WO2015199617A1 - A monoclonal antibody with t-cell receptor-like specificity that targets epstein-barr infected human tumour cells based on their expression of ebna1 - Google Patents

Abstract

Compositions and methods related to monoclonal antibodies that target Epstein-Barr virus (EBV) associated tumor cells due to binding to HLA/EBNA1 peptide complexes for the detection and targeting of EBV infected tumor cells are provided.

Description

A MONOCLONAL ANTIBODY WITH T-CELL RECEPTOR-LIKE SPECIFICITY THAT TARGETS EPSTEIN-BARR INFECTED HUMAN TUMOUR CELLS BASED

ON THEIR EXPRESSION OF EBNA1

CROSS-REFERENCE

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 62/016,530, filed June 24, 2014, entitled "A MONOCLONAL ANTIBODY WITH T-CELL RECEPTOR-LIKE SPECIFICITY THAT TARGETS EPSTEIN-BARR INFECTED

HUMAN TUMOUR CELLS BASED ON THEIR EXPRESSION OF EBNA1," the entire disclosure of which is hereby incorporated herein by reference for all purposes.

FIELD

[0002] The disclosure relates to monoclonal antibodies that target Epstein-Barr virus (EBV) associated tumor cells by binding to HLA/EBNA1 peptide complexes.

SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on June 18, 2015, is named 97520-947923_SL.txt and is 12,748 bytes in size.

BACKGROUND

[0004] Epstein-Barr Virus (EBV) 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. Although the association of EBV with cancers such as gastric carcinoma (-15%), Hodgkin's lymphoma (20-40%), and sporadic Burkitt's lymphoma (-30%) is not complete, assignment of a causal role to EBV has been based in part on the observation that these diseases are clonal expansions of a single EBV- infected cell 7 ' 8. At the protein level, the EBV gene products consistently observed in NPC biopsies and other EBV malignancies include BRLF1, EBNA1, LMP1 and LMP2A⁹'¹⁰.

[0005] Given the prevalence of EBV infection and its causative role in various cancers, it would be useful to have reagents that are able to detect and target EBV infected tumor cells based on the intracellular expression of EBV proteins. The present disclosure satisfies these and other needs.

SUMMARY

[0006] Described herein are compositions and methods related to monoclonal antibodies that target Epstein-Barr virus (EBV) associated tumor cells by binding to HLA/EBNA1 peptide complexes for the detection and targeting of EBV infected tumor cells.

[0007] In particular, as disclosed herein, we have exploited the fact that a defined EBV epitope from EBNA1 is expressed on the surface of EBV infected cells in the context of MHC class I ³'^{4 10} to develop novel monoclonal antibodies, based on a technical innovation of standard hybridoma technology, that binds to the HLA-0201/EBNA1 complexes on the surface of EBV infected cells and targets them for immune mediated lysis. We have shown that these types of monoclonal antibodies have a high affinity for HLA-A0201/EBNA1 peptide complexes, can recognize all clinically observed variants of the epitope and can bind to EBV-infected human tumour biopsy.

[0008] Accordingly, in one aspect, the present disclosure provides recombinant antibodies or fragments thereof that bind to a HLA/peptide complex.

[0009] In some embodiments, the peptide is derived from Epstein-Barr virus nuclear antigen 1 (EBNA1). In some embodiments, the peptide has the sequence FMVFLQTHI (SEQ ID NO: 1). In some embodiments, the HLA is HLA-A0201.

[0010] In some embodiments, 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-A02/EBNAl hybridoma described herein.

[0011] In some embodiments, 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.

[0012] In some embodiments, 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. [0013] In some embodiments, 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.

[0014] In some embodiments, the antibody or fragment thereof comprises a heavy chain comprising at least one CDR selected from the group of CDR sequences having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 32-34.

[0015] In some embodiments, 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: 37-39.

[0016] In some embodiments, the antibody or fragment thereof comprises a heavy chain comprising three CDR sequences selected from the group consisting of SEQ ID NOs: 32-34, or a sequence having at least 95% identity thereto.

[0017] In some embodiments, the antibody or fragment thereof comprises a light chain comprising three CDR sequences selected from the group consisting of SEQ ID NOs: 37-39, or a sequence having at least 95% identity thereto.

[0018] In some embodiments, the antibody or fragment thereof comprises a heavy chain sequence at least 95% identical to SEQ ID NO: 31.

[0019] In some embodiments, the antibody or fragment thereof comprises a light chain sequence at least 95% identical to SEQ ID NO: 36.

[0020] In some embodiments, the antibody or fragment thereof binds to an antigen with an affinity constant ( han -8

K_D) of less t 1 x 10^" 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.

[0021] In a further aspect, the present disclosure provides 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.

[0022] In a further aspect, 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.

[0023] In a further aspect, 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. [0024] In some embodiments, the antibody is administered intravenously (IV), subcutaneously (SC), intramuscularly (IM), transdermally, or orally.

[0025] In some embodiments, the antibody is administered in an amount in the range of 1 to 100 milligrams per kilogram of the subject's body weight.

[0026] In a further aspect, 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).

[0027] In some embodiments, the isolation in step (c) is by immuno-magnetic selection.

[0028] In some embodiments, the peptide is derived from Epstein-Barr virus nuclear antigen 1 (EBNA1).

[0029] In some embodiments, the peptide has the sequence FMVFLQTHI (SEQ ID NO: 1). In some embodiments, the HLA is HLA-A0201.

[0030] In a further aspect, the present disclosure provides an isolated nucleic acid encoding the antibody or fragment thereof according to any of the aspects and embodiments above.

[0031] In some embodiments, an expression vector comprising the above nucleic acid is provided. In some embodiments, a host cell comprising the expression vector above is provided. In some embodiments, the host cell is a bacterial cell, a eukaryotic cell, or a mammalian cell.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1: Schematic illustrating the experimental procedures used for the production of TCR-like monoclonal antibodies.

[0033] Figure 2: Expression and purification of HLA heavy and light chains.

[0034] Figure 3: Pre-selection of B-lymphocytes from immunised mice using

immunomagenetic Selection.

[0035] Figure 4: Pre-selection of B cells for required specificity significantly enhances the percentage of HLA-A02/EBNA1 specific Hybridomas versus Unselected splenocytes.

[0036] Figure 5: Screening B cell hybridomas for TCR-like HLA-A201/EBNA1 specificity. Figure 5 discloses SEQ ID NOS 1, 3, 2, 10, 12-14, 6, 7, 40 and 11, respectively, in order of appearance.

[0037] Figure 6: Immunoglobulin isotype test for anti-HLA-A0201/EBNAl specific monoclonal.

[0038] Figure 7: Affinity determination of TCR-like monoclonals HLA-A0201/EBNA1 against increasing concentrations of antigen using Biacore™.

[0039] Figure 8: Fine mapping of interaction between HLA-A0201/EBNA1 complex with TCR-like monoclonal antibody by alanine walking.

[0040] Figure 9: Deoxyribonucleic acid (top) and corresponding translated amino acid sequences (bottom) (heavy (SEQ ID NOS 30 and 31, respectively, in order of appearance with bolded CDR peptides disclosed as SEQ ID NOS 32-34, respectively, in order of appearance) and light chains (SEQ ID NOS 35 and 36, respectively, in order of appearance with bolded CDR peptides disclosed as SEQ ID NOS 37-39, respectively, in order of appearance) variable regions) derived from murine hybridoma single cell clones with specificity for HLA-A0201/EBNA1 (peptide FMVFLQTHI (SEQ ID NO: 1)).

[0041] Figure 10: Anti-HLA- A02/EBNA 1 recognizes EBNA1 epitope presented on HLA- A0201, HLA-A203, HLA-A0206 and HLA-A0207.

[0042] Figure 11: Binding activity of anti-HLA- A02/EB NA 1 antibody to cells naturally infected with EBV.

[0043] Figure 12: Quantification of HLA-A0201/EBNA1 complexes on the cell surface of tumour cells that are positive for Epstein-Barr virus infection.

[0044] Figure 13: Immunological staining of EBV infected-HLA-A0201 positive nasopharyngeal carcinoma (NPC) biopsy with anti-HLA-A0201/EBNAl antibody. DETAILED DESCRIPTION

[0045] Provided herein are new monoclonal antibodies that can target Epstein-Barr virus (EBV) associated tumour cells based on their surface expression of EBV derived peptides in association with human HLA-A0201. This represents a significant technical breakthrough that underlies the development of a new family of monoclonal antibodies with the potential to target infected cells and tumour cells based on their intracellular expression of signature antigens. This is based on the fact that all endogenous cellular proteins (host or pathogen) are processed into short peptides for display at the cell surface in association with HLA molecules¹.

[0046] The advent of Major Histocompatability Complex (MHC) based tetramer technology has enabled the detection and analysis of rare populations of antigen- specific T lymphocytes . However, existing methodologies cannot be utilized to determine the distribution of T cell receptor (TCR) ligands on tumor cells or to visualize antigen-presenting cells (APC) in tissues. As disclosed herein, we have made a significant technological breakthrough that allows for the development of TCR- like anti-HLA/peptide specific monoclonal antibodies using an adapted hybridoma approach, with a higher degree of reliability than has been previously possible. As proof of principle, we have developed a monoclonal antibody specific for a peptide (amino acid sequence FMVFLQTHI (SEQ ID NO: 1)) derived from the tumour virus antigen, Epstein-Barr virus nuclear antigen 1 (EBNAl)³'⁴ 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). As EBNAl is found in all EBV-associated malignancies, it makes the epitopes generated from this protein a good target for therapy⁴. Hence, 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 EBNAl. Given that 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 cancer⁵ 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.

DEFINITIONS

[0047] It is to be understood that this disclosure is not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural references unless the content clearly dictates otherwise.

[0048] The term "about" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of +20% or +10%, +5%, +1%, or +0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0049] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. Any methods and materials similar or equivalent to those described herein can be used in the practice of the present disclosure.

[0050] "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.

[0051] "Treating" or "treatment" 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.

[0052] "Preventing" or "prevention" refers to prophylactic administration with compositions described herein.

[0053] "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. Likewise, the terms "treat" and "treating" in reference to Epstein-Barr virus infection, as used herein, 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.

[0054] "Passive immunity" refers generally to the transfer of active humoral immunity in the form of pre-made antibodies from one individual to another. Thus, 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

intramuscular (IG) use, as high-titer animal or human IVIG or IG from immunized subjects or from donors recovering from a disease, and as monoclonal antibodies. Passive transfer can be used prophylactically for the prevention of disease onset, as well as, in the treatment of several types of acute infection. Typically, 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.

ANTIBODIES

[0055] As used herein, the term "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, Fab, Fab', F(ab)' fragments and/or F(v) portions of the whole antibody and variants thereof. All isotypes are encompassed by this term, including IgA, IgD, IgE, IgG, and IgM.

[0056] As used herein, the term "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. Examples of 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.

[0057] 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_H) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHi, CH₂ and CH₃. 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). 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. Examples of binding 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).

[0058] In some embodiments, the antibody (e.g., a mouse monoclonal 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. For example, if 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.

[0059] As used herein, the term "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_R. 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 ah, Science, 242:423-426 (1988); and Huston et al, Proc Natl Acad Sci USA, 85:5879-5883 (1988)). Such single chain antibodies are included by reference to the term "antibody" fragments and can be prepared by recombinant techniques or enzymatic or chemical cleavage of intact antibodies.

[0060] As used herein, the term "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. However, 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).

[0061] Also, 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).

[0062] As used herein, 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. Accordingly, 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. In one aspect, 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.

[0063] As used herein, 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". In the strict sense, 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. 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).

[0064] As used herein, the term "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. [0065] 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. For example, 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.

[0066] In addition, techniques have been developed for the production of humanized antibodies (see, e.g., U.S. Patent No. 5,585,089 and U.S. Patent No. 5,225,539, which are incorporated herein by reference in their entirety). 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). Briefly, 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.

[0067] Alternatively, techniques described for the production of single chain antibodies can be adapted to produce single chain antibodies against an immunogenic conjugate of the present disclosure. 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.

[0068] An "antigen binding protein" ("ABP") as used herein means any protein that binds a specified target antigen (e.g., a peptide derived from the EBNA1 protein). "Antigen binding protein" includes but is not limited to antibodies and binding parts thereof, such as immunologically functional fragments.

[0069] Peptibodies are another example of antigen binding proteins. The term

"immunologically functional fragment" (or simply "fragment") of an antibody or

immunoglobulin chain (heavy or light chain) antigen binding protein, as used herein, 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. It is further contemplated that a functional portion of the antigen binding proteins disclosed herein, for example, one or more CDRs, could be covalently bound to a second protein or to a small molecule to create a therapeutic agent directed to a particular target in the body, possessing bifunctional therapeutic properties, or having a prolonged serum half-life. As will be appreciated by one of skill in the art, an antigen binding protein can include nonprotein components.

[0070] The term "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). In some embodiments, 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

immunologically functional fragment thereof, 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). In some embodiments, the neutralizing ability is characterized and/or described via a competition assay. In some embodiments, the neutralizing ability is described in terms of an IC50 or EC50 value. The term "compete" 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 the EBNA1 protein). Numerous types of competitive binding assays can be used to determine if one antigen binding protein competes with another, for example: solid phase direct or indirect

radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), 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.

137:3614-3619) 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.

Immunol. 32:77-82). Typically, 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 (competing antigen binding proteins) 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. Additional details regarding methods for determining competitive binding are provided in the examples herein. Usually, when a competing antigen binding protein 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. ANTIGEN BINDING PROTEIN VARIANTS

[0071] Other antibodies that are provided are variants of the ABPs described in this application and 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). In some instances, 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.

[0072] In some aspects, 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. In an aspect, 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.

[0073] In some aspects, 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. In an aspect, 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.

[0074] In light of the present disclosure, a skilled artisan will be able to determine suitable variants of the ABPs as set forth herein using well-known techniques. In certain

embodiments, 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. In certain embodiments, one can identify residues and portions of the molecules that are conserved among similar polypeptides. In certain embodiments, 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.

[0075] In certain embodiments, 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. In some embodiments, 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. In contrast, in certain embodiments, 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.

[0076] For example, 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. Furthermore, 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. In certain embodiments, amino acid substitutions can be used to identify important residues of antibodies, or to increase or decrease the affinity of the antibodies described herein.

[0077] Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues which are important for activity or structure in similar proteins. One skilled in the art can opt for chemically similar amino acid substitutions for such predicted important amino acid residues.

[0078] 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.

[0079] Moreover, one skilled in the art can generate 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.

[0080] A number of scientific publications have been devoted to the prediction of secondary structure. See Moult J., Curr. Op. in Biotech., 7(4):422-427 (1996), Chou et al.,

Biochemistry, 13(2):222-245 (1974); Chou et al., Biochemistry, 113(2):211-222 (1974); Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol., 47:45-148 (1978); Chou et al., Ann. Rev. Biochem., 47:251-276 and Chou et al., Biophys. J., 26:367-384 (1979). Moreover, computer programs are currently available to assist with predicting secondary structure. 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.

[0081] Additional methods of predicting secondary structure include "threading" (Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87 (1997); Sippl et al., Structure, 4(1): 15-19 (1996)), "profile analysis" (Bowie et al., Science, 253: 164-170 (1991); Gribskov et al., Meth. Enzym., 183: 146-159 (1990); Gribskov et al., Proc. Nat. Acad. Sci. USA, 84(13):4355-4358 (1987)), and "evolutionary linkage" (See Holm, supra (1999), and Brenner, supra (1997)).

[0082] In certain embodiments, 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. In certain embodiments, 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

glycosylation sites (typically those that are naturally occurring) are eliminated and one or more new N-linked sites are created. 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.

[0083] According to certain embodiments, 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. According to certain embodiments, 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). In certain embodiments, 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). 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.

[0084] In some embodiments, the variants are variants of the nucleic acid sequences of the ABPs disclosed herein. One of skill in the art will appreciate that the above discussion can be used for identifying, evaluating, and/creating ABP protein variants and also for nucleic acid sequences that can encode for those protein variants. Thus, nucleic acid sequences encoding for those protein variants are contemplated. For example, 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.

[0085] In some embodiments, 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. In one embodiment, 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.SSC; followed by washing twice at 65 C for 20 minutes with each of 2.times., 0.5.times., and 0.2.times.SSC containing 0.1% SDS. Such 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. In some embodiments, 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 described herein 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. Generally, the nucleic acid sequence homology between the polynucleotides, oligonucleotides, and fragments described herein 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. For example, 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.

Alternatively, 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. In contradistinction, 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. For illustration, the nucleotide sequence "TATAC" corresponds to a reference sequence "TATAC" and is complementary to a reference sequence "GTATA".

ANTIBODY ASSAYS

[0086] 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.

[0087] The terms "specific binding" or "specifically binding" refer 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.

[0088] Once antibodies are produced, they 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. However, 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.

[0089] 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 Fundamental Immunology, Paul, W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby, Janis Immunology, W. H. Freeman and Company: New York, N.Y. (1992); and methods described herein). The measured affinity of a particular antibody- antigen interaction can vary if measured under different conditions (e.g., salt concentration, pH). Thus, measurements of affinity and other antigen -binding parameters (e.g., KD, K_a, K_d) are preferably made with standardized solutions of antibody and antigen, and a standardized buffer.

[0090] The affinity binding constant (K_aff) can be determined using the following formula: a^tt 2 (n [mAb'] _t -[mAb] _t )

in which:

[0091] [mAb] is the concentration of free antigen sites, and [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)).

[0092] As disclosed herein, surface plasmon resonance (SPR) can be used for detection and measurement of antibody-antigen affinity and kinetics. (See, e.g. , Hearty, S ., et ah, Methods Mol. Biol., 907:411-42 (2012); Malmqvist, M., Current Opinion in Immunology, 5: 282-286 (1993); Chatellier, J, et al , J. Molecular Recognition, 9: 39-51 (1996); Margulies, D. H., et ah, Current Opinion in Immunology, 8: 262-270 (1996); Forbes, B.E., et ah , Eur. J.

Biochem., 269:961-968 (2002).)

[0093] The term "high affinity" for an antibody refers to an equilibrium association constant

7 8

(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 10⁹ liters/mole, or at least about 1 x 10¹⁰ liters/mole, or at least about 1 x 10¹¹

12 13

liters/mole, or at least about 1 x 10 liters/mole, or at least about 1 x 10 liters/mole, or at least about 1 x 10¹⁴ 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.

[0094] KD, the equilibrium dissociation constant, is a term that is also used to describe antibody affinity and is the inverse of K_aff. If K_D is used, the term "high affinity" for an antibody refers to an equilibrium dissociation constant (KD) of less than about 1 x 10^"

-8 -9

mole/liters, or less than about 1 x 10^" mole/liters, or less than about 1 x 10^" 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 1 x 10^" mole/liters, or less than about 1 x 10^" mole/liters, or less than about 1 x 10^" mole/liters or lower.

[0095] The production of 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.

[0096] 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. In some embodiments, monoclonal antibodies produced in accordance with the present disclosure have neutralizing activity, where the antibody can neutralize at a concentration of 10^"9M or lower (e.g. 10^"10M, 10^"nM, 10^"12M or lower).

[0097] The immunoglobulin molecules described herein 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. In some embodiments, 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

combination with the entirety or a portion of the following: hinge region, CHI, CH2, and CH3 domains. Also included in the present disclosure are antigen-binding fragments comprising any combination of variable region(s) with a hinge region, CHI, CH2, and CH3 domains.

RECOMBINANT EXPRESSION

[0098] 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.

[0099] In some embodiments, 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. In some embodiments, 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.

[00100] Production of antibodies using recombinant DNA methods is described, for example, in U.S. Pat. No. 4,816,567. For recombinant production of the antibody, the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning

(amplification of the DNA) or for expression. DNA encoding a monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). 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).

PHARMACEUTICAL COMPOSITIONS

[00101] The presently disclosed subject matter provides pharmaceutical compositions comprising the antibodies produced in accordance with the present disclosure. In some embodiments, a pharmaceutical composition can comprise one or more monoclonal antibodies produced in using the methods disclosed herein. In some embodiments, a panel of monoclonal antibodies produced according to the present disclosure can be included in a pharmaceutical composition. In some embodiments, 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.

[00102] In some embodiments a pharmaceutical composition can also contain a

pharmaceutically acceptable carrier or adjuvant for administration of the antibody. In some embodiments, 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. [00103] 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.

[00104] 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.

[00105] The compositions of the presently disclosed subject matter can further comprise a carrier to facilitate composition preparation and administration. Any 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. 5,786,387), collagen (U.S. Pat. No. 5,922,356), a polysaccharide or derivative thereof (U.S. Pat. No. 5,688,931), a nanosuspension (U.S. Pat. No. 5,858,410), a polymeric micelle or conjugate (Goldman et al. (1997) Cancer Res 57: 1447-1451 and U.S. Pat. Nos. 4,551,482, 5,714,166, 5,510,103, 5,490,840, and 5,855,900), and a polysome (U.S. Pat. No. 5,922,545).

[00106] 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

Therapy. In Current Protocols in Human Genetics, John Wiley & Sons, New York; Park et al. (1997) Adv. Pharmacol. 40:399-435; Pasqualini et al. (1997) Nat. Biotechnol. 15:542-546; Bauminger & Wilchek (1980) Meth. Enzymol. 70: 151-159; U.S. Pat. No. 6,071,890; and European Patent No. 0 439 095).

[00107] A therapeutic composition described herein comprises in some embodiments 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, bacteriostats, 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. 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. In some

embodiments, the carrier is pharmaceutically acceptable. In some embodiments the carrier is pharmaceutically acceptable for use in humans.

[00108] Pharmaceutical 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.

[00109] Pharmaceutical compositions can include an effective amount of one or more antibodies as described herein. In some embodiments, 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.

TREATMENT REGIMENS: PHARMACOKINETICS

[00110] The pharmaceutical compositions described herein 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

concentration of active agent, and the like. In calculating the dosage regimen for a patient, 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

Remington's; Egleton, Peptides 18: 1431-1439, 1997; Langer, Science 249: 1527-1533, 1990.

[00111] For purposes of the present disclosure, a therapeutically effective amount of a composition comprising an antibody, contains about 0.05 to 1500 μg protein, about 10 to 1000 μg protein about 30 to 500 μg or about 40 to 300 pg, or any integer between these values. For example, antibodies described herein can be administered to a subject at a dose of about 0.1 μg to about 200 mg, e.g., from about 0.1 μg to about 5 μg, from about 5 μg to about 10 μg, from about 10 μg to about 25 μg, from about 25 μg to about 50 μg, from about 50 μg to about 100 μg, from about 100 μg to about 500 μg, from about 500 μg 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. It is understood that 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.

[00112] Routes of administration include, but are not limited to, oral, topical, subcutaneous, intramuscular, intravenous, subcutaneous, intradermal, transdermal and subdermal.

Depending on the route of administration, the volume per dose is about 0.001 to 10 ml, about 0.01 to 5 ml, or about 0.1 to 3 ml. Compositions can be administered in a single dose treatment or in multiple dose treatments on a schedule and over a time period appropriate to the age, weight and condition of the subject, the particular antibody formulation used, and the route of administration.

KITS

[00113] Also provided are 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.

The following examples of specific aspects are offered for illustrative purposes only, and are not intended to limit the scope of the present disclosure in any way.

EXAMPLES

Example 1: Overview of Experimental Procedures used for the Production of TCR-like Monoclonal antibodies.

[00114] As shown in Figure 1, 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 the antigenic peptide- FMVFLQTHI (SEQ ID NO: 1) from EBNA1. 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

polyethylene glycol (PEG) mediated fusion with the Myeloma cells line NS 1. The resulting hybridoma clones are screened by flow cytometry using peptide pulsed HLA expressing human cell lines.

Example 2: Expression and purification of HLA heavy and light chains.

[00115] As shown in Figure 2, recombinant HLA-A201 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 EBNA-1 peptide (FMVFLQTHI (SEQ ID NO: 1)) were refolded into complete HLA-complexes in vitro. Anion-exchange chromatography was used to purify the monomers-see FPLC profiles. B. SDS-PAGE analysis of purified fractions- positive fractions contain both Heavy and light chains from immunoglobulin. C. The collected fractions are analysed on a non-denaturing native gel immunoblotted with the anti- HLA conformation specific monoclonal w6/32. This confirmed that the purified monomer was correctly folded.

[00116] To perform Examples 1 and 2, the following methods were used.

[00117] A single inoculant of the successfully transformed cell containing the pET30a- HLA-A0201 plasmid was inoculated into 15 ml of LB media containing 50 μg/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.

[00118] 10 ml of starter culture was inoculated into 1 L of LB media containing 50 μg/ml of kanamycin selective antibiotics and incubated at 37 °C for 1.5 hr with shaking. Subsequently, the OD₆oo was measured every half an hour to determine the mid-log phase of the culture (OD₆oo between 0.6 to 0.8) and protein expression was induced with 1 mM of isopropyl β-D- I-thiogalactopyranoside (IPTG) (Sigma-Aldrich). 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 μΐ 1 mg/ml pepstatin A (Sigma- Aldrich). The resuspended pellet was stored at -80 °C overnight.

[00119] 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. After sonication, 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 μΐ 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 μΐ 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 μΐ 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 μΐ 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 mM PMSF and 10 μΐ 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.

FPLC purification

Anion exchange chromatography

[00120] Pumps A and B were pre-washed with RO water at flow rate of 1 ml/min and the HiPrep 16/10 DEAE FF column (AKTA, GE Healthcare) was attached onto the AKTA FPLC System (GE Healthcare). The column was equilibrated with 5 column volumes (CV) of RO water followed by 5 CV of buffer A, 5 CV of buffer B and 5 CV of buffer A. Buffer A is a solution of 20 mM Tris (pH 8.0) while buffer B is 20 mM Tris/ 1 M NaCl (pH 8.0). The dialyzed solution was loaded into the equilibrated column at a flow rate of 5 ml/min and run according to a stepwise gradient program. 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 μΐ using the Centricon Centrifugal Filter Unit with Ultracel YM-30 membrane (Millipore Corporation). The purified fraction was used for further purification.

Superdex (size-exclusion column)

[00121] The Superdex HR 10/30 75 column (GE Healthcare) was equilibrated with 5 CV of RO water and 5 CV of buffer A before use. Approximately 200 μΐ of the anion exchanged purified HLA monomers were injected into the sample loop using a 1 ml syringe with no introduction of air bubbles. The monomers would be subsequently loaded into the column and eluted using an isocratic gradient with a flow rate of 0.8 ml/min in 20 mM Tris (pH 8.0). Fractions were collected in 0.5 ml aliquots and analyzed on SDS-PAGE gel. Fractions that contain both the heavy chain (35 kDa) and beta-2 microglobulin (12 kDa) were pooled together and further concentrated to 500 μΐ using the Centricon YM-10 (Millipore

Corporation). The purified monomers were quantitated and stored at -80 0C for further use. Biotinylation of HLA monomers

[00122] Approximately 70 μΐ of purified monomer was biotinylated with 10 μΐ of biomix A, 10 μΐ of biomix B and 10 μΐ of supplemental biotin, 0.2 μΐ of 3 mg/ml BirA enzyme (Avidity LLC), 0.2 μΐ of 0.1 M PMSF and 0.1 μΐ of 1 mg/ml pepstatin A at 28°C for 18 hrs. 1 ml of 10 mM Tris was added to the biotinylated mixture, transferred to a Centricon YM-30, and centrifuged at 3500 rpm for 10 mins to wash out the mixture to leave behind the biotinylated monomers.

[00123] The amount of biotinylated product was quantitated by immunoprecipitation using streptavidin beads (Sigma- Aldrich Inc.). Approximately 10 μΐ 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 μg of previously biotinylated monomers were added with 500 μΐ of 10 mM Tris, 2 μΐ of 0.1 M PMSF and 1 μΐ 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. The biotinylated monomers were subsequently aliquot and stored at -80 °C for further use.

[00124] To enrich specific splenocytes, 50 μg of 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 μΐ 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. After 30 mins, 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.

[00125] 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. Following incubation, 3 ml of RPMI medium was added at a rate of 1 ml/ min while consistently rotating the tube gently. Subsequently, the suspension was topped up with 5 ml of RPMI medium and centrifuging at 1100 rpm for 3 mins pelleted the cells down. The pellet was then resuspended with 20 ml of HAT medium prior to incubation at 37 °C in a C0₂ incubator for 2 hrs. After 2 hrs, the cell suspension was topped up with an appropriate volume with HAT medium and 100 μΐ of the suspension was aliquoted into each well of a 96-well plate that had been previously been incubated with similar volume of feeder layer. The plates were incubated at 37 °C in a C0₂ incubator and each well was further topped up with 100 μΐ 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.

Example 3: Pre-selection of B -lymphocytes from Immunised Mice using

Immunomagenetic Selection.

[00126] As shown in Figure 3, 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.

[00127] To perform Example 3, the following methods were used.

Biotinylation of HLA monomers and selection of antigen specific B cells

[00128] Approximately 70 μΐ of purified monomer was biotinylated with 10 μΐ of biomix A, 10 μΐ of biomix B and 10 μΐ of supplemental biotin, 0.2 μΐ of 3 mg/ml BirA enzyme (Avidity LLC), 0.2 μΐ of 0.1 M PMSF and 0.1 μΐ of 1 mg/ml pepstatin A at 28°C for 18 hrs. 1 ml of 10 mM Tris was added to the biotinylated mixture, transferred to a Centricon YM-30, and centrifuged at 3500 rpm for 10 mins to wash out the mixture to leave behind the biotinylated monomers.

[00129] The amount of biotinylated product was quantitated by immunoprecipitation using streptavidin beads (Sigma- Aldrich Inc.). Approximately 10 μΐ 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 μg of previously biotinylated monomers were added with 500 μΐ of 10 mM Tris, 2 μΐ of 0.1 M PMSF and 1 μΐ 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. The biotinylated monomers were subsequently aliquot and stored at -80 C for further use.

[00130] To enrich specific splenocytes, 50 μg of 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 μΐ 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. After 30 mins, 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: Pre-selection of B cells for required specificity significantly enhances the percentage of HLA-A02/EBNA1 specific Hybridomas versus Unselected splenocytes.

[00131] As shown in Figure 4: A. We compared unselected splenocytes with splenocytes selected on the basis of their binding to HLA-A02/EBNA1 monomers prior to fusion.

Unselected splenocytes generated small percentage of anti-HLA-A02 specific hybridomas compared to splenocytes selected for their binding capacity to the immunogen. And only with selection, anti-HLA-A02/EBNAl specific hybridomas can be isolated. Hence, selection of splenocytes enhanced the percentage of targeted hybridomas. B. The binding of antibodies in hybridoma clone supernatant to T2 cells pulsed with EBNA1 peptide (Blue Histogram) was compared by flow cytometry with T2 cells pulsed with the HLA-A0201 restricted Influenza A peptide GILGFVFTL (SEQ ID NO: 11) (Red Histogram) and just cells stained with anti- mouse IgG antibody control (EBNA1 peptide: Purple Histogram; Ml peptide: Pink histogram).

Example 5: Screening B cell Hybridomas for TCR-like HLA-A201/EBNA1 specificity.

[00132] As shown in Figure 5: A. The anti-HLA-A02/EBNAl antibody was screened against a panel of HLA-A0201 restricted peptides from Epstein-Barr virus— LMP1

(YLLEMLWRL (SEQ ID NO: 2)), LMP2 (CLGGLLTMV (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 (see legend in figure). The TCR-like anti-HLA-A0201/EBNAl antibody is exquisitely specific for only EBV-EBNA-1 (FMVFLQTHI (SEQ ID NO: 1)) associated with HLA-A0201 (Red

Histogram). B. Titration of peptide FMVFLQTHI (SEQ ID NO: 1) on T2 cells from ΙΟμιη to

0.01 μιη. Specific binding is detected by flow cytometry down to 14pM concentration of peptide FMVFLQTHI (SEQ ID NO: 1).

[00133] To perform Example 5, the following methods were used.

Screening of clones by flow cytometry

[00134] The supernatant of scored clones were screened by flow cytometry using T2 cells. 100 μΐ 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 LMP-2A₄₂₆-₄₃4 (CLGGLLTMV (SEQ ID NO: 3)) (Mimotope) for 30 mins at 37 °C in a C0₂ 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. The supernatant was discarded and 10 μΐ of diluted goat anti-mouse IgG (H+L) RPE conjugated antibody (Dako) was added to the pelleted cells. The tubes were incubated in the dark at 4 °C for 30 mins. After incubation, 4 ml of previously described wash buffer was added and centrifuged at 350 g for 5 mins to wash out the unbound secondary antibodies. The cells were resuspended and fixed with 500 μΐ of 4 % Para formaldehyde (PFA) (Thermo Fisher Scientific) before being analyzed with CellQuest Pro Software on a BD FACsCalibur machine (BD Biosciences).

[00135] The exquisite specificity of the TCR-like mAbs were determined by exogenously pulsing T2 cells with 5 μΜ of each HLA-A0201 restricted peptides from various pathogens including their respective targeted peptide. As 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

1. 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). [00136] The antibodies were screened against a panel of HLA-A0201 restricted peptides that include EBNAl₅₆2-57o (FMVFLQTHI (SEQ ID NO: 1)) (red), LMP1125-133 (YLLEMLWRL (SEQ ID NO: 2)) (purple), LMP2A₄₂₆-₄3₄ (CLGGLLTMV (SEQ ID NO: 3)) (brown), Influenza virus Ml₅₈_₆₆ (GILGFVFTL (SEQ ID NO: 11)) (dark green), Mycobacterium tuberculosis Ag85Bi₄₃-i₅₂ (FIYAGSLSAL (SEQ ID NO: 10)) (pink), Hepatitis B virus sAgi₈₃_i9i (FLLTRILTI (SEQ ID NO: 12)) (light blue), Human immunodeficiency virus Pol₄₇₆-₄₈₄ (ILKEPVHGV (SEQ ID NO: 13)) (blue), gpl20i₂₀-i₂₈ (KLTPLCVTL (SEQ ID NO: 14)) (yellow), Gag₇₇_₈₅ (SLYNTIAVL (SEQ ID NO: 15)) (grey), CMV IE1₃₁₆_₃₂₄

(VLEETSVML (SEQ ID NO: 6)) (orange),IEl₈i_₈₉ (VLAELVKQI (SEQ ID NO: 7)) (black), pp65₄₉₅-503 (NLPVMVATV (SEQ ID NO: 16)) (light brown) and cells stained with anti- murine IgG (H+L) antibody (light green). It was observed that the TCR-like mAb was exquisitely specific only for its respective peptide associated with HLA-A0201 and not the rest of HLA-A0201 peptide complexes.

[00137] 1 x 10⁶ cells were pulsed with increasing concentrations of peptide, ranging from InM to 10 OOOnM (final concentration) and incubated with 0.5 g of TCR-like mAb at 4°C for 30 min, followed by detection with 0.5μ1 of 10μg/ml RPE goat anti-mouse IgG (Dako) and analysis by flow cytometry.

[00138] To determine the sensitivity of the TCR-like monoclonal antibody, a titration of the peptides was tested with T2 cells. T2 cells were pulsed with various concentrations of the peptides and negative control for the TCR-like monoclonal antibody, namely EBNAI₅₆₂-570 (FMVFLQTHI (SEQ ID NO: 1)). As seen from the plot, 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/EBNAl specific monoclonal.

[00139] As shown in Figure 6, the isotype profile for clone anti-HLA-A02/EBNAl is IgGl and kappa light chain. HC=Heavy Chains, LC=Light Chains.

[00140] To perform Example 6, the following methods were used.

Antibody isotyping

[00141] Supernatant of positively scored hybridoma was collected and placed into each development tube. The tube was incubated at room temperature for 30 sec and vortexed briefly to resuspend the colored latex. The isotyping strip of the Isostrip Mouse Monoclonal Isotyping kit (Roche) was placed into each tube and incubated until the distinctive blue bands appear. The bands indicate the class or subclass and light chain composition of the antibody found in the supernatant.

Example 7: Affinity determination of TCR-like monoclonals HLA- A0201/EBNA1 against increasing concentrations of antigen using Biacore™.

[00142] As shown in Figure 7, antigen concentrations ranged from 0 to 160nM. The dissocation constant KD was determined by measuring the binding kinetics of the HLA- A0201/EBNA1 mononers to anti-HLA-A02/EBNAl antibody. Serially diluted concentrations of the HLA-A0201/EBNA1 monomers (160nM to ΙΟηΜ) were flowed over the immobilized antibody to determine the K_on and K₀ff .The binding response were analyzed using a global fit algorithms (BIA evaluations 3.1) assuming 1 : 1 Langmuir binding. The KD of antibody to HLA-A0201/EBNA1 monomers was determined to be 6.02nM. The data points were fitted using four-parameter logistics. The affinity for the antibody produced by the hybridoma is significantly higher than that generated by BB7.2 (K_D =162nM).

[00143] To perform Example 7, the following methods were used.

Surface Plasmon Resonance (SPR)

[00144] Kinetic studies were evaluated by surface plasmon resonance using a BIAcore 3000™ (GE Healthcare). The monoclonal antibodies were immobilized covalently onto the surface of sensor chip CM5 by amine coupling. To determine the dissociation constant (Κ_Ό) of the antibodies, the respective MHC class I/peptide were flowed at different concentrations ranging from 160 nM to 10 nM over the relevant flow cells at a rate of 30 μΙ_, min^"1 at 25°C. Responses were recorded and analyzed using BIAevaluation software 3.2.

[00145] The affinity of each of the antibodies to their respective HLA-A0201 /peptide was determined using surface plasmon resonance (SPR) using the Biacore 3000 optical biosensor (GE Healthcare). 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/EBNA1 peptide monomer over the surface of the respective antibody coated chip. Dilutions of 160 nM to 10 nM of monomers were injected at 30 μΐ/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 (K₀ff) values to determine the dissociation constant or affinity constant C¾)- The of the TCR- like mAb against HLA-A02/EBNA1 is 6.02 nM. Example 8: Fine mapping of interaction between HLA-A0201/EBNA1 complex with TCR-like monoclonal antibody by alanine walking.

[00146] As shown in Figure 8, each of the peptides was mutated with alanine at the position indicated by the peptide's number allocation. The histogram showed the flow cytometry results of staining with the monoclonal antibody on various T2 cells pulsed with the peptide allocated as stated. The interaction between the antibody and antigen (FMVFLQTHI (SEQ ID NO: 1)) is sensitive between positions 1 to 9 as any mutation in the amino acids resulted in a change in the ability of the antibody to recognize the antigen and resulted in a change in the mean fluorescence index (MFI).

[00147] To perform Example 8, the following methods were used.

Alanine scanning

[00148] 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 β-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.

[00149] This method is significantly less laborious than saturation mutagenesis substitutes with all 20 naturally occurring amino acids and similar results are observed from both methodology. Using alanine substitution of each amino acid on the peptide, we can interrogate the sites necessary for antibody recognition. These peptides are pulsed onto TAP- deficient T2 cells exogenously to associate with HLA-A0201 on the cell surface for antibody detection. With flow cytometry, the mean fluorescence index (MFI) of each antibody binding on each mutated peptide can be determined and the effect of positional amino acid can be examined.

Example 9: DNA and corresponding translated amino acid sequences of heavy and light chains variable regions derived from murine hybridoma single cell clones with specificity for HLA-A0201/EBNA1 (peptide FMVFLQTHI (SEQ ID NO: 1)).

[00150] Shown in Figure 9 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/EBNA1 (peptide FMVFLQTHI (SEQ ID NO: 1)).

Example 10: Anti-HLA-A02/EBNA1 recognizes EBNAl epitope presented on HLA-A0201, HLA-A203, HLA-A0206 and HLA-A0207.

[00151] As shown in Figure 10: A. Schematic detailing position of amino acid substitutions that define different forms of HLA-A02 plus their relative frequences in different human populations. B. Flow cytometry was used to analyse binding to 8 BLCL cell lines

homozygous for the different forms of HLA-A02 pulsed with EBNAl peptide FMVFLQTHI (SEQ ID NO: 1) versus controls pulsed with the HLA-A02 restricted Influenza A peptide GILGFVFTL (SEQ ID NO: 11). A small but significant shift was detected in all cell lines pulsed with the EBNAl peptide versus controls.

[00152] To perform Example 10, the following methods were used.

Application of TCR-like mAbs in the context of HLA-A02 haplotype polymorphism

[00153] The majority of 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 al and a.2 chains. The MHC class I diagram illustrates the anchor amino acids at positions 2 and 9 of a typical A0201 restricted peptide.

[00154] Differential recognition of the TCR-like mAb against EBNAI₅₆₂-570 epitope presented on HLA-A0201, -A0203, -A0206 and -A0207. Flow cytometry was used to analyze binding to 8 BLCLs (CF 986, CM 800, CF960, CM392, CM304, CM371, CF1007 and CSH) pulsed with the EBNA- I₅₆₂-570 peptides and its clinical variant and stained with the TCR-like mAb. These BLCLs were obtained from various patients and volunteers. This was in comparison of pulsed BLCLs stained with an isotype antibody.

Example 11: Binding activity of anti-HLA-A02/EBNAl antibody to cells naturally infected with EBV.

[00155] As shown in Figure 11: A. Flow cytometry analysis of peptide pulsed, EBV transformed BLCL reveals significant background staining to control cell lines that were pulsed with Influenza A peptide GILGFVFTL (SEQ ID NO: 11) (upper histograms). This background binding was not observed in HLA-A02+ve cell lines that are not EBV infected (T2, THP-1 and U937.A2)(lower histograms) suggesting that our antibody recognises endogenous HLA-A02/EBNA1 complexes generated through EBV infection. B. This hypothesis was confirmed using EBV-infected tumour cell lines (CCRF-SB) and RPMI6666. Left histograms show flow cytometry staining patterns for all HLA-A02 complexes using the pan-HLA-A02 monoclonal antibody - BB7.2 (Light blue histogram). Right histograms indicate binding of our anti-HLA-A02/EBNAl antibody (purple histogram) to endogenous surface complexes on these EBV infected cells.

[00156] To perform Example 11, the following methods were used.

Binding of endogenously produced epitopes

[00157] The cells were pulsed with EBNAl peptide before the addition of the monoclonal, stained with FITC and analysed with flow cytometry. In BLCLs, peptide loading is facilitated by the exchange of endogenously derived peptides with EBNA-1 peptides that were introduced externally. Our results show that the monoclonal was able to detect HLA-

A0201/EBNA1 complexes on the surface of the BLCLs. Interestingly, all three BLCLs also showed a degree of binding when pulsed with the irrelevant Ml peptide (Red) compared with isotype controls of both Ml and EBNAl peptide pulsed cells (filled histograms). This could be that since BLCLs are EBV transformed, they would express EBV latent proteins (EBNA- 1). Hence it can be deduced that our monoclonal is able to detect constitutive levels of HLA- A0201/EBNA1 complexes.

[00158] To test this further, the monoclonal was tested on cell lines that is HLA-A201 expressing but not EBV transformed. Hence, there should not be any EBV proteins present constitutively. The three cell lines used were T2, THP-1 and U937-A2 (bottom panel). T2 cells are human lymphoblast suspension cells, THP-1 are human monocytic leukemic suspension cells and U937-A2 are human monocytic suspension cells that are stably transfected with HLA-A0201. The results show that when the cells were pulsed with Ml influenza peptide, incubated with the monoclonal and analyzed by flow cytometry, no shift was observed. However, when the cells were pulsed with EBNAl peptide, a shift was observed and this shows that the monoclonal is able to bind specifically to HLA-

A0201/EBNA1 peptide complexes on the cell surface and not to HLA-A0201 or the irrelevant peptide. It also confirms the previous observation that the monoclonal is able to detect constitutive levels of HLA-A0201/EBNA1 complexes. This is in comparison with the Ml and EBNA-1 pulsed BLCLs on the top panel.

[00159] To test the binding of the monoclonal to naturally processed EBNA-1 epitopes in tumour cells, two tumour cell lines were chosen. CCRF-SB is a human lymphoblast suspension cell line derived from acute lymphoblastic leukaemia while RPMI-6666 is a human lymphoblast suspension cell line derived from Hodgkin's lymphoma. Both cell lines are EBNAl positive and express HLA-A0201 as seen by their staining with BB7.2 which is a HLA-A2 monoclonal antibody. These cells give a more accurate representation of the normal levels of HLA-A0201/EBNA-1 complexes that are expected to be present on the cell surface as compared to the peptide loaded samples. The cells were incubated with the monoclonal, stained with goat anti-mouse FITC and analysed by flow cytometry. The results showed a clear binding of the monoclonal to EBNA1 epitopes in a natural state without the addition of exogenous peptide.

Example 12: Quantification of HLA-A0201/EBNA1 complexes on the cell surface of tumour cells that are positive for Epstein-Barr virus infection.

[00160] As shown in Figure 12, the two tumour cell lines examined were CCRF-SB and RPMI6666 and were obtained from patients who were diagnosed with acute lymphoblastic leukemia and Hodgkin lymphoma respectively. The haplotype of both cell lines were also HLA-0201. The number of HLA-A0201/EBNA1 complexes presented on these cell surfaces was determined using the anti-HLA-A02/EBNAl antibody. This was carried out using flow cytometry and the fluorescent intensity was measured in comparison with that of calibrated beads that has known numbers of fluorphores provided in a Qifikit (Dako, Denmark).

[00161] To perform Example 12, the following methods were used.

Quantitation of HLA-A0201/EBNA1 complexes of endogenously produced epitopes

[00162] Epstein-Barr virus infected HLA-A02 cells were stained with the various TCR-like mAbs followed by RPE-conjugated goat anti-mouse IgG (Dako). The level of fluorescence intensity was compared with that of calibrated beads containing pre-determined quantity of mouse monoclonal antibody per bead (QIFIKIT® calibration beads; Dako). By means of a standard curve based upon the calibration beads, the number of complexes on the cell surface can be determined or inferred from the fluorescence intensity of each TCR-like rnAb. The amount of complexes on the surface of the respective cells could also be determined using other antibodies such as W6/32 to determine the number of properly folded MHC class I complexes on the cell surface and BB7.2 to determine the number of HLA-A0201 heavy chains on the cell surface.

Example 13: Immunological staining of EBV infected-HLA-A0201 positive nasopharyngeal carcinoma (NPC) biopsy with anti-HLA-A0201/EBNAl antibody.

[00163] As shown in Figure 13, the control micrograph was stained with secondary antibodies only (upper). The biopsy was also stained with BB7.2 antibody that is specific for HLA-A02 (middle). This biopsy was observed to stain positive for HLA-A0201/EBNA1 complexes in a number of cells. Positive staining was observed to be reddish brown in colour (lower). [00164] To perform Example 13, the following methods were used.

Immunohistochemistry

[00165] Frozen PC biopsies were sectioned (4 μιη) and embedded onto slides. Slides were fixed and subsequently stained with TCR-like mAbs or BB7.2. Peroxidase block (Dako) was used prior to treatment with Dako labeled polymer. After further washing, immunoperoxidase staining was developed with DAB (Dako).

Biopsy and blood collection

[00166] Informed consent from healthy volunteers and patients were taken before biopsy or blood extraction. NPC patients at the National University Hospital were enrolled with informed consent. The respective IRBs approved this study and conformed to the WMA Declaration of Helsinki and the NIH Belmont Report (IRB no: 07-043E).

Discussion

[00167] While existing tetramer technology can be utilized to study EBV-specific T- lymphocytes generated as a result of natural infection, there are no current methodologies that allow the visualization, quantification and analysis of the TCR ligands expressed on the surface of EBV infected cells and tumors. This severely impedes the ability of EBV researchers to directly address the impact of candidate immune evasion mechanisms on the processing and presentation of EBV antigens in infected cells and APC. This also impairs targeting of tumor cells in situ based on their association with EBV. In our laboratory, we have developed techniques that allow for the production of novel monoclonal antibodies with fine specificity for EBV epitopes expressed in the context of HLA on the surface of EBV infected cells (Figure 1 -outline of new methodology). The EBV antigen that we have successfully targeted is EBV nuclear antigen 1 (EBNA1: FMVFLQTHI (SEQ ID NO:

1)/HLA-A0201). These monoclonals represent unique immunological targeting reagents that will enable a thorough investigation of EBV-EBNA1 antigen processing/presentation during latency and tumouragenesis and the targeting of EBNA1 expressing EBV-associated tumour cells in situ.

[00168] As disclosed herein, we have designed, expressed and FPLC purified recombinant, membrane free, fully folded, heterotrimeric complexes of HLA-A0201 heavy and light chains plus an antigenic peptide derived from the EBV nuclear antigen 1 (EBNA1) protein of

Epstein Barr virus (amino acid sequence: FMVFLQTHI (SEQ ID NO: 1)). This complex was used as a source of antigen to stimulate antibody responses in immunized Balb/C mice (see Figure 1 and 2). Spleens were removed from the immunized mice after 45 days and used as a source of B-lymphocytes for myeloma fusion/hybridoma generation. Antigen specific antibody producing hybridomas were screened by flow cytometry using peptide pulsed human B-cell lines.

[00169] We have also developed a technical innovation of standard hybridoma approaches that addresses the technical challenge of generating monoclonal antibodies with fine specificity for peptide/HLA-A0201 complexes. A standard monoclonal antibody protocol involves immunizing mice with the antigen of choice then fusing immune splenocytes from the immunized mice with a myeloma fusion partner to generate immortalized antibody producing hybridomas. However, we found that this 'classical' approach does not work. No hybidomas generated using the classical approach was specific for human HLA-A0201 plus the EBV derived EBNA1 peptide. Hence, we developed a technical innovation of standard hybridoma technology that involves pre-purifying the antigen specific B cells prior to myeloma fusion from the immunized mice, using a biotinylated form of the monomer linked to an immunomagnetic bead (see Figure 3). This greatly enables the obtaining of hybridomas that have the correct specificity compared to standard hybridoma approaches (see Figure 4A).

[00170] We have also designed a screening methodology to test for monoclonal hybridomas producing antibodies that are HLA-A0201/EBNA1 specific. This involved using Flow Cytometry to compare the binding activities of the antibodies in multiple hybridoma cell line supernatants for an HLA-A0201 expressing human cell line (T2) that have been pulsed with the EBNA1 peptide (FMVFLQTHI (SEQ ID NO: 1)) or with an HLA-A0201 restricted peptide from Influenza A (GILGFVFTL (SEQ ID NO: 11)). Hybridoma supernatants with clear specificity for the EBNA1 peptide on HLA-A0201 can be identified using this methodology (see Figure 4B).

[00171] We have also demonstrated the importance of our adaptation to standard hybridoma technology by comparing the numbers of specific hybridoma clones generated from the same splenocyte pool using immunomagnetic pre-selection of antigen specific cells versus no preselection prior to myeloma fusion and immortalization (see Figure 4A).

[00172] We have also shown that the antibody secreted by the immortalized hybridoma clone, has a mouse IgGl heavy chain and a Kappa light chain (see Figure 6).

[00173] We have also shown that high affinity antibodies can be made with specificity for HLA-A0201/EBNA1 peptide complexes (see Figure 5). We measured the affinity binding constant of our anti-HLA-A0201/EBNAl monoclonal and can show that it has a higher binding affinity (KD = 6.02nM) for these complexes than a pan-HLA-A02 antibody (162nM) (BB7.2) (see Figure 7). [00174] We have also analyzed the interaction of antigen-antibody between HLA- A0201/EBNA1 (FMVFLQTHI (SEQ ID NO: 1)) and the antibody targeting it. The fine interaction between the antibody and antigen was carried out using alanine walking, where each amino acid of the antigen was substituted with alanine. This enables the determination of the essential sites of binding between antibody and the antigen (FMVFLQTHI (SEQ ID NO: 1)) (see Figure 8).

[00175] We have also sequenced the variable regions of the antibody heavy and light chains. 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 9).

[00176] We have also analyzed the ability of our antibody to detect HLA-A02/EBNA1 complexes and its clinically observed variants in human cell lines expressing the Caucasian form of HLA-A02 (HLA-A0201) versus the Asian variants of HLA-A02, HLA-A0203, HLA-A0206 and HLA-A0207¹¹. 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 10A. The ability of antibody to recognize these complexes by flow cytometry is shown in Figure 10B.

[00177] We have also analyzed the ability of our antibody to detect EBNA1 epitopes HLA- A02 positive cells that were naturally infected with EBV (see Figure 11) and quantitate the number of HLA-A0201/EBNA1 complexes expressed on the surface of these cells (see Figure 12).

[00178] We have also shown that the antibody is able to detect HLA-A0201/EBNA1 complexes in biopsy of HLA-A0201 and EBV-positive nasopharyngeal carcinoma in Figure 13.

[00179] To our knowledge, there are no antibodies that have been made previously with specificity for EBV derived peptides expressed in association with human HLA on the surface of EBV infected cells. Hence, this antibody represents the first antibody worldwide that has the potential to be used to target EBV infected cells based on their HLA expression profile.

[00180] Moreover, in order to make this very specialized monoclonal, we developed an improved method for making specific antibodies based on a prior selection of the antigen specific B cells that should be applicable to all known HLA/peptide combinations from tumor, virus, bacteria, and parasite derived protein antigens. Whilst antigen specific T lymphocyte cell lines can be made in 1-2 laboratories worldwide with a 'similar' specificity, these cell lines require a high degree of technical proficiency to make. [00181] Other laboratories have successfully produced TCR-like monoclonal antibodies using a standard hybridoma approach or by employing phage antibody libraries. However, the numbers of this type of antibody remain extremely low and none of them have been designed for targeting EBV epitopes. The affinities of the antibodies made by phage display are predictably low (see Table 1). Hence, our methodology represents a way of making the generation of these antibodies easier. Given that EBNA1 is expressed by 100% of EBV- associated tumours and the prevalence of these tumours worldwide (for instance, NPC represents 18% of all newly diagnosed cancers in China)³'⁴, this makes a reagent that can be used to target these living tumour cells in situ highly exploitable.

Table 1: Tabulation of TCR-like mAbs raised via classical hybridoma technique and phage display library. The table depicts the haplotype, antigen targeted, affinity, method of isolation and strain of mice and adjuvant used for immunization.

fused with SP2/0

Human TCR-like mAb (Phage display)

[00182] Additional embodiments of the present disclosure include:

[00183] 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.

[00184] 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.

[00185] 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.

[00186] 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.

[00187] Humanized anti-HLA-A2/EBNAl antibody can be used as a diagnostic to directly quantify the numbers of infected cells ex vivo or in vivo. REFERENCES

1) Yewdell, J. W. Plumbing the sources of endogenous MHC class I peptide ligands. Curr Opin Immunol. 19, 179-86 Feb, 2007.

2) Altman, J. D.Moss, P. A.Goulder, P. J.Barouch, D. H.McHeyzer-Williams, M. G.Bell, J. I.McMichael, A. J. Davis, M. M. Phenotypic analysis of antigen- specific T lymphocytes.

Science. 274, 5284, 94-6, Oct 4, 1996.

3) Stuber, G, et al., HLA-A0201 and HLA-B7 binding peptides in the EBV-encoded EBNA- 1, EBNA-2 and BZLF-1 proteins detected in the MHC class I stabilization assay. Low proportion of binding motifs for several HLA class I alleles in EBNA-1. Int Immunol. 7,4, 653-663, 1995.

4) Chen, M.R., et al., The major immunogenic epitopes of 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

5) Young, L. S. Rickinson, A. B. Epstein-Barr virus: 40 years on. Nat Rev Cancer. 4, 10, 757-68. Oct 2004.

6) Rickinson, A.B., and E. Kieff. Epstein-Barr virus, p. 2575-2627 (Lippincott Williams and Wilkins, Philadelphia, Pa, 2001).

7) Brown, N.A., Liu, C.R., Wang, Y.F. & Garcia, C.R. B-cell lymphoproliferation and lymphomagenesis are associated with clonotypic intracellular terminal regions of the Epstein- Barr virus. J Virol 62, 962-9 (1988).

8) Raab-Traub, N. & Flynn, K. The structure of the termini of the Epstein-Barr virus as a marker of clonal cellular proliferation. Cell 47, 883-9 (1986).

9) Hamilton-Dutoit, S.J. et al. Epstein-Barr virus-latent gene expression and tumor cell phenotype in acquired immunodeficiency syndrome-related non-Hodgkin's lymphoma.

Correlation of lymphoma phenotype with three distinct patterns of viral latency. Am J Pathol 143, 1072-85 (1993).

10) Young, L.S. et al. Epstein-Barr virus gene expression in nasopharyngeal carcinoma. J Gen Virol 69 (Pt 5), 1051-65 (1988).

11) Middleton, D., et al., Analysis of the distribution of HLA-A alleles in populations from five continents. Hum Immunol 61, 1048-1052 (2000).

[00188] While specific aspects have been described and illustrated herein, such aspects should be considered illustrative only and not as limiting the disclosure as construed in accordance with the accompanying claims. [00189] All publications, patent applications, and sequence accession numbers (e.g., Genbank accession numbers) cited in this specification are herein incorporated by reference in their entirety for all purposes as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference for all purposes.

[00190] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this disclosure that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Claims

CLAIMS What is Claimed:

1. A recombinant antibody or fragment thereof that binds to a HLA/peptide complex, wherein the peptide has the sequence FMVFLQTHI (SEQ ID NO: 1).

2. The recombinant antibody or fragment thereof according to claim 1, wherein the peptide is derived from Epstein-Barr virus nuclear antigen 1 (EBNA1).

3. The recombinant antibody or fragment thereof according to claim 1, wherein the HLA is HLA-A0201.

4. The recombinant antibody or fragment thereof according to claims 1-3, wherein the recombinant antibody or fragment thereof is capable of competitively inhibiting specific binding to the peptide of SEQ ID NO: 1 or binding by a monoclonal antibody produced by an anti-HLA-A02/EBNA 1 hybridoma.

5. The recombinant antibody or fragment thereof according to claim 1, wherein 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.

6. The antibody or fragment thereof according to claim 1, which 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.

7. The antibody or fragment thereof according to claim 1, which 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.

8. The antibody or fragment thereof according to claim 1, wherein 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: 32-34.

9. The antibody or fragment thereof according to claim 1, wherein 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: 37-39.

10. The antibody or fragment thereof according to claim 1, wherein the antibody or fragment thereof comprises a heavy chain comprising three CDR sequences selected from the group consisting of SEQ ID NOs: 32-34, or a sequence having at least 95% identity thereto.

11. The antibody or fragment thereof according to claim 1, wherein the antibody or fragment thereof comprises a light chain comprising three CDR sequences selected from the group consisting of SEQ ID NOs: 37-39, or a sequence having at least 95% identity thereto.

12. The antibody or fragment thereof according to claim 1, wherein the antibody comprises a heavy chain sequence at least 95% identical to SEQ ID NO: 31.

13. The antibody or fragment thereof according to claim 1, wherein the antibody comprises a light chain sequence at least 95% identical to SEQ ID NO: 36.

14. The antibody or fragment thereof according to claim 1, wherein the antibody or fragment thereof binds to an antigen with an affinity constant (K_D) of less than 1 x 10 -^"8

M.

15. The antibody or fragment thereof according to claim 1, wherein the antibody or fragment thereof binds to an antigen with an affinity constant (K_D) of less than 1 x 10^"9 M.

16. A pharmaceutical composition comprising the antibody or fragment thereof according to any one of claims 1-15 and a pharmaceutically acceptable carrier effective to reduce EBV infected tumor cells in a subject.

17. A method of passive immunization comprising administration to a subject an effective amount of the antibody or fragment thereof according to any one of claims 1-15.

18. 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 one of claims 1- 15, effective to reduce or prevent the disease.

19. The method of claim 18, wherein the antibody is administered intravenously (IV), subcutaneously (SC), intramuscularly (IM), transdermally, or orally.

20. The method of claim 18, wherein the antibody is administered in an amount in the range of 1 to 100 milligrams per kilogram of the subject's body weight.

21. 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).

22. The method of claim 21, wherein the isolation in step (c) is by immuno -magnetic selection.

23. The method of claim 21, wherein the peptide is derived from Epstein-Barr virus nuclear antigen 1 (EBNA1).

24. The method of claim 21, wherein the peptide has the sequence FMVFLQTHI (SEQ ID NO: 1).

25. The method of claim 21 , wherein the HLA is HLA-A0201.

26. An isolated nucleic acid encoding the antibody or fragment thereof according to any one of claims 1-15.

27. An expression vector comprising the nucleic acid of claim 26.

28. A host cell comprising the expression vector of claim 27.

29. The host cell of claim 28, wherein the host cell is a bacterial cell.

30. The host cell of claim 28, wherein the host cell is a eukaryotic cell.

31. The host cell of claim 28, wherein the host cell is a mammalian cell.