer 6 .
  • NPC nasopharyngeal carcinoma
  • BL Burkitt's lymphoma
  • HL Hodgkin's lymphoma
  • gastric carcinoma T-cell lymphomas
  • leiomyosarcoma and breast cancer 6 .
  • EBV EBV-infected cell 7 ' 8
  • EBV gene products consistently observed in NPC biopsies and other EBV malignancies include BRLF1, EBNA1, LMP1 and LMP2A 9 ' 10 .
  • EBV Epstein-Barr virus
  • the present disclosure provides a recombinant antibody or fragment thereof that binds to a HLA/peptide complex.
  • the peptide is derived from Epstein-Barr virus Latent Membrane Protein 2 (LMP2). In some embodiments, the peptide has the sequence
  • the HLA is HLA-A0201.
  • the recombinant antibody or fragment thereof is capable of competitively inhibiting specific binding to the peptide of SEQ ID NO: 1, or competitively inhibiting specific binding by a monoclonal antibody produced by an anti-HLA-
  • the recombinant antibody or fragment thereof is capable of competitively inhibiting specific binding by a monoclonal antibody produced by an anti-HLA-A0201/LMP2 hybridoma clone #243.
  • the antibody or fragment thereof is selected from the group consisting of: (a) a whole immunoglobulin molecule; (b) an scFv; (c) a Fab fragment; (d) an F(ab')2; and (e) a disulfide linked Fv.
  • the antibody or fragment thereof comprises a heavy chain immunoglobulin constant domain selected from the group consisting of: (a) a human IgM constant domain; (b) a human IgGl constant domain; (c) a human IgG2 constant domain; (d) a human IgG3 constant domain; (e) a human IgG4 constant domain; and (f) a human IgAl/2 constant domain.
  • the antibody or fragment thereof comprises a light chain immunoglobulin constant domain selected from the group consisting of: (a) a human Ig kappa constant domain; and (b) a human Ig lambda constant domain.
  • the antibody or fragment thereof comprises a heavy chain comprising at least one CDR having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 31-33.
  • the antibody or fragment thereof comprises a light chain comprising at least one CDR having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 36-38.
  • the antibody or fragment thereof comprises a heavy chain comprising three CDR sequences selected from the group consisting of SEQ ID NOs: 31-33, or a sequence having at least 95% identity thereto.
  • the antibody or fragment thereof comprises a light chain comprising three CDR sequences selected from the group consisting of SEQ ID NOs: 36-38, or a sequence having at least 95% identity thereto.
  • the antibody or fragment thereof comprises a heavy chain sequence at least 95% identical to SEQ ID NO: 30.
  • the antibody or fragment thereof comprises a light chain sequence at least 95% identical to SEQ ID NO: 35.
  • the antibody or fragment thereof binds to an antigen with an affinity constant (K D ) of less than 1 x 10 - " 8 M. In some embodiments, the antibody or fragment thereof binds to an antigen with an affinity constant (K D ) of less than 1 x 10 "9 M.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or fragment thereof according any of the aspects and embodiments above and a pharmaceutically acceptable carrier effective to reduce EBV infected tumor cells in a subject.
  • the present disclosure provides a method of passive immunization comprising administration to a subject an effective amount of the antibody or fragment thereof according to any of the aspects and embodiments above.
  • the present disclosure provides a method of treatment of EBV virus infection comprising administration to a subject in need thereof an amount of antibody or fragment thereof according to any of the aspects and embodiments above, effective to reduce or prevent the disease.
  • the antibody is administered intravenously (IV), subcutaneously (SC), intramuscularly (IM), transdermally, or orally.
  • the antibody is administered in an amount in the range of 1 to 100 milligrams per kilogram of the subject's body weight.
  • the present disclosure provides a method of generating a recombinant antibody or fragment thereof that binds to a HLA/peptide complex, the method comprising the steps of: (a) generating a complex of HLA associated with a peptide; (b) immunizing an animal with the complex of step (a); (c) isolating B cells specific to the complex; and (d) immortalizing the B-cells from (c).
  • the isolation in step (c) is by immuno-magnetic selection.
  • the peptide is derived from Epstein-Barr virus Latent Membrane Protein 2 (LMP2).
  • LMP2 Epstein-Barr virus Latent Membrane Protein 2
  • the peptide has the sequence CLGGLLTMV (SEQ ID NO: 1).
  • the HLA is HLA- A0201.
  • the present disclosure provides an isolated nucleic acid encoding the antibody or fragment thereof according to any of the aspects and embodiments above.
  • an expression vector comprising the above nucleic acid.
  • a host cell comprising the expression vector above is provided.
  • the host cell is a bacterial cell, a eukaryotic cell, or a mammalian cell.
  • Figure 1 Schematic illustrating the experimental procedures used for the production of TCR-like monoclonal antibodies.
  • Figure 2 Expression and purification of HLA heavy and light chains.
  • Figure 4 Screening B cell hybridomas for TCR-like HLA-A0201/LMP2 specificity.
  • Figure 4 discloses SEQ ID NOS 1, 2, 10, 3, 9, 6, 39, 8, 4, 5 and 11, respectively, in order of appearance.
  • Figure 5 Pre-selection of B cells for required specificity significantly enhances the percentage of A0201/LMP2 specific hybridomas versus unselected spenocytes.
  • Figure 6 Immunoglobulin isotype test for anti-HLA-A0201/LMP2 specific monoclonal.
  • Figure 7 Affinity determination of TCR-like monoclonals HLA-A0201/LMP2 #243 against increasing concentrations of antigen using BiacoreTM.
  • Figure 8 Fine mapping of interaction between HLA-A0201/LMP2 complex with #243 TCR-like monoclonal antibody by alanine walking.
  • Figure 8B discloses SEQ ID NOS 1, 17-25 and 11, respectively, in order of appearance.
  • Figure 9 Variants of HLA-A0201 restricted LMP2 peptide (SEQ ID NO: 1) of interest.
  • Figure 9 table discloses SEQ ID NOS 1 and 26-28, respectively, in order of appearance.
  • Figure 10 Surface binding of TCR-like monoclonal to T2 cells pulsed with peptides with various clinically observed peptides variant to CLGGLLTMV (SEQ ID NO: 1).
  • Figure 11 Anti-HLA- A02/LMP 1 recognises LMP2 epitope presented on HLA- A0201, HLA-A0206 and HLA-A0207.
  • Figure 12 Deoxyribonucleic acid (top) and corresponding translated amino acid sequences (bottom) (heavy (SEQ ID NOS 29 and 30, respectively, in order of appearance with bolded CDR peptides disclosed as SEQ ID NOS 31-33, respectively, in order of appearance) and light chains (SEQ ID NOS 34 and 35, respectively, in order of appearance with bolded CDR peptides disclosed as SEQ ID NOS 36-38, respectively, in order of appearance) variable regions) of #243 derived from murine hybridoma single cell clones with specificity for HLA-A0201/LMP2 (peptide CLGGLLTMV (SEQ ID NO: 1)).
  • Figure 13 Immunological staining of EBV infected-HLA-A0201 positive nasopharyngeal carcinoma (NPC) biopsy with TCR-like monoclonal antibodies.
  • EBV Epstein-Barr virus
  • TCR T cell receptor
  • APC antigen-presenting cells
  • a monoclonal antibody specific for a peptide (amino acid sequence CLGGLLTMV (SEQ ID NO: 1)) derived from the tumour virus antigen, Latent Membrane Protein 2 (LMP2) of Epstein-Barr Virus (EBV) that is expressed on the surface of EBV infected cells in association with MHC class I (specifically on a form termed Human Leukocyte Antigen-HLA-A0201).
  • LMP2 Latent Membrane Protein 2
  • EBV Epstein-Barr Virus
  • This epitope is also more immunogenic than other HLA-0201 restricted LMP2 epitopes in terms of donor responses and the number of specific CTL clones isolated 4 .
  • this monoclonal represents a unique new reagent that can be utilized to detect and target EBV infected tumor cells based on their intracellular expression of LMP2.
  • Epstein-Barr virus infection has been linked to the development of several important forms of human cancer including nasopharyngeal carcinoma (NPC), Burkitt's lymphoma (BL), Hodgkin's lymphoma (HL), gastric carcinoma, T-cell lymphomas, leiomyosarcoma, and breast cancer 5
  • NPC nasopharyngeal carcinoma
  • BL Burkitt's lymphoma
  • HL Hodgkin's lymphoma
  • gastric carcinoma gastric carcinoma
  • T-cell lymphomas T-cell lymphomas
  • leiomyosarcoma and breast cancer 5
  • this antibody can be utilized as a targeted delivery system for chemotherapeutic drugs, cytokines, pro-inflammatory mediators and toxins that will target the tumors based on their infection with EBV.
  • “Vertebrate,” “mammal,” “subject,” “mammalian subject,” or “patient” are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, rats, and mice, cows, horses, goats, and other animals.
  • Animals include all vertebrates, e.g. , mammals and non-mammals, such as mice, sheep, dogs, cows, avian species, ducks, geese, pigs, chickens, amphibians, and reptiles.
  • Treating refers generally to either (i) the prevention of infection or reinfection, e.g., prophylaxis, or (ii) the reduction or elimination of symptoms of a disease of interest, e.g. , therapy. Treating a subject with the compositions described herein can prevent or reduce the risk of infection from Epstein-Barr virus. Treatment can be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease.
  • Preventing or “prevention” refers to prophylactic administration with compositions described herein.
  • “Therapeutically-effective amount” or “an amount effective to reduce or eliminate infection” or “an effective amount” refers to an amount of an antibody composition that is sufficient to prevent Epstein-Barr virus infection or to alleviate (e.g. , mitigate, decrease, reduce) at least one of the symptoms associated with such an infection. It is not necessary that the administration of the composition eliminate the symptoms of Epstein-Barr virus infection, as long as the benefits of administration of the composition outweigh the detriments.
  • the terms “treat” and “treating” in reference to Epstein-Barr virus infection are not intended to mean that the subject is necessarily cured of infection or that all clinical signs thereof are eliminated, only that some alleviation or improvement in the condition of the subject is effected by administration of the composition.
  • Passive immunity refers generally to the transfer of active humoral immunity in the form of pre-made antibodies from one individual to another.
  • passive immunity is a form of short-term immunization that can be achieved by the transfer of antibodies, which can be administered in several possible forms, for example, as human or animal blood plasma or serum, as pooled animal or human immunoglobulin for intravenous (IVIG) or
  • IG intramuscular
  • Passive transfer can be used prophylactically for the prevention of disease onset, as well as, in the treatment of several types of acute infection.
  • immunity derived from passive immunization lasts for only a short period of time, and provides immediate protection, but the body does not develop memory, therefore the patient is at risk of being infected by the same pathogen later.
  • antibody refers to any immunoglobulin or intact molecule as well as to fragments thereof that bind to a specific epitope.
  • Such antibodies include, but are not limited to polyclonal, monoclonal, chimeric, humanized, single chain,
  • IgA IgA, IgD, IgE, IgG, and IgM.
  • antibody fragment refers specifically to an incomplete or isolated portion of the full sequence of the antibody which retains the antigen binding function of the parent antibody.
  • antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • An intact "antibody” comprises at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or V R ) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CHi, CH 2 and CH 3 .
  • Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or V L ) and a light chain constant region.
  • the light chain constant region is comprised of one domain, C L -
  • the V H and V L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • the term antibody includes antigen-binding portions of an intact antibody that retain capacity to bind.
  • binding examples include (i) a Fab fragment, a monovalent fragment consisting of the V L , V H , C L and CHI domains; (ii) a F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, Nature, 341:544-546 (1989)), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).
  • a Fab fragment a monovalent fragment consisting of the V L , V H , C L and CHI domains
  • F(ab') 2 fragment a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region
  • the antibody e.g., a mouse monoclonal antibody
  • the antibody comprises CDR sequences that were generated by affinity maturation, and thus would not naturally be found in the organism from which the immune response was generated.
  • affinity maturation e.g., a mouse was immunized with an antigen or immunogen described herein, one of skill in the art would understand that, after affinity maturation, the CDR sequences of the antibody produced by a hybridoma described herein would differ from the germline sequences of the immunized mouse, and therefore would not be found in nature.
  • single chain antibodies or “single chain Fv (scFv)” refers to an antibody fusion molecule of the two domains of the Fv fragment, V L and V H - Although the two domains of the Fv fragment, V L and V H , are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al, Science, 242:423-426
  • human sequence antibody includes antibodies having variable and constant regions (if present) derived from human germline immunoglobulin sequences.
  • the human sequence antibodies described herein can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site- specific mutagenesis in vitro or by somatic mutation in vivo).
  • Such antibodies can be generated in non-human transgenic animals, e.g., as described in PCT App. Pub. Nos. WO 01/14424 and WO 00/37504.
  • the term "human sequence antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (e.g., humanized antibodies).
  • recombinant immunoglobulins can be produced. See, Cabilly, U.S. Patent No. 4,816,567, incorporated herein by reference in its entirety and for all purposes; and Queen et al, Proc Natl Acad Sci USA, 86: 10029-10033 (1989).
  • the term "monoclonal antibody” refers to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • the term “human monoclonal antibody” refers to antibodies displaying a single binding specificity which have variable and constant regions (if present) derived from human germline immunoglobulin sequences.
  • the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.
  • the term "antigen” refers to a substance that prompts the generation of antibodies and can cause an immune response. It can be used interchangeably in the present disclosure with the term "immunogen".
  • immunogens are those substances that elicit a response from the immune system, whereas antigens are defined as substances that bind to specific antibodies.
  • An antigen or fragment thereof can be a molecule (i.e., an epitope) that makes contact with a particular antibody.
  • a protein or a fragment of a protein When a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein can induce the production of antibodies (i.e., elicit the immune response), which bind specifically to the antigen (given regions or three-dimensional structures on the protein).
  • humanized antibody refers to at least one antibody molecule in which the amino acid sequence in the non-antigen binding regions and/or the antigen-binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
  • Humanized antibodies include those antibodies that, while initially starting off containing antibody amino acid sequences that are not human, have had at least some of these nonhuman antibody amino acid sequences replaced with human antibody sequences. This is in contrast with human antibodies, in which the antibody is encoded (or capable of being encoded) by genes possessed a human.
  • chimeric antibodies In addition, techniques developed for the production of "chimeric antibodies" (Morrison, et ah, Proc Natl Acad Sci, 81:6851-6855 (1984), incorporated herein by reference in their entirety) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used.
  • the genes from a mouse antibody molecule specific for an autoinducer can be spliced together with genes from a human antibody molecule of appropriate biological activity.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region.
  • An immunoglobulin light or heavy chain variable region consists of a "framework" region interrupted by three hypervariable regions, referred to as complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • humanized antibodies are antibody molecules from non-human species having one or more CDRs from the non- human species and a framework region from a human immunoglobulin molecule.
  • Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.
  • Fab and F(ab')2 portions of antibody molecules can be prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibody molecules by methods that are well-known. See e.g., U.S. Patent No. 4,342,566.
  • Fab' antibody molecule portions are also well-known and are produced from F(ab')2 portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide.
  • ABSP antigen binding protein
  • target antigen e.g., a peptide derived from LMP2
  • Antigen binding protein includes but is not limited to antibodies and binding parts thereof, such as immunologically functional fragments.
  • Peptibodies are another example of antigen binding proteins.
  • immunologically functional fragment (or simply “fragment") of an antibody or
  • immunoglobulin chain (heavy or light chain) antigen binding protein is a species of antigen binding protein comprising a portion (regardless of how that portion is obtained or synthesized) of an antibody that lacks at least some of the amino acids present in a full-length chain but which is still capable of specifically binding to an antigen.
  • Such fragments are biologically active in that they bind to the target antigen and can compete with other antigen binding proteins, including intact antibodies, for binding to a given epitope. In some embodiments, the fragments are neutralizing fragments.
  • These biologically active fragments can be produced by recombinant DNA techniques, or can be produced by enzymatic or chemical cleavage of antigen binding proteins, including intact antibodies.
  • Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, a diabody (heavy chain variable domain on the same polypeptide as a light chain variable domain, connected via a short peptide linker that is too short to permit pairing between the two domains on the same chain), Fab', F(ab')2, Fv, domain antibodies and single-chain antibodies, and can be derived from any mammalian source, including but not limited to human, mouse, rat, camelid or rabbit.
  • an antigen binding protein can include nonprotein components.
  • neutralizing antigen binding protein or “neutralizing antibody” refers to an antigen binding protein or antibody, respectively, that binds to a ligand and prevents or reduces the biological effect of that ligand. This can be done, for example, by directly blocking a binding site on the ligand or by binding to the ligand and altering the ligand's ability to bind through indirect means (such as structural or energetic alterations in the ligand).
  • the term can also denote an antigen binding protein that prevents the protein to which it is bound from performing a biological function. In assessing the binding and/or specificity of an antigen binding protein, e.g., an antibody or
  • an antibody or fragment can substantially inhibit binding of a ligand to its binding partner when an excess of antibody reduces the quantity of binding partner bound to the ligand by at least about 1-20, 20-30%, 30-40%, 40- 50%, 50-60%, 60-70%, 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-98%, 98-99% or more (as measured in an in vitro competitive binding assay).
  • the neutralizing ability is characterized and/or described via a competition assay.
  • the neutralizing ability is described in terms of an IC50 or EC50 value.
  • antigen binding proteins e.g., antigen binding proteins or antibodies
  • competition when used in the context of antigen binding proteins (e.g., antigen binding proteins or antibodies) that compete for the same epitope means competition between antigen binding proteins as determined by an assay in which the antigen binding protein (e.g., antibody or immunologically functional fragment thereof) being tested prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., a ligand, or a reference antibody) to a common antigen (e.g., a peptide derived from LMP2).
  • a reference antigen binding protein e.g., a ligand, or a reference antibody
  • RIA solid phase direct or indirect radioimmunoassay
  • EIA solid phase direct or indirect enzyme immunoassay
  • sandwich competition assay see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242- 253
  • solid phase direct biotin-avidin EIA see, e.g., Kirkland et al., 1986, J. Immunol.
  • solid phase direct labeled assay solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using 1-125 label (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer et al., 1990, Scand. J.
  • Such an assay involves the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabelled test antigen binding protein and a labeled reference antigen binding protein. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen binding protein. Usually the test antigen binding protein is present in excess.
  • Antigen binding proteins identified by competition assay include antigen binding proteins binding to the same epitope as the reference antigen binding proteins and antigen binding proteins binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antigen binding protein for steric hindrance to occur.
  • a competing antigen binding protein when it is present in excess, it will inhibit (e.g., reduce) specific binding of a reference antigen binding protein to a common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or 75% or more. In some instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more. In some instances, binding is inhibited by at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or more.
  • variants of the ABPs described in this application comprise variable light and/or variable heavy chains that each have at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or above 99% identity to the amino acid sequences of ABPs described herein (either the entire sequence or a subpart of the sequence, e.g., one or more CDR).
  • such antibodies include at least one heavy chain and one light chain; whereas in other instances the variant forms contain two identical light chains and two identical heavy chains (or subparts thereof). For example, by comparing similar sequences, one can identify those sections (e.g., particular amino acids) that can be modified and how they can be modified while still retaining (or improving) the functionality of the ABP.
  • an ABP can comprise a polypeptide comprising an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence described herein.
  • an ABP can comprise a polypeptide comprising an amino acid sequence having at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or 500 (or any integer within these numbers) contiguous amino acids of the amino acid sequences described herein.
  • an ABP can comprise a polypeptide encoded by a nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleotide sequence described herein.
  • an ABP can comprise a polypeptide encoded by a nucleotide sequence having at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or 500 (or any integer within these numbers) contiguous nucleotides of the nucleotide sequences described herein.
  • a skilled artisan will be able to determine suitable variants of the ABPs as set forth herein using well-known techniques.
  • one skilled in the art can identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be important for activity.
  • even areas that can be important for biological activity or for structure can be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.
  • conservative modifications to the heavy and light chains of antibodies will produce antibodies having functional and chemical characteristics similar to those of the antibodies from the cell lines described herein.
  • amino acid substitutions are selected such that the side chains have similar size, charge,
  • hydrophobicity and/or polar groups and would be expected to produce antibodies having functional and chemical characteristics similar to those of the antibodies described herein.
  • substantial modifications in the functional and/or chemical characteristics of antibodies can be accomplished by selecting substitutions in the amino acid sequence of the heavy and light chains that differ significantly in their effect on maintaining (a) the structure of the molecular 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.
  • a “conservative amino acid substitution” can involve a substitution of a native amino acid residue with a normative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position.
  • any native residue in the polypeptide can also be substituted with alanine, as has been previously described for "alanine scanning mutagenesis.” Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired.
  • amino acid substitutions can be used to identify important residues of antibodies, or to increase or decrease the affinity of the antibodies described herein.
  • One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar ABPs. In view of such information, one skilled in the art can predict the alignment of amino acid residues of an antibody with respect to its three dimensional structure. In certain embodiments, one skilled in the art can choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues can be involved in important interactions with other molecules.
  • test variants containing a single amino acid substitution at each desired amino acid residue.
  • the variants can then be screened using activity assays known to those skilled in the art.
  • Such variants can be used to gather information about suitable variants. For example, if one discovered that a change to a particular amino acid residue resulted in destroyed, undesirably reduced, or unsuitable activity, variants with such a change can be avoided. In other words, based on information gathered from such routine experiments, one skilled in the art can readily determine the amino acids where further substitutions should be avoided either alone or in combination with other mutations.
  • One method of predicting secondary structure is based upon homology modeling. For example, two polypeptides or proteins which have a sequence identity of greater than 30%, or similarity greater than 40% often have similar structural topologies.
  • the recent growth of the protein structural database (PDB) has provided enhanced predictability of secondary structure, including the potential number of folds within a polypeptide's or protein's structure. See Holm et al., Nucl. Acid. Res., 27(l):244-247 (1999). It has been suggested (Brenner et al., Curr. Op. Struct. Biol., 7(3):369-376 (1997)) that there are a limited number of folds in a given polypeptide or protein and that once a critical number of structures have been resolved, structural prediction will become dramatically more accurate.
  • Additional methods of predicting secondary structure include “threading” (Jones,
  • antigen binding protein variants include glycosylation variants wherein the number and/or type of glycosylation site has been altered compared to the amino acid sequences of a parent polypeptide.
  • protein variants comprise a greater or a lesser number of N-linked glycosylation sites than the native protein.
  • An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X can be any amino acid residue except proline.
  • the substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions which eliminate this sequence will remove an existing N-linked carbohydrate chain. Also provided is a rearrangement of N-linked carbohydrate chains wherein one or more N-linked
  • Additional antibody variants include cysteine variants wherein one or more cysteine residues are deleted from or substituted for another amino acid (e.g., serine) as compared to the parent amino acid sequence. Cysteine variants can be useful when antibodies must be refolded into a biologically active conformation such as after the isolation of insoluble inclusion bodies. Cysteine variants generally have fewer cysteine residues than the native protein, and typically have an even number to minimize interactions resulting from unpaired cysteines.
  • amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and/or (4) confer or modify other physiocochemical or functional properties on such polypeptides.
  • single or multiple amino acid substitutions in certain embodiments, conservative amino acid substitutions can be made in the naturally-occurring sequence (in certain embodiments, in the portion of the polypeptide outside the domain(s) forming intermolecular contacts).
  • a conservative amino acid substitution typically may not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence).
  • a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence.
  • Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden & J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al., Nature, 354: 105 (1991), which are each incorporated herein by reference.
  • the variants are variants of the nucleic acid sequences of the ABPs disclosed herein.
  • ABP nucleic acid sequences of the ABPs disclosed herein.
  • nucleic acid sequences encoding for those protein variants are contemplated.
  • an ABP variant can have at least 80, 80-85, 85-90, 90-95, 95-97, 97-99 or greater percent identity (or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to at least one nucleic acid sequence described herein or at least one to six (and various combinations thereof) of the CDR(s) encoded by the nucleic acid sequences described herein.
  • the antibody (or nucleic acid sequence encoding it) is a variant if the nucleic acid sequence that encodes the particular ABP (or the nucleic acid sequence itself) can selectively hybridize to any of the nucleic acid sequences that encode the proteins described herein under stringent conditions.
  • suitable moderately stringent conditions include prewashing in a solution of 5.times.SSC; 0.5% SDS, 1.0 mM EDTA (pH 8:0); hybridizing at 50 C, -65 C, 5.times.SSC, overnight or, in the event of cross- species homology, at 45 C with 0.5. times.
  • hybridizing DNA sequences are also within the scope of this disclosure, as are nucleotide sequences that, due to code degeneracy, encode an antibody polypeptide that is encoded by a hybridizing DNA sequence and the amino acid sequences that are encoded by these nucleic acid sequences.
  • variants of CDRs include nucleic acid sequences and the amino acid sequences encoded by those sequences, that hybridize to one or more of the CDRs within the sequences noted above.
  • the phrase "selectively hybridize" referred to in this context means to detectably and selectively bind.
  • Polynucleotides, oligonucleotides and fragments thereof in accordance with the disclosure selectively hybridize to nucleic acid strands under hybridization and wash conditions that minimize appreciable amounts of detectable binding to nonspecific nucleic acids.
  • High acids High stringency conditions can be used to achieve selective hybridization conditions as known in the art and discussed herein.
  • the nucleic acid sequence homology between the polynucleotides, oligonucleotides, and fragments of the disclosure and a nucleic acid sequence of interest will be at least 80%, and more typically at least 85%, 90%, 95%, 99%, and 100%. Two amino acid sequences are homologous if there is a partial or complete identity between their sequences.
  • 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching, for example gap lengths of 5 or less or 2 or less.
  • two protein sequences (or polypeptide sequences derived from them) are homologous, as this term is used herein, if they have an alignment score of at more than 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater. See Dayhoff, M. O., in Atlas of Protein Sequence and Structure, pp. 101-110 (Volume 5, National Biomedical Research Foundation (1972)) and Supplement 2 to this volume, pp. 1-10.
  • the two sequences or parts thereof are homologous if their amino acids are greater than or equal to 50% identical when optimally aligned using the ALIGN program.
  • the term "corresponds to” is used herein to mean that a polynucleotide sequence is homologous (i.e., is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence.
  • the term “complementary to” is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence.
  • the nucleotide sequence "TAT AC” corresponds to a reference sequence "TAT AC” and is complementary to a reference sequence "GTATA”.
  • a number of screening assays are known in the art for assaying antibodies of interest to confirm their specificity and affinity and to determine whether those antibodies cross-react with other proteins.
  • binding refers to the interaction between the antigen and their corresponding antibodies. The interaction is dependent upon the presence of a particular structure of the protein recognized by the binding molecule (i.e., the antigen or epitope). In order for binding to be specific, it should involve antibody binding of the epitope(s) of interest and not background antigens.
  • antibodies are assayed to confirm that they are specific for the antigen of interest and to determine whether they exhibit any cross reactivity with other antigens.
  • One method of conducting such assays is a sera screen assay as described in U.S. App. Pub. No. 2004/0126829, the contents of which are hereby expressly incorporated herein by reference.
  • other methods of assaying for quality control are within the skill of a person of ordinary skill in the art and therefore are also within the scope of the present disclosure.
  • Antibodies, or antigen-binding fragments, variants or derivatives thereof of the present disclosure can also be described or specified in terms of their binding affinity to an antigen.
  • the affinity of an antibody for an antigen can be determined experimentally using any suitable method. (See, e.g., Berzofsky et al, "Antibody- Antigen Interactions," In
  • the measured affinity of a particular antibody- antigen interaction can vary if measured under different conditions ⁇ e.g., salt concentration, pH).
  • affinity and other antigen -binding parameters ⁇ e.g., K D , K a , K d
  • K D , K a , K d are preferably made with standardized solutions of antibody and antigen, and a standardized buffer.
  • the affinity binding constant (K aff ) can be determined using the following formula:
  • [mAb] is the concentration of free antigen sites
  • [mAg] is the concentration of free monoclonal binding sites as determined at two different antigen concentrations ⁇ i.e., [mAg] t and [mAg'] t ) (Beatty et al, J Imm Meth, 100: 173-179 (1987)).
  • SPR surface plasmon resonance
  • K aff of at least about 1 x 10 liters/mole, or at least about 1 x 10 liters/mole, or at least about 1 x 10 9 liters/mole, or at least about 1 x 10 10 liters/mole, or at least about 1 x 10 11 liters/mole, or at least about 1 x 10 12 liters/mole, or at least about 1 x 1013 liters/mole, or at least about 1 x 10 14 liters/mole or greater.
  • "High affinity" binding can vary for antibody isotypes.
  • K D the equilibrium dissociation constant, is a term that is also used to describe antibody affinity and is the inverse of K aff .
  • K D the equilibrium dissociation constant
  • the term "high affinity" for an antibody refers to an equilibrium dissociation constant (K D ) of less than about 1 x 10 " mole/liters, or less than about 1 x 10 - " 8 mole/liters, or less than about 1 x 10 - " 9 mole/liters, or less than about 1 x 10 "10 mole/liters, or less than about 1 x 10 "11 mole/liters, or less than about
  • antibodies according to the present disclosure provides for antibodies with the characteristics of those produced in the course of a physiological human immune response, i.e. antibody specificities that can only be selected by the human immune system. In the present case, this includes a response to the human pathogen Epstein-Barr virus. In some embodiments, antibodies of the present disclosure possess the characteristics of those produced in the course of a response to infection by Epstein-Barr virus. These antibodies can be used as prophylactic or therapeutic agents upon appropriate formulation.
  • a neutralizing antibody In relation to a particular pathogen, a “neutralizing antibody”, “broadly neutralizing antibody”, or “neutralizing monoclonal antibody”, all of which are used interchangeably herein, is one that can neutralize the ability of that pathogen to initiate and/or perpetuate an infection in a host.
  • monoclonal antibodies produced in accordance with the present disclosure have neutralizing activity, where the antibody can neutralize at a concentration of 10 "9 M or lower (e.g. 10 "10 M, 10 "U M, 10 "12 M or lower).
  • the immunoglobulin molecules of the present disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), or subclass of immunoglobulin molecule.
  • the antibodies are antigen- binding antibody fragments (e.g., human) and include, but are not limited to, Fab, Fab' and F(ab') 2 , Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a V L or V H domain.
  • Antigen-binding antibody fragments, including single-chain antibodies can comprise the variable region(s) alone or in
  • antigen-binding fragments comprising any combination of variable region(s) with a hinge region, CHI, CH2, and CH3 domains.
  • the methods of the present disclosure also provide for obtaining and/or sequencing a nucleic acid for an antibody from a selected B cell clone; and utilizing the nucleic acid to generate a host cell that can express the antibody of interest.
  • the nucleotide sequence encoding a desired antibody can be sequenced and thereafter employed in a heterologous expression system, e.g. 293 cells or CHO cells.
  • an antibody can be recombinantly expressed by obtaining one or more nucleic acids (e.g. heavy and/or light chain genes) from the a B cell clone that encodes the antibody of interest and inserting the nucleic acid into a host cell in order to permit expression of the antibody of interest in that host.
  • Vectors that can be used generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Examples of such expression system components are disclosed in, for example, U.S. Pat. No. 5,739,277.
  • Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells (see, e.g., U.S. Pat. No. 5,739,277).
  • compositions comprising the antibodies produced in accordance with the present disclosure.
  • a pharmaceutical composition can comprise one or more monoclonal antibodies produced in using the methods disclosed herein.
  • a panel of monoclonal antibodies produced according to the present disclosure can be included in a pharmaceutical composition.
  • the monoclonal antibodies produced according to the present disclosure can be included with one or more additional agents, for example, antiviral or anticancer drugs or analgesics.
  • a pharmaceutical composition can also contain a
  • the carrier is pharmaceutically acceptable for use in humans.
  • the carrier or adjuvant should not itself induce the production of antibodies harmful to the individual receiving the composition and should not be toxic.
  • Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, ammo acid copolymers and inactive virus particles.
  • Pharmaceutically acceptable salts can be used, for example mineral acid salts, such as hydrochlorides, hydrobromides, phosphates and sulphates, or salts of organic acids, such as acetates, propionates, malonate and benzoates.
  • Pharmaceutically acceptable carriers in therapeutic compositions can additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents or pH buffering substances, can be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by the patient.
  • compositions of the presently disclosed subject matter can further comprise a carrier to facilitate composition preparation and administration.
  • a suitable delivery vehicle or carrier can be used, including but not limited to a microcapsule, for example a microsphere or a nanosphere (Manome et al. (1994) Cancer Res 54:5408-5413; Saltzman & Fung (1997) Adv Drug Deliv Rev 26:209-230), a glycosaminoglycan (U.S. Pat. No. 6,106,866), a fatty acid (U.S. Pat. No. 5,994,392), a fatty emulsion (U.S. Pat. No. 5,651,991), a lipid or lipid derivative (U.S. Pat. No.
  • Antibody sequences can be coupled to active agents or carriers using methods known in the art, including but not limited to carbodiimide conjugation, esterification, sodium periodate oxidation followed by reductive alkylation, and glutaraldehyde crosslinking (Goldman et al. (1997) Cancer Res. 57: 1447-1451 ; Cheng (1996) Hum. Gene Ther. 7:275- 282; Neri et al. (1997) Nat. Biotechnol. 15: 1271-1275; Nabel (1997) Vectors for Gene
  • a therapeutic composition of the present disclosure comprises in some
  • a pharmaceutical composition that includes a pharmaceutically acceptable carrier.
  • suitable formulations include aqueous and non-aqueous sterile injection solutions which can contain anti-oxidants, buffers, bacterio stats, bactericidal antibiotics and solutes which render the formulation isotonic with the bodily fluids of the intended recipient; and aqueous and non-aqueous sterile suspensions which can include suspending agents and thickening agents.
  • the formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a frozen or freeze-dried
  • lyophilized condition requiring only the addition of sterile liquid carrier, for example water for injections, immediately prior to use.
  • sterile liquid carrier for example water for injections
  • Some exemplary ingredients are SDS in the range of in some embodiments 0.1 to 10 mg/ml, in some embodiments about 2.0 mg/ml; and/or mannitol or another sugar in the range of in some embodiments 10 to 100 mg/ml, in some embodiments about 30 mg/ml; and/or phosphate -buffered saline (PBS). Any other agents conventional in the art having regard to the type of formulation in question can be used.
  • the carrier is pharmaceutically acceptable.
  • the carrier is pharmaceutically acceptable for use in humans.
  • compositions of the present disclosure can have a pH between 5.5 and 8.5, between 6 and 8, or about 7.
  • the pH can be maintained by the use of a buffer.
  • the composition can be sterile and/or pyrogen free.
  • the composition can be isotonic with respect to humans.
  • Pharmaceutical compositions of the presently disclosed subject matter can be supplied in hermetically- sealed containers.
  • compositions can include an effective amount of one or more antibodies as described herein.
  • a pharmaceutical composition can comprise an amount that is sufficient to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic effect.
  • Therapeutic effects also include reduction in physical symptoms.
  • the precise effective amount for any particular subject will depend upon their size and health, the nature and extent of the condition, and therapeutics or combination of therapeutics selected for administration. The effective amount for a given situation is determined by routine experimentation as practiced by one of ordinary skill in the art.
  • compositions of the disclosure can be administered in a variety of unit dosage forms depending upon the method of administration. Dosages for typical antibody pharmaceutical compositions are well known to those of skill in the art. Such dosages are typically advisory in nature and are adjusted depending on the particular therapeutic context or patient tolerance. The amount antibody adequate to accomplish this is defined as a "therapeutically effective dose.”
  • the dosage schedule and amounts effective for this use, i.e., the "dosing regimen,” will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical status, age, pharmaceutical formulation and
  • the mode of administration also is taken into consideration.
  • the dosage regimen must also take into consideration the pharmacokinetics, i.e., the pharmaceutical composition's rate of absorption, bioavailability, metabolism, clearance, and the like. See, e.g., the latest
  • a therapeutically effective amount of a composition comprising an antibody contains about 0.05 to 1500 ⁇ g protein about 10 to 1000 ⁇ g protein, about 30 to 500 ⁇ g or about 40 to 300 pg, or any integer between these values.
  • antibodies described herein can be administered to a subject at a dose of about 0.1 ⁇ g to about 200 mg, e.g., from about 0.1 ⁇ g to about 5 ⁇ g, from about 5 ⁇ g to about 10 ⁇ g, from about 10 ⁇ g to about 25 ⁇ g, from about 25 ⁇ g to about 50 ⁇ g, from about 50 ⁇ g to about 100 ⁇ g, from about 100 ⁇ g to about 500 ⁇ g, from about 500 ⁇ g to about 1 mg, from about 1 mg to about 2 mg, with optional boosters given at, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, two months, three months, 6 months and/or a year later.
  • the specific dose level for any particular patient depends upon a variety of factors including the activity of the specific antibody employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
  • Routes of administration include, but are not limited to, oral, topical,
  • compositions can be administered in a single dose treatment or in multiple dose treatments on a schedule and over a time period
  • kits comprising antibodies produced in accordance with the present disclosure which can be used, for instance, for therapeutic applications described above.
  • the article of manufacture comprises a container with a label.
  • Suitable containers include, for example, bottles, vials, and test tubes.
  • the containers can be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which includes an active agent that is effective for therapeutic applications, such as described above.
  • the active agent in the composition can comprise the antibody.
  • the label on the container indicates that the composition is used for a particular therapy or non-therapeutic application, and can also indicate directions for either in vivo or in vitro use, such as those described above.
  • Example 1 Overview of Experimental Procedures used for the Production of TCR-like Monoclonal antibodies.
  • human MHC class-I heavy chains (HC) and light chains (LC) are expressed as recombinant proteins and refolded into HLA monomers in the presence of an antigenic peptide CLGGLLTMV (SEQ ID NO: 1) from LMP2.
  • CLGGLLTMV SEQ ID NO: 1
  • the monomers are purified by FPLC and used as immunogens in Balb/C mice.
  • Splenic B lymphocytes with desired antigenic specificities are purified using immuno-magnetic beads prior to
  • PEG polyethylene glycol
  • Example 2 Expression and purification of HLA heavy and light chains.
  • HLA-A0201 heavy and light chains were expressed in BL21 E.coli.
  • the heavy and light chains were isolated as inclusion bodies and dissolved in 8M urea.
  • Protein content of heavy chain (HC) and light chain (LC) inclusion bodies was analysed using SDS-Page (A). Purification and analysis of folded monomers. Heavy chain, light chain and the LMP2 peptide (CLGGLLTMV (SEQ ID NO: 1)) were refolded into complete HLA-complexes in vitro. Anion-exchange chromatography was used to purify the monomers.
  • B FPLC profiles (i) followed by SDS-PAGE analysis (ii).
  • Peak A at 19mins contained light chain ( ⁇ 2 ⁇ ) only whereas peak B at 36-37mins contained both heavy chain (44kD) and light chain (12kD).
  • Fractions collected from peak B were pooled and analyzed and further purified using a size-exclusion column.
  • the peak was collected from the chromatography (Ci) before being analysed on a non-denaturing native gel (Cii), lane 1.
  • the gel was immunoblotted with the anti-HLA conformation specific monoclonal w6/32 and compared with two previously successfully folded monomers (lane 2 and 3). This confirmed that the purified monomer was correctly folded.
  • a single inoculant of the successfully transformed cell containing the pET30a- HLA-A0201 plasmid was inoculated into 15 ml of LB media containing 50 ⁇ g/ml of kanamycin selective antibiotics. This inoculant was incubated at 37°C for up to 16 hrs with shaking to serve as a starter culture.
  • the OD 6 oo was measured every half an hour to determine the mid-log phase of the culture (OD 6 oo between 0.6 to 0.8) and protein expression was induced with 1 mM of isopropyl ⁇ -D-I-thiogalactopyranoside (IPTG) (Sigma- Aldrich).
  • IPTG isopropyl ⁇ -D-I-thiogalactopyranoside
  • the culture was further incubated in the dark at 37 °C for 4 hrs with shaking. Thereafter, the culture was centrifuged at 4 °C, 5000 rpm for 10 mins to pellet down the cells. The pellet was resuspended in 10ml of resuspension buffer with 10 mM dithiothreitol (DTT) (Sigma-Aldrich), 0.2 mM
  • phenylsulphonylfluoride (PMSF) (Sigma-Aldrich), and 5 ⁇ 1 mg/ml pepstatin A (Sigma- Aldrich).
  • PMSF phenylsulphonylfluoride
  • pepstatin A Sigma- Aldrich
  • the inclusion bodies were thawed at room temperature. For every 10 ml of resuspended pellet, 25 ml of lysis buffer with 1 ml 1 mg/ml DNase I (Sigma Aldrich), 10 mM DTT and 5 mM MgC12 was added. The mixture was shaken vigorously on ice for 20 mins followed by addition of 10 mM sodium ethylenediaminetetraacetate (NaEDTA, pH8.0). Sonication on the cells was carried out for 5 cycles, each consisting of 30 s pulsing with 30 s breaks.
  • the mixture was pelleted down at 10 000 rpm, 4 °C for 15 mins followed by resuspending it in 30 ml of wash buffer with 1 mM DTT, 0.2 mM PMSF and 30 ⁇ 1 mg/ml pepstatin A.
  • the mixture was further homogenized for 30 s and centrifuged down at 10 000 rpm, 4 °C for 15 mins. This step was repeated twice.
  • the pellet was further resuspended in 20 ml of wash buffer with 1 mM DTT, 0.2 mM PMSF and 20 ⁇ 1 mg/ml pepstatin A.
  • the mixture was further homogenized for 30 s and centrifuged down at 10 000 rpm, 4 °C for 15 mins.
  • the pellet was resuspended with 400 ⁇ of water to form a white paste.
  • 20 ml of 8 M urea buffer supplemented with 0.1 mM DTT, 0.2 mM PMSF and 20 ⁇ 1 mg/ml pepstatin A was added to the white paste.
  • the mixture were shaken for 1 hr at room temperature and centrifuged down at 10 000 rpm, 4 °C for 1 hr.
  • the supernatant containing either the HLA-A0201 or beta 2 microglobulin constructs were collected into 50 ml falcon tube and 0.2 niM PMSF and 10 ⁇ lmg/ml pepstatin A were added into each tube. These construct where then aliquot into 1.5 ml eppendorf tubes and stored at -80 °C.
  • Protein was eluted in an increasing salt concentrations using buffer B at a flow rate of 5 ml/min and fractions were collected in 1 ml samples.
  • the refolded HLA monomers were eluted with approximately 15 % of buffer B and the selected samples were subsequently analyzed on a SDS-PAGE gel.
  • Fractions that contain both the heavy chain (35 kDa) and beta-2 microglobulin (12 kDa) were pooled together and concentrated to approximately 500 ⁇ using the Centricon Centrifugal Filter Unit with Ultracel YM-30 membrane (Millipore Corporation). The purified fraction was used for further purification.
  • biotinylated product was quantitated by immunoprecipitation using streptavidin beads (Sigma- Aldrich Inc.). Approximately 10 ⁇ of beads were washed with 1 ml of 10 mM Tris, 150 mM NaCl, 0.1% Triton-XlOO and pulsed down at not more than 8000 rpm. The supernatant was discarded and 20 ⁇ g of previously biotinylated monomers were added with 500 ⁇ of 10 mM Tris, 2 ⁇ of 0.1 M PMSF and 1 ⁇ of 1 mg/ml of pepstatin A. The mixture was left to shake at 4 °C for 16 hrs. After incubation, the beads were washed similarly as stated above twice.
  • the supernatant were subsequently discarded and the sample was ran on 15 % SDS-PAGE gel and visualized with Coomassie blue staining.
  • the percentage of successfully biotinylated HLA monomers was determined by comparing against non-immunoprecipiated HLA monomers. A negative control of streptavidin beads only was used. The quantity of monomers was determined by visual comparison with known concentrations of bovine serum albumin (BSA) standards (Sigma- Aldrich) that were ran on the same SDS-PAGE gel.
  • BSA bovine serum albumin
  • biotinylated monomer was incubated with half the splenocytes harvested at 4 °C for 20 mins. After which 5ml of cold RPMI was added and centrifuged at 1100 rpm for 5mins to wash out the unbound biotinylated monomers. The pelleted cells were incubated with 100 ⁇ of anti-biotin microbeads (Miltenyi Biotec GmbH) and incubated at 4 °C for a further 30 mins. In the meantime, the LS columns (Miltenyi Biotec GmbH) were pre- wet with 1 ml of cold PBS followed by 1 ml of RPMI medium.
  • the unbound anti-biotin microbeads (Miltenyi Biotec GmbH) were washed out by centrifuging the mixture of pelleted cells and anti-biotin microbeads at 1100 rpm for 5 mins with 3 ml of cold RPMI medium. About 1 million of the pelleted cells were then added directly onto the each pre-wet LS column. After the appropriate cells were adsorbed onto the column, 3 x 1 ml of cold RPMI medium was used to wash out the unbound cells. After which, the LS column with the bound cells was removed from the magnetic board and cells were flushed out onto a 15 ml Falcon tube with cold 3 ml of RPMI medium. This process was repeated with another 3 ml of RPMI medium to elute out the remaining cells. The 15 ml Falcon tube now contains splenocytes enriched with specificity of interest.
  • the non-enriched and enriched splenocytes were subsequently treated similarly for hybridoma fusion.
  • the splenocytes were added directly onto the NS 1 myeloma cells and centrifuged at 1100 rpm for 3 mins to pellet the cells into close proximity. The supernatant was discarded and 1 ml of warm polyethylene glycol 1500 (PEG) (Sigma- Aldrich Inc.) was added slowly over 1 min with a glass pipette. The suspension was incubated for 1 min at 37 °C for cell fusion between the splenocytes and the myeloma cells.
  • PEG polyethylene glycol 1500
  • the plates were incubated at 37 °C in a C0 2 incubator and each well was further topped up with 100 ⁇ of HAT medium on day 7. The plates were observed 8-10 days later macroscopically for visible colonies and on day 14, the medium was changed to HT media. The clones were scored from day 14 onwards and supernatant of scored clones were harvested for flow cytometry screening.
  • biotynlated HLA Monomers bind to specific B cell Receptors.
  • Anti-biotin coated Miltenyi MACs beads used to isolate bound B cells on magnetic columns prior to myeloma fusion.
  • Example 3 To perform Example 3, the following methods were used.
  • biotinylated product was quantitated by immunoprecipitation using streptavidin beads (Sigma- Aldrich Inc.). Approximately 10 ⁇ of beads were washed with 1 ml of 10 mM Tris, 150 mM NaCl, 0.1% Triton-XlOO and pulsed down at not more than 8000 rpm. The supernatant was discarded and 20 ⁇ g of previously biotinylated monomers were added with 500 ⁇ of 10 mM Tris, 2 ⁇ of 0.1 M PMSF and 1 ⁇ of 1 mg/ml of pepstatin A. The mixture was left to shake at 4 C for 16 hrs. After incubation, the beads were washed similarly as stated above twice.
  • the supernatant were subsequently discarded and the sample was ran on 15 % SDS-PAGE gel and visualized with Coomassie blue staining.
  • the percentage of successfully biotinylated HLA monomers was determined by comparing against non-immunoprecipiated HLA monomers. A negative control of streptavidin beads only was used. The quantity of monomers was determined by visual comparison with known concentrations of bovine serum albumin (BSA) standards (Sigma- Aldrich) that were ran on the same SDS-PAGE gel.
  • BSA bovine serum albumin
  • the unbound anti-biotin microbeads (Miltenyi Biotec GmbH) were washed out by centrifuging the mixture of pelleted cells and anti-biotin microbeads at 1100 rpm for 5 mins with 3 ml of cold RPMI medium. About 1 million of the pelleted cells were then added directly onto the each pre-wet LS column. After the appropriate cells were adsorbed onto the column, 3 x 1 ml of cold RPMI medium was used to wash out the unbound cells. After which, the LS column with the bound cells was removed from the magnetic board and cells were flushed out onto a 15 ml Falcon tube with cold 3 ml of RPMI medium. This process was repeated with another 3 ml of RPMI medium to elute out the remaining cells. The 15 ml Falcon tube now contains splenocytes enriched with specificity of interest.
  • Example 4 Screening B cell Hybridomas for TCR-like HLA-A0201/LMP2 specificity.
  • GILGFVFTL Green Histogram
  • cells stained with anti-mouse IgG antibody only Green Histogram
  • the antibody of clone #243 were also screened against a panel of HLA-A0201 restricted peptides from Epstein-Barr virus-LMPl (YLLEMLWRL (SEQ ID NO: 2)), LMP2 (CLGGLLTMV (SEQ ID NO: 1)), EBNA1 (FMVFLQTHI (SEQ ID NO: 3)),
  • Cytomegalovirus-pp65 (NLVPMVATV (SEQ ID NO: 4)), pp65 (ILARNLVPM (SEQ ID NO: 5)), IE1 (VLEETSVML (SEQ ID NO: 6)), IE1 (VLAELVKQI (SEQ ID NO: 7)), IE1 (YILGADPLRV (SEQ ID NO: 8)), Hepatitis B virus-core (FLPSDFFPS (SEQ ID NO: 9)), BCG ⁇ Ag85 (FIYAGSLSAL (SEQ ID NO: 10)) and Influenza matrix A-Ml (GILGFVFTL (SEQ ID NO: 11)). These peptides were pulsed onto T2 cells for staining.
  • the legend of the histogram is stated on the figure 4B.
  • the TCR-like anti-HLA-A0201/LMP2 #243 is astonishingly specific for only EBV-LMP2 (CLGGLLTMV (SEQ ID NO: 1)) associated with HLA-A0201 (Red Histogram).
  • Example 5 Pre-selection of B cells for required specificity significantly enhances the percentage of A0201/LMP2 specific Hybridomas versus Unselected spenocytes.
  • Unselected splenocytes generated no HLA-A0201/LMP2 specific hybridomas compared to splenocytes selected for their binding capacity to the immunogen.
  • the supernatant of scored clones were screened by flow cytometry using T2 cells. 100 ⁇ of the supernatant of scored clones was pipetted out of the well and transferred into two labeled flow cytometry tubes (BD Biosciences). Appropriate number of T2 cells were harvested and one set was incubated with HLA-A0201 restricted peptide (Mimotope) while the other set was incubated with our peptide of interest HLA-A0201 restricted peptide of
  • Epstein-Barr virus LMP2A 426 - 434 (CLGGLLTMV (SEQ ID NO: 1)) (Mimotope) for 30 mins at 37 °C in a C0 2 incubator. After incubation, an equal amount of peptide-pulsed cells were aliquoted into tubes containing the supernatant. The tubes with the supernatant were subsequently incubated at 4 °C for 30 mins. To wash away the excess supernatant and unbound antibodies, 4 ml of cold PBS supplemented with 1 % FBS was added into each tube and centrifuged at 350 g for 5 mins.
  • TCR-like mAbs were determined by exogenously pulsing T2 cells with 5 ⁇ of each HLA-A0201 restricted peptides from various pathogens including their respective targeted peptide.
  • T2 cell lacks both transporter associated with antigen processing-TAPl and TAP2, it is unable to express endogenously processed peptides on its MHC class I and will express peptides that are exogenously pulsed onto its MHC class I.
  • the bindings of the various TCR-like mAbs were analyzed by flow cytometry after staining with anti-murine IgG (H+L)-conjugated with RPE (Dako).
  • the antibodies were screened against a panel of HLA-A0201 restricted peptides that include EBNAl 56 2-570 (FMVFLQTHI (SEQ ID NO: 3)) (red), LMPI125-133
  • Mycobacterium tuberculosis Ag85Bi 43 _i 52 (FIYAGSLSAL (SEQ ID NO: 10)) (pink), Hepatitis B virus sAgi 83 _i 9 i (FLLTRILTI (SEQ ID NO: 12)) (light blue), Human
  • immunodeficiency virus Pol 476 _ 484 (ILKEPVHGV (SEQ ID NO: 13)) (blue), gpl20i 20 -i28 (KLTPLCVTL (SEQ ID NO: 14)) (yellow), Gag 77 _ 85 (SLYNTIAVL (SEQ ID NO: 15)) (grey), CMV IEl 3 i 6 -324 (VLEETSVML (SEQ ID NO: 6)) (orange),IEl 8 i_ 89 (VLAELVKQI (SEQ ID NO: 7)) (black), pp65 495 -503 (NLPVMVATV (SEQ ID NO: 16)) (light brown) and cells stained with anti-murine IgG (H+L) antibody (light green). It was observed that theTCR-like mAb was astonishingly specific only for its respective peptide associated with HLA-A0201 and not the rest of HLA-A0201 peptide complexes .
  • T2 cells were pulsed with various concentrations of the peptides and negative control for the TCR-like monoclonal antibody, namely EBNAI 562 -570 (FMVFLQTHI (SEQ ID NO: 3)).
  • EBNAI 562 -570 FMVFLQTHI (SEQ ID NO: 3)
  • the monoclonal antibodies were used to detect their respective HLA-A0201 bound peptides.
  • the TCR-like mAbs targeting HLA-A0201/EBNA1 was able to detect its targeted peptide pulsed down to 14pM.
  • Example 6 Immunoglobulin Isotype Test for anti-HLA-A0201/LMP2 specific monoclonal.
  • the isotype profile for clone LMP2#243 shown above is of IgGl and kappa light chain.
  • HC Heavy Chains
  • LC Light Chains.
  • Example 7 Affinity determination of TCR-like monoclonals HLA-A0201/LMP2 #243 against increasing concentrations of antigen using BiacoreTM.
  • antigen concentrations ranged from 0 to 320nM.
  • the TCR-like monoclonal antibody was immobilized to a Biacore CM5 sensor chip via amine group linkage up to 1200 RU.
  • the kinetic measurements of the binding of the antibody were determined by flowing various concentrations of HLA-A02/LMP-2A peptide monomer over the surface of the respective antibody coated chip. Dilutions of 320 nM to 10 nM of monomers were injected at 30 ⁇ /min and the binding response was calculated assuming 1: 1 Langmuir binding.
  • the data were analyzed using a global fit algorithm (BIA evaluation 3.1) to calculate association rate (K on ) and dissociation rate (K 0 ff) values to determine the dissociation constant or affinity constant C3 ⁇ 4)-
  • the of the TCR- like mAb against HLA-A02/LMP2A is 6.98 nM.
  • Example 8 Fine mapping of interaction between HLA-A0201/LMP2 complex with #243 TCR-like monoclonal antibody by alanine walking.
  • each of the peptides were mutated with alanine at the position indicated by the peptide's number allocation within the HLA-A0201 restricted LMP2 epitope sequence CLGGLLTMV (SEQ ID NO: 1).
  • the antibody staining of each peptide pulsed on T2 cells were visualized using flow cytometry.
  • the histogram showed the flow cytometry results of staining with #243 monoclonal antibody on various T2 cells pulsed with the peptide allocated as stated (A).
  • the positions mutated to alanine at various positions are shown on the table B.
  • the measured mean fluorescence index (MFI) for the readings from flow cytometry were plotted in a histogram (C).
  • the interaction between #243 antibody and antigen (CLGGLLTMV (SEQ ID NO: 1)) is sensitive between positions 5 to 9 as any mutation in the amino acids resulted in a decrease in the ability of the antibody to recognize the antigen.
  • Alanine scanning mutagenesis of the peptide of interest is a method of systematic alanine substitution for the identification of sites or amino acid for proper conformation. This form of mutagenesis enables the fine mapping of antibody-epitope interaction with non-polar hydrophobic amino acid (alanine). With the substitution of alanine, all side chain atoms beyond the ⁇ -carbon are removed and the role of the side chain functional groups of the original amino acids can be inferred from these alanine mutations. Alanine with methyl side chain lacks the unusual backbone dihedral angel preferences. Although glycine with an H side chain similarly nullifies the side chain, it enables conformational flexibility due to other non-mutated amino acids within the peptide.
  • Example 9 Variants of HLA-A0201 restricted LMP2 peptide of interest.
  • Example 10 Surface binding of TCR-like monoclonal to T2 cells pulsed with peptides with various clinically observed peptides variant to CLGGLLTMV (SEQ ID NO: 1).
  • the bar chart represents the mean fluorescent index of the various peptides pulsed T2 cells followed by detection with anti-HLA-A0201/LMP2 antibody #243.
  • the flow cytometry histogram is a representative data from each of the T2 cells pulsed with the peptides indicated by the arrows before being stained with the TCR-like monoclonal antibody against HLA-A0201/LMP2 #243.
  • the allocation of the peptides is based on Figure 9.
  • the antibody #243 binds to all clinical variants of the peptide, CLGGLLTMV (SEQ ID NO: 1).
  • Example 11 Anti-HLA-A02/LMP2 recognises LMP2 epitope presented on HLA-A0201, HLA-A0206 and HLA-A0207.
  • TCR-like mAbs in the context of HLA-A02 haplotype polymorphism
  • HLA-A02 positive Caucasian consists of A0201 and A02
  • Southern Chinese population consist of four major groups namely A0201, A0203, A0206 and A0207.
  • the polymorphism of these 4 subfamilies is due to amino acid mutations in the l and o2 chains.
  • the MHC class I diagram illustrates the anchor amino acids at positions 2 and 9 of a typical A0201 restricted peptide.
  • Differential recognition of the TCR-like mAb against LMP-2A 426 - 4 34 epitope presented on HLA-A0201, -A0203, -A0206 and -A0207.
  • Flow cytometry was used to analyze binding to 4 BLCL pulsed with the LMP-2A 42 6- 434 peptides and its clinical variant and stained with the TCR-like mAb. This was in comparison of pulsed BLCLs stained with an isotype antibody.
  • Example 12 DNA and corresponding translated amino acid sequences of heavy and light chains variable regions of #243 derived from murine hybridoma single cell clones with specificity for HLA-A0201/LMP2 (peptide CLGGLLTMV (SEQ ID NO: 1)).
  • FIG. 12 Shown in Figure 12 is the deoxyribonucleic acid (top) and corresponding translated amino acid sequences (bottom) (heavy and light chains variable regions) derived from murine hybridoma single cell clones with specificity for HLA-A0201/LMP2 (peptide CLGGLLTMV (SEQ ID NO: 1)).
  • Example 13 Immunological staining of EBV infected-HLA-A0201 positive
  • NPC nasopharyngeal carcinoma
  • the control panel was stained with secondary antibodies only.
  • the biopsy was also stained with BB7.2 antibody that is specific for HLA-A02 .
  • the rest of the panels showed the staining of the biopsy with HLA-A0201/LMP2 and HLA- A0201/EBNA1 antibodies and the combination staining for the last panel is a combination of both TCR-like monoclonal antibodies.
  • Both biopsies were observed to stain positive for HLA-A0201/LMP2 and HLA-A0201/EBNA1 complexes in a number of cells. Positive staining was seen by reddish brown colour.
  • Example 13 To perform Example 13, the following methods were used.
  • LMP2 Latent Membrane Protein 2
  • CLGGLLTMV SEQ ID NO: 1
  • HLA-A0201 Latent Membrane Protein 2
  • HLA-A0206 and HLA-A0207 12 The changes in amino acid sequence expressed by the different variants of HLA-A02 plus their frequency in Caucasian and Asian populations are illustrated in Figure 11 A.
  • the ability of antibody #243 to recognize these complexes by Flow cytometry is shown in Figure 11B.
  • variable regions of the antibody heavy and light chains are sequenced. These can now be combined with the known sequence of the conserved constant regions of murine IgGl to yield a complete IgG sequence (see Figure 12 for sequenced variable regions).
  • antibody #243 is able to detect HLA-A0201/LMP2 complexes in biopsy of HLA-0201 and EBV-positive nasopharyngeal carcinoma in Figure 13.
  • Additional embodiments of the present disclosure include: [00179]
  • the monoclonal antibodies can be humanized and used to elicit an immune attack against cells and tissues infected with Epstein-Barr virus in human patients.
  • the disease applications include EBV-linked lymphoproliferative disease, infectious mononucleosis, Nasopharyngeal Carcinoma, Burkitt's Lymphoma, Hodgkins lymphoma.
  • the monoclonal antibodies can be tagged with toxins and chemotherapeutic reagents to be used as an 'immunotoxin' that better targets these toxic agents to tumour cells.
  • These monoclonal antibodies can be tagged with radionuclides to improve targeting of EBV infected tumour cells in vivo in both a diagnostic and therapeutic capacity. For instance, detecting EBV infected nasopharyngeal carcinoma cells in disparate lymph nodes after the tumour has metastasized by PET would be one approach. This will enable otolaryngologist to better target the diseased lymph nodes for surgery and/or radiotherapy.
  • a possible major advantage of this type of antibody is the ability to employ it in combination with other similar antibodies targeting different EBV peptides in association with different HLA-types.
  • Most human beings express up to 25 different peptide/HLA combinations from EBV proteins on their infected cells. Hence, this allows us to potentially employ up to 25 different antibodies simultaneously, all of which are EBV tumour specific. This could be hugely advantageous in ensuring that the tumour cells and viruses are given no opportunity to adapt to this form of therapy.
  • Humanized anti-HLA-A2/LMP2 antibody can be used as a diagnostic to directly quantify the numbers of infected cells ex vivo or in vivo.
  • Epstein-Barr virus (EBV) nuclear antigen 1 are encoded by sequence domains which vary among nasopharyngeal carcinoma biopsies and EBV-associated cell lines. J Gen Virol 80, 447-455, 1999.

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Abstract

L'invention concerne des compositions et des procédés relatifs à des anticorps monoclonaux qui ciblent des cellules tumorales associées au virus d'Epstein-Barr (VEB) en raison de la liaison à des complexes peptidiques HLA/LMP2 pour la détection et le ciblage de cellules tumorales infectées par le VEB.
PCT/SG2015/050181 2014-06-24 2015-06-24 Anticorps spécifique de lmp2 du virus d'epstein-barr et ses utilisations Ceased WO2015199618A1 (fr)

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WO2016201124A3 (fr) * 2015-06-09 2017-03-30 Memorial Sloan Kettering Cancer Center Anticorps ressemblant aux récepteurs des lymphocytes t spécifiques de la protéine membranaire latente 2a de l'ebv présentée par le hla humain
WO2019165326A1 (fr) * 2018-02-23 2019-08-29 REMD Biotherapeutics, Inc Anticorps antagonistes du peptide lié au gène de la calcitonine (cgrp)
CN111647564A (zh) * 2020-05-18 2020-09-11 李欣 抗eb病毒lmp1的单克隆抗体及其细胞株和应用
CN111690617A (zh) * 2020-05-18 2020-09-22 李欣 抗eb病毒lmp2a的单克隆抗体及其细胞株和应用
WO2021158073A1 (fr) 2020-02-06 2021-08-12 아주대학교산학협력단 Anticorps de fusion permettant la présentation d'un épitope d'antigène de lymphocyte t dérivé d'un antigène ou d'un peptide le contenant sur une surface cellulaire, et composition le comprenant

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WO2016201124A3 (fr) * 2015-06-09 2017-03-30 Memorial Sloan Kettering Cancer Center Anticorps ressemblant aux récepteurs des lymphocytes t spécifiques de la protéine membranaire latente 2a de l'ebv présentée par le hla humain
US10501559B2 (en) 2015-06-09 2019-12-10 Memorial Sloan Kettering Cancer Center T cell receptor-like antibody agents specific for EBV latent membrane protein 2A peptide presented by human HLA
US11168150B2 (en) 2015-06-09 2021-11-09 Memorial Sloan Kettering Cancer Center T cell receptor-like antibody agents specific for EBV latent membrane protein 2A peptide presented by human HLA
WO2019165326A1 (fr) * 2018-02-23 2019-08-29 REMD Biotherapeutics, Inc Anticorps antagonistes du peptide lié au gène de la calcitonine (cgrp)
US11629184B2 (en) 2018-02-23 2023-04-18 Remd Biotherapeutics, Inc. Calcitonin gene-related peptide (CGRP) antagonist antibodies
WO2021158073A1 (fr) 2020-02-06 2021-08-12 아주대학교산학협력단 Anticorps de fusion permettant la présentation d'un épitope d'antigène de lymphocyte t dérivé d'un antigène ou d'un peptide le contenant sur une surface cellulaire, et composition le comprenant
US12590156B2 (en) 2020-02-06 2026-03-31 Ajou University Industry-Academic Cooperation Foundation Fusion antibody for presenting antigen-derived T cell antigen epitope or peptide containing same on cell surface, and composition comprising same
CN111647564A (zh) * 2020-05-18 2020-09-11 李欣 抗eb病毒lmp1的单克隆抗体及其细胞株和应用
CN111690617A (zh) * 2020-05-18 2020-09-22 李欣 抗eb病毒lmp2a的单克隆抗体及其细胞株和应用
CN111647564B (zh) * 2020-05-18 2023-07-04 李欣 抗eb病毒lmp1的单克隆抗体及其细胞株和应用

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