US20210115113A1

US20210115113A1 - Epstein-barr virus antibodies and uses thereof

Info

Publication number: US20210115113A1
Application number: US16/940,304
Authority: US
Inventors: Javier Gordon Ogembo; Lorraine Zvichapera MUTSVUNGUMA
Original assignee: City of Hope
Current assignee: City of Hope
Priority date: 2017-04-28
Filing date: 2020-07-27
Publication date: 2021-04-22
Also published as: US12012446B2; US20230056712A1

Abstract

Disclosed herein are antibodies or immunogenic fragments thereof that specifically bind to Epstein-Barr virus (EBV) glycoprotein 350 (gp350) or 220 or one or more immunogenic peptides. Also disclosed are immunogenic peptides comprising fragments of gp350 amino acid sequence, EBV antibody-small molecule conjugates and pharmaceutical compositions comprising the antibody or an immunogenic fragment thereof, one or more immunogenic peptides, or the EBV antibody-small molecule conjugate. The antibodies, immunogenic peptides, conjugates, and pharmaceutical compositions can be used to treat or prevent EBV infections and EBV-associated conditions and diseases.

Description

PRIORITY CLAIM

The present invention claims the benefit of U.S. Provisional Patent Application No. 62/880,024, filed on Jul. 29, 2019. The present invention is also a continuation-in-part of U.S. patent application Ser. No. 16/609,078, filed on Oct. 28, 2019, which is a national phase entry of International Application No. PCT/US2018/30030, filed on Apr. 27, 2018, which claims priority to U.S. Provisional Patent Application No. 62/491,945, filed on Apr. 28, 2017. The contents of all priority applications are incorporated herein by reference in their entireties.

STATEMENT OF GOVERNMENT INTEREST

The present invention was made with government support under Grant Nos. R21CA205106 and R21CA232275, awarded by the National Institutes of Health. The Government has certain rights in the invention.

SEQUENCE LISTING

This disclosure includes a sequence listing, which is submitted in ASCII format via EFS-Web, and is hereby incorporated by reference in its entirety. The ASCII copy, created on Dec. 31, 2020, is named 8171US03_SequenceListing.txt and is 83 kilobytes in size.

BACKGROUND

Epstein-Barr virus (EBV) infection is the causal agent of acute infectious mononucleosis (62, 63). Persistent EBV infection in immunodeficient individuals is associated with numerous epithelial and lymphoid malignancies, such as nasopharyngeal carcinoma, gastric carcinoma, Burkitt lymphoma, Hodgkin lymphoma, and post-transplant lymphoproliferative diseases (PTLD) (1). Transplantation is the treatment of choice for a variety of patients with end-stage organ failure or hematologic malignancies, or in need of reconstructive transplantation (1). Transplantation success depends entirely on potent immunosuppressive drugs to prevent stem cell/organ rejection. However, these drugs impose several serious side effects, including an increased risk of infection with or reactivation of Epstein-Barr virus (EBV), and the resultant development of PTLDs, which are aggressive, life-threatening complications (2, 3). Through the early 2000s, PTLD patients who had been EBV-naïve prior to transplantation showed mortality rates of 50-90% for stem cell and solid organ transplants; while recent data suggest outcomes have improved, challenges remain. PTLDs usually develop in EBV-naïve patients, particularly pediatric patients, who receive organs from EBV+ donors. A variety of non-standardized, non-specific treatments are used to treat EBV+ PTLD cases (4-9). Initial clinical management typically involves reduction of immunosuppression; however, this can lead to graft-versus-host disease. Other treatments including radiation/chemotherapy and excision of PTLD lesions all have undesirable side effects. Second-line treatment often includes antibodies (Abs) against the B cell antigen, CD20; however, this also targets healthy B cells, further weakening the immune system and exposing patients to other opportunistic infections.
In over 50 years of EBV vaccine research, few candidates have demonstrated partial clinical efficacy, and none have been efficacious enough to elicit sterilizing immunity and be licensed (24). Antibodies, whether elicited in the host naturally or via passive immunization, provide an effective first-line of defense against viral infection.
Thus, there is an urgent need for a novel EBV-specific therapy that targets EBV+ cells to neutralize EBV infection and prevent subsequent PTLD development in EBV-naïve patients.

SUMMARY

In one aspect, this disclosure relates to an Epstein-Barr virus (EBV) antibody or an immunogenic fragment thereof. In some embodiments, the EBV antibody or an immunogenic fragment thereof specifically binds to EBV glycoprotein 350/220. In some embodiments, the EBV antibody comprises a VH region comprising CDR-1, CDR-2, and CDR-3 represented by SEQ ID NOs: 5-19, 21-35, and 37-51, respectively. In some embodiments, the EBV antibody comprises a VL region comprising CDR-1, CDR-2, and CDR-3 represented by SEQ ID NOs: 53-67, 69-83, and 85-99, respectively. In some embodiments, the EBV antibody is a monoclonal antibody. In some embodiments, the EBV antibody is a chimeric antibody, a human antibody, or a humanized antibody. In some embodiments, the EBV antibody is a neutralizing antibody. In some embodiments, the EBV antibody is humanized 72A1 or humanized E1D1. For example, the EBV antibody comprises one or more CDRs of antibody clone 72A1. In another example, the EBV antibody comprises one or more CDRs of antibody clone E1D1. In some embodiments, the EBV antibody comprises a heavy chain having an amino acid sequence of SEQ ID NO: 180, SEQ ID NO: 185, or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 180 or SEQ ID NO: 185. In some embodiments, the EBV antibody comprises a light chain having an amino acid sequence of SEQ ID NO: 181, SEQ ID NO: 186, or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 181 or SEQ ID NO: 186.
In another aspect, this disclosure relates to an immunogenic peptide comprising the amino acid sequence of a fragment of EBV350 such as AA1-101, AA102-201, or AA402-501, or an amino acid sequence identical to or sharing at least 60% similarity to the fragment. In some embodiments, the immunogenic peptide further comprises a known immunogenic peptide such as keyhole limpet hemocyanin (KLH) peptide.
In another aspect, this disclosure relates to an EBV antibody-small molecule conjugate. The EBV antibodies disclosed herein can be conjugated to small molecules having activities against EBV-transformed cells. For example, the small molecules have anti-proliferative activities against EBV-transformed B lymphoma cells. In some embodiments, the small molecules are growth inhibitors of EBV infected B cells. In some embodiments, the small molecule is L₂P₄, 2-butynediamide, or a derivative thereof. In some embodiments, the small molecule is conjugated to the antibody via a linker or an adaptor. In some embodiments, the small molecule is conjugated to the constant region of the heavy chain or the light chain of the antibody.
In a related aspect, this disclosure relates to a pharmaceutical composition comprising one or more EBV antibodies disclosed herein or one or more immunogenic fragments thereof, one or more immunogenic peptides disclosed herein, or the EBV antibody-small molecule conjugate disclosed herein. The pharmaceutical composition can further comprise one or more pharmaceutically acceptable excipients. The pharmaceutical composition can be formulated into any suitable formulation depending on the administration route. In some embodiments, the pharmaceutical composition comprising a humanized 72A1, a humanized E1D1, or both.
In another aspect, this disclosure relates to a method of neutralizing EBV infection. The method includes administering to a subject infected with EBV a therapeutically effective amount of one or more EBV antibodies disclosed herein or one or more immunogenic fragments thereof, the EBV antibody-small molecule conjugate, one or more immunogenic peptides disclosed herein, or the pharmaceutical composition described above. In some embodiments, the subject is human. In some embodiments, the subject suffers from or at an elevated risk of suffering from EBV infection, such as EBV+ post-transplant lymphoproliferative diseases (PTLDs).
In another aspect, this disclosure relates to a method of treating or preventing EBV infection. The method includes administering to a subject at an elevated risk of EBV infection a therapeutically effective amount of one or more EBV antibodies disclosed herein or an immunogenic fragment thereof, the EBV antibody-small molecule conjugate, one or more immunogenic peptides disclosed herein, or the pharmaceutical composition described above. In some embodiments, the subject is human. In some embodiments, the EBV antibody is a humanized 72A1, or a humanized E1D1.
In another aspect, this disclosure relates to a method of preventing a post-transplant lymphoproliferative disease (PTLD). PTLD is associated with EBV infection of B cells, either as a consequence of reactivation of the virus post transplantation or from primary EBV infection. The method includes administering to a subject who is a transplant recipient a prophylactically or therapeutically effective amount of one or more EBV antibodies disclosed herein or an immunogenic fragment thereof, the EBV antibody-small molecule conjugate, one or more immunogenic peptides disclosed herein, or the pharmaceutical composition described above. The administration can be before, during, and/or after the transplant. In some embodiments, the subject is a pediatric transplant recipient who is EBV naïve. In some embodiments, the subject is an adult transplant recipient. In some embodiments, the subject is human.
In another aspect, this disclosure relates to a method of treating an EBV-associated cancer. The method includes administering to a subject suffering from an EBV-associated cancer a prophylactically or therapeutically effective amount of one or more EBV antibodies disclosed herein or an immunogenic fragment thereof, the EBV antibody-small molecule conjugate, one or more immunogenic peptides disclosed herein, or the pharmaceutical composition described above. In some embodiments, the examples of EBV-associated cancer include but are not limited to Hodgkin lymphoma, Burkitt lymphoma, gastric cancer, and nasopharyngeal carcinoma. In some embodiments, the subject is human. In some embodiments, the EBV antibody is a humanized 72A1, or a humanized E1D1.
In another aspect, this disclosure relates to a method of immunizing or vaccinating a subject against an EBV infection. The method includes administering to a subject suffering from an EBV infection a therapeutically effective amount of one or more EBV antibodies disclosed herein or an immunogenic fragment thereof, one or more immunogenic peptides disclosed herein, or the pharmaceutical composition thereof as described above. In some embodiments, the subject is human.
In another aspect, this disclosure relates to a method of inducing the production of neutralizing antibodies against an EBV in a subject. The method includes administering to a subject an effective amount of one or more immunogenic peptides disclosed herein. In some embodiments, the subject is human.

BRIEF DESCRIPTION OF THE DRAWINGS

This application contains at least one drawing executed in color. Copies of this application with color drawing(s) will be provided by the Office upon request and payment of the necessary fees.

FIGS. 1A-1E show specificity of the novel anti-gp350 mAbs. FIG. 1A: SDS-PAGE analysis of anti-EBV gp350 antibodies purified from indicated hybridoma (HB) supernatants. FIG. 1B: ELISA screening of hybridoma (HB) supernatants for anti-gp350-specific antibodies. Soluble EBV gp350 protein was used as the target antigen at 0.5 μg/ml. m72A1 at 10 μg/ml and KSHV anti-gH/gL (54A1) were used as positive and negative (not shown) controls, respectively. Bound antibodies were detected using HRP-conjugated anti-mouse IgG (1:2,000). Twenty-three HB clones with ELISA signals two times greater than those of 54A1 control were considered to be positive/reactive to gp350. FIG. 1C: Immunoblot analysis with gp350-transfected stable CHO lysate to determine specificity of anti-gp350-producing HB supernatants. FIG. 1D: Flow cytometric analysis of surface expression of gp350 protein on gp350 expressing CHO cells. Cells were stained with indicated anti-gp350 mAb (1:250), followed by secondary goat anti-mouse conjugated to AF488. FIG. 1E: Flow cytometric analysis of HB5, HB17 and HB19.

FIG. 2 shows agarose gel analysis of PCR products of heavy chain of select novel anti-gp350 antibodies (HB1, HB4, HB7, HB13, and HB15) and m72A1 was used as a positive control.

FIGS. 3A and 3B show PROMALS3D multiple sequence alignment of VH (FIG. 3A) and VL (FIG. 3B) regions of 15 mAbs and nAb-72A1 (SEQ ID NOS:100-131). The highly variable complementarity determining regions (CDR) 1-3, indicated by black boxes, define the antigen binding specificity. The conserved framework regions (FR) 1-4 flank the CDRs. Consensus amino acid (AA) are in bold and upper case. Consensus predicted secondary structure (ss) symbols: alpha-helix: h and beta-strand: e.

FIGS. 4A-4D show the comparison of murine 72A1 (m72A1) and humanized 72A1 (h72A1). FIG. 4A: Sequence comparison of murine (m72A1) and humanized (h72A1) 72A1. ClustalW alignment of heavy chain (i) and light chain (ii) variable region amino acid sequences. Murine 72A1 heavy chain and light chain AA sequences are represented by SEQ ID NOs: 178 and 179, respectively, and humanized 72A1 heavy chain and light chain AA sequences are represented by SEQ ID NOs: 180 and 181, respectively. Regions of identical sequence are represented by *. Regions of similarity are represented by: FIG. 4B: ELISA comparison screening of m72A1 and h72A1 for anti-gp350-specificity. Soluble EBV gp350 protein was used as the target antigen at 0.5 μg/ml. m72A1 and h72A1 were serially diluted (5-0.062 μg/ml) and 1× phosphate buffered saline (PBS) was used as a negative control (data not shown). Bound h72A1 and m72A1 antibodies were detected using HRP-conjugated anti-mouse IgG and anti-human IgG (1:2,000) as relevant. FIG. 4C: ELISA determining the reactivity of humanized 72A1 to murine IgG. Soluble EBV gp350 protein was used as the target antigen at 0.5 μg/ml. Plates were incubated with 10 μg/ml of m72A1 and h72A1, followed by three washes. Bound antibodies were detected using HRP-conjugated anti-mouse IgG or anti-human IgG (1:2,000). FIG. 4D: Flow cytometric analysis of m72A1 and h72A1 gp350 specificity. CHO wild-type cells and gp350-expressing CHO cells were stained with m72A1 and h72A1, followed by secondary goat anti-mouse or anti-human conjugated to AF488. Unstained cells and cells stained with secondary goat anti-mouse or anti-human conjugated to AF488 alone were used as negative controls.

FIGS. 5A-5C show neutralization activity of novel anti-gp350 mAbs against EBV-eGFP in Raji cells. FIG. 5A: EBV-eGFP titration in Raji cells to determine optimal dose of infection. FIG. 5B: EBV-eGFP was pre-incubated with 15 indicated serial diluted (12.5-100 μg/ml), maxispin column-purified anti-gp350 mAbs, followed by incubation with 10⁵Raji cells for 48 hours. EBV-eGFP+ cells were enumerated using flow cytometry. Anti-gp350 (m72A1) nAb served as positive control and non-neutralizing anti-gp350 (2L10) mAb and anti-KSHV gH/gL mAb (54A1) served as negative controls. FIG. 5C: EBV-eGFP was pre-incubated with 7 indicated serially diluted (12.5-100 μg/ml) protein G affinity chromatography- and size-exclusion chromatography-purified anti-gp350 mAbs, followed by incubation with 10⁵Raji cells for 48 hours. EBV-eGFP+ cells were enumerated using flow cytometry. Anti-gp350 (m72A1 and h72A1) nAbs served as positive controls and anti-KSHV gH/gL mAb (54A1) served as negative control.

FIGS. 6A-6I show the linear peptide epitope mapping of gp350 of various antibodies. ELISA was used to detect the responses of the indicated antibodies against each linear peptide. ELISA plates were coated overnight with 10 μg/ml of each of the indicated 45 linear peptides and 0.5 μg/ml of recombinant purified gp350 protein was used as a positive control. Plates were blocked for 1 hour, washed three times, and incubated with 10 μg/ml of each antibody for 2 hours. Bound antibodies were detected using HRP-conjugated anti-mouse IgG or anti-human IgG (1:2,000).

FIGS. 7A-7B show the schematic diagram (FIG. 7A) and the amino acid sequence (FIG. 7B, SEQ ID NO:182) of EBV gp350 protein, illustrating the ectodomain and the splice site (AA 501-699) for making gp220, the transmembrane domain, TM (AA 841-897) and the cytoplasmic domain, CT (AA 898-907). To analyze and classify binding of anti-gp350 mAbs to linear epitopes on the protein, EBV gp350 was separated into 9 regions of ˜100 AA.

FIGS. 8A-8C show construction of chimeric gp350 nAbs. FIG. 8A: Construction of chimeric Ab. FIG. 8B: Example of the cloning strategy of heavy chain and light chain variable regions into expression vectors. FIG. 8C: PCR amplification of heavy chain and light chain variable regions. 72A1 and clone 19 were used as examples of PCR amplification.

FIG. 9 illustrates an antibody-L₂P₄conjugate.

FIGS. 10A-10B show the comparison of murine E1D1 (mE1D1) and humanized E1D1 (hE1D1). FIG. 10A: Sequence comparison of mE1D1 and hE1D1. Alignment of heavy chain (i) and light chain (ii) variable region amino acid sequences by the Clustal W. Murine E1D1 heavy chain and light chain AA sequences are represented by SEQ ID NOs: 183 and 184, respectively, and humanized E1D1 heavy chain and light chain AA sequences are represented by SEQ ID NOs: 185 and 186, respectively. Regions of identical sequence are represented by *. Regions of similarity are represented by: FIG. 10B: Flow cytometric analysis of mE1D1 and hE1D1 gH/gL specificity. CHO wild type cells and gH/gL-expressing CHO cells were stained with mE1D1 and hE1D1, followed by secondary goat anti-mouse or anti-human conjugated to AF488. Unstained cells and cells stained with secondary goat anti-mouse or anti-human conjugated to AF488 alone, were used as negative controls.

FIGS. 11A-11F demonstrate biochemical characterization of chimeric and humanized antibodies. FIG. 11A is a schematic diagram of murine (m), chimeric (ch), humanized (h) and human antibodies. FIG. 11B shows SDS-PAGE analysis of antibodies under reducing conditions after purification using size exclusion chromatography. FIG. 11C shows ELISA determining the reactivity of chimeric and humanized antibodies to murine IgG. FIG. 11D shows Western blot analysis of anti-gp350 antibodies detecting liner epitopes. FIG. 11E shows ELISA binding of (i) anti-gp350 and (ii) anti-gH/gL to soluble gp350 and gH/gL proteins. FIG. 11F shows flow cytometric analysis of (i) gp350 and (ii) gH/gL specificity.

FIGS. 12A-12C show the EBV inhibitory effect of anti-gp350 and anti-gH/gL neutralizing antibodies in epithelial and B cells in vitro. Neutralization activity of single and combination anti-gp350 and anti-gH/gL in HEK 293 cells (12A), SVKCR2 cells (12B) and Raji cells (12C). Black dotted line represents 50% neutralization activity.

DETAILED DESCRIPTION

Epstein-Barr virus (EBV) predominantly infects epithelial cells and B cells, reflecting the viral tropism and cellular ontogeny characteristic of most EBV-associated malignancies (1). Despite the fact that EBV infection is associated with more than 200,000 cases of a variety of human malignancies every year, and has significant public health impacts, there is no licensed vaccine to date (20). The EBV glycoprotein gp350/220 (gp350) is a known target for a host's virus neutralizing antibody (nAb) response upon natural EBV infection (52, 67, 71) or immunization, and thus has been tested as a viable target for vaccines and therapeutics in five clinical trials to prevent B cell infection (30, 32-35). However, not all of the potential nAb epitopes on gp350 have been identified or fully characterized.
EBV infects at least 90% of the human population globally, irrespective of geographical location. Currently, there are two models describing how initial EBV infection of human host cells occurs in vivo (72). In the first infection model, the incoming virus first targets epithelial cells and engages with host ephrin receptor tyrosine kinase A2 via heterodimeric glycoproteins gH/gL (73, 74) or with host integrins via BMRF-2 (75, 76). This triggers fusion of EBV glycoprotein gB with the host epithelial cell membrane to enhance viral entry into the cytoplasm. This interaction is thought to occur in the oral mucosa; there, EBV undergoes lytic replication in epithelial cells to release virions that subsequently infect resting B cells in tonsillar crypts or circulating naïve B cells. In the alternative infection model, the incoming virus binds to B cells in the oral mucosa via host CD35 (45) and/or CD21 through its major immunodominant glycoprotein, gp350 (55, 65). The interaction between gp350 and CD35 and/or CD21 triggers viral adsorption, capping, and endocytosis into B cells (66). This subsequently leads to the heterotrimeric EBV glycoprotein complex gp42/gH/gL binding to host HLA class II molecules to activate gB membrane fusion and infection of B cells (17). Once infected, B cells typically remain latent and harbor the virus for life, but may also traffic back to the oropharynx, where EBV is amplified by lytic replication in epithelial cells, and shed into the saliva (72). Thus, B cells are the main reservoirs for EBV reactivation and for the development of virus-related malignancies (77). Novel strategies that could block interactions between EBV glycoproteins and cellular receptors that mediate viral infection could be beneficial in the development of effective antiviral therapies.
Antibodies are the first line of defense against viral infection and nearly all EBV-infected individuals develop nAbs directed to the ectodomain of EBV gp350 (52, 67, 71). A recent study showed that polyclonal serum antibodies against gp350 from naturally infected individuals or immunized animals block EBV infection of B cells in vitro better than antibodies against EBV gH/gL or gp42 (78). Thus, gp350 is a promising candidate for development of EBV vaccines against B cell infection; however, to make effective vaccines, the nAbs epitopes on the gp350 ectodomain must be identified and fully characterized.
Disclosed herein are EBV antibodies or immunogenic fragments thereof that specifically bind to gp350/gp220, immunogenic peptides, and EBV antibody-small molecule conjugates for treating or preventing EBV infection, in particular, in subjects receiving a transplant. In some embodiments, chimeric (human/mouse) anti-gp350 nAbs or humanized antibodies or functional fragments thereof are conjugated to L₂P₄to obtain an EBV-specific ADC that improves the therapeutic efficacy of treating EBV-associated PTLDs. L2P4 described by Jiang et al., Nature Biomedical Engineering 1: 0042 (2017), is an example of the small molecules encompassed by this disclosure.
The term “antibody” as used herein refers to an immunoglobulin molecule or an immunologically active portion thereof that specifically binds to, or is immunologically reactive with a particular antigen, for example, EBV gp350/gp220, or a particular domain or fragment of gp350/gp220 such as AA1-101, 102-201, and 402-501.
In certain embodiments an antibody for use in the present methods or compositions is a full-length immunoglobulin molecule, which comprises two heavy chains and two light chains, with each heavy and light chain containing three complementary determining regions (CDRs). The CDRs of various antibodies are identified and listed in Table 1 below.

TABLE 1

CDR Sequences

VH

VL

Antibodies	CDR-1	CDR-2	CDR-3	CDR-1	CDR-2	CDR-3

m72A1	GSSFTDY	INPYNGG	GGLRRVNWFAYW	TGAVTTSNY	GTN	VLWFISNHWV
	(SEQ ID NO: 4)	(SEQ ID NO: 20)	(SEQ ID NO: 36)	(SEQ ID NO: 52)	(SEQ ID NO: 68)	(SEQ ID NO: 84)

h72A1	GSSFTDY	INPYNGG	GGLRRVNWFAYW	TGAVTTSNY	GTN	VLWFISNHWV
	(SEQ ID NO: 4)	(SEQ ID NO: 20)	(SEQ ID NO: 36)	(SEQ ID NO: 52)	(SEQ ID NO: 68)	(SEQ ID NO: 84)

HB-1	GFLLTTY	IWAGGS	RDRGYGYLYAMDYW	QNVGTN	STD	QQYNTYPYT
	(SEQ ID NO: 5)	(SEQ ID NO: 21)	(SEQ ID NO: 37)	(SEQ ID NO: 53)	(SEQ ID NO: 69)	(SEQ ID NO: 85)

HB-2	GYTFTAY	INYKTGE	PYGYALDYW	SSVNY*	ATS*	QQWSSNPPT*
	(SEQ ID NO: 6)	(SEQ ID NO: 22)	(SEQ ID NO: 38)	(SEQ ID NO: 54)	(SEQ ID NO: 70)	(SEQ ID NO: 86)

HB-3	GYTFASY	INPNNGH*	RNLYYYGRPDYW*	QDIGNY*	YTS*	QQGNTLPPT*
	(SEQ ID NO: 7)	(SEQ ID NO: 23)	(SEQ ID NO: 39)	(SEQ ID NO: 55)	(SEQ ID NO: 71)	(SEQ ID NO: 87)

HB-5	GYTFTNH	INPYNDY	RSEGWLRRGAWFAY	QSIGTS	YAS	QQSNSWPMLT
	(SEQ ID NO: 8)	(SEQ ID NO: 24)	(SEQ ID NO: 40)	(SEQ ID NO: 56)	(SEQ ID NO: 72)	(SEQ ID NO: 88)

HB-6	GYTFTDY*	INTRTGE	PYGYALDYW	SSVNY*	ATS*	QQWSSNPPT*
	(SEQ ID NO: 9)	(SEQ ID NO: 25)	(SEQ ID NO: 41)	(SEQ ID NO: 57)	(SEQ ID NO: 73)	(SEQ ID NO: 89)

HB-7	GYTFTDY*	ISPGRSG	RYGHPSYLDVW	QSVGNA	SAS	QQYSSYPLT
	(SEQ ID NO: 10)	(SEQ ID NO: 26)	(SEQ ID NO: 42)	(SEQ ID NO: 58)	(SEQ ID NO: 74)	(SEQ ID NO: 90)

HB-8	GYSFTNY*	INTYTGE	RYYYGSVYSAWFAYW	QSIVHSNGNTY*	KVS*	FQGSHVPYT*
	(SEQ ID NO: 11)	(SEQ ID NO: 27)	(SEQ ID NO: 43)	(SEQ ID NO: 59)	(SEQ ID NO: 75)	(SEQ ID NO: 91)

HB-9	GFTFSSY	ISSGGSY	REDFYYGSSYGFFDVW	QSIVHSNGNTY*	KVS*	FQGSHVPYT*
	(SEQ ID NO: 12)	(SEQ ID NO: 28)	(SEQ ID NO: 44)	(SEQ ID NO: 60)	(SEQ ID NO: 76)	(SEQ ID NO: 92)

HB-10	GYTFTSY*	INPSNGH	RNLYYYGRPDYW	QDIGNY*	YTS*	QQNTLPPT
	(SEQ ID NO: 13)	(SEQ ID NO: 29)	(SEQ ID NO: 45)	(SEQ ID NO: 61)	(SEQ ID NO: 77)	(SEQ ID NO: 93)

HB-11	GDSITSG	ISYSGS	RGNGGNYDWYFDVW	SSVNF	YIS	QQFTSSPSWT
	(SEQ ID NO: 14)	(SEQ ID NO: 30)	(SEQ ID NO: 46)	(SEQ ID NO: 62)	(SEQ ID NO: 78)	(SEQ ID NO: 94)

HB-12	GYTFTNY*	INPNNGH*	RNLYYYGRPDYW*	QSLVHSNGNTY	KVS*	SQSTHVPLT
	(SEQ ID NO: 15)	(SEQ ID NO: 31)	(SEQ ID NO: 47)	(SEQ ID NO: 63)	(SEQ ID NO: 79)	(SEQ ID NO: 95)

HB-14	GYTFTDY*	IHPRRGG	RYGYPVVYFDVW	QSIVHDNGNTY	KVS*	FQGSHVPPT
	(SEQ ID NO: 16)	(SEQ ID NO: 32)	(SEQ ID NO: 48)	(SEQ ID NO: 64)	(SEQ ID NO: 80)	(SEQ ID NO: 96)

HB-17	GYTFTSY*	INPNNGH*	RNLFYYSRPDYW	QDIGNY*	YTS*	QQGNTLPPT*
	(SEQ ID NO: 17)	(SEQ ID NO: 33)	(SEQ ID NO: 49)	(SEQ ID NO: 65)	(SEQ ID NO: 81)	(SEQ ID NO: 97)

HB-20	GYTFTSY*	INPTNGH	RNLYYYGRPDYW*	QDIGNY*	YTS*	QQGNALPPT
	(SEQ ID NO: 18)	(SEQ ID NO: 34)	(SEQ ID NO: 50)	(SEQ ID NO: 66)	(SEQ ID NO: 82)	(SEQ ID NO: 98)

HB-22	GFSLTNY	IWSDGS	RNYYGNSYPAWFAYW	QSIVHSNGNTY	KVS*	FQGSHVPWT
	(SEQ ID NO: 19)	(SEQ ID NO: 35)	(SEQ ID NO: 51)	(SEQ ID NO: 67)	(SEQ ID NO: 83)	(SEQ ID NO: 99)

The term “antibody,” in addition to natural antibodies, also includes genetically engineered or otherwise modified forms of immunoglobulins, such as synthetic antibodies, intrabodies, chimeric antibodies, fully human antibodies, humanized antibodies, peptibodies and heteroconjugate antibodies (e.g., bispecific antibodies, multispecific antibodies, dual-specific antibodies, anti-idiotypic antibodies, diabodies, triabodies, and tetrabodies). For example, humanized bispecific nAbs comprising E1D1 and 72A1 targeting two EBV gps, gp350 and gH/gL complex, respectively, can be produced for use as a prophylactic agent against EBV infection or re-infection. In some embodiments, bispecific or multispecific antibodies comprising a combination of the mAbs identified herein or immunogenic fragments thereof can be produced. The bispecific or multispecific antibodies can be humanized according to known technologies. Alternatively, the bispecific or multispecific antibodies can be chimeric antibodies. The antibodies disclosed herein can be monoclonal antibodies or polyclonal antibodies. In those embodiments wherein an antibody is an immunologically active portion of an immunoglobulin molecule, the antibody may be, for example, a Fab, Fab′, Fv, Fab′ F(ab′)₂, disulfide-linked Fv, single chain Fv antibody (scFv), single domain antibody (dAb), or diabody. The antibodies disclosed herein, including those that are immunologically active portion of an immunoglobulin molecule, retain the ability to bind a specific antigen such as EBV gp350/220, or to bind a specific fragment of gp350/gp220 such as AA1-101, AA102-201, and AA402-501.
In some embodiments, the EBV antibodies disclosed herein have undergone post-translational modifications such as phosphorylation, methylation, acetylation, ubiquitination, nitrosylation, glycosylation, or lipidation associated with expression in a mammalian cell line, including a human or a non-human host cell. Techniques for producing recombinant antibodies and for in vitro and in vivo modifications of recombinant antibodies are known in the art.
Provided in certain embodiments herein are chimeric, and/or humanized EBV antibodies. Various techniques are known in the art for humanizing antibodies from non-human species such that the antibodies are modified to increase their similarity to antibodies naturally occurring in humans. Six CDRs are present in each antigen binding domain of a natural antibody. These CDRs are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen binding domain as the antibody assumes its three-dimensional configuration. CDR sequences of certain antibodies identified herein are shown in Table 1. The remainder of the amino acids in the antigen binding domains, referred to as “framework” regions, show less inter-molecular variability and form a scaffold to allow correct positioning of the CDRs. This disclosure also relates to antibodies comprising VH and VL regions comprising the CDRs shown in Table 1.
“Treating” or “treatment” of a disease or a condition may refer to preventing the disease or condition, slowing the onset or rate of development of the disease or condition, reducing the risk of developing the disease or condition, preventing or delaying the development of symptoms associated with the disease or condition, reducing or ending symptoms associated with the disease or condition, generating a complete or partial regression of the disease or condition, or some combinations thereof.
As used herein, the term “subject” refers to mammalian subject, preferably a human. The phrases “subject” and “patient” are used interchangeably herein.
The method for treating a condition or a viral infection includes administering a therapeutically effective amount of a therapeutic agent or a pharmaceutical composition. An “effective amount,” “therapeutically effective amount” or “effective dose” is an amount of a composition (e.g., a therapeutic agent or a pharmaceutical composition) that produces a desired therapeutic effect in a subject, such as preventing or treating a target disease or condition, or alleviating symptoms associated with the disease or condition. The precise therapeutically effective amount is an amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic agent (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, namely by monitoring a subject's response to administration of a compound and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy 21^stEdition, Univ. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, Pa., 2005.
The pharmaceutical composition may include, among other things, one or more EBV antibodies disclosed herein or one or more immunogenic fragments thereof, one or more immunogenic peptides disclosed herein, or an EBV antibody-small molecule conjugate disclosed herein.
The pharmaceutical composition may also include one or more pharmaceutically acceptable carriers. A “pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or some combination thereof. Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation. It also must be suitable for contact with any tissue, organ, or portion of the body that it may encounter, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.
The pharmaceutical compositions described herein may be administered by any suitable route of administration. A route of administration may refer to any administration pathway known in the art, including but not limited to aerosol, enteral, nasal, ophthalmic, oral, parenteral, rectal, transdermal (e.g., topical cream or ointment, patch), or vaginal. “Transdermal” administration may be accomplished using a topical cream or ointment or by means of a transdermal patch. “Parenteral” refers to a route of administration that is generally associated with injection, including infraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. In some embodiments, the therapeutic compositions described herein are administered by intravenous injection or intraperitoneal injection.
In certain embodiments, disclosed herein is a method of treating or preventing EBV infection in a subject, comprising administering a therapeutically effective amount of one or more anti-gp350 antibodies disclosed herein or one or more immunogenic fragments thereof, one or more immunogenic peptides described herein, an EBV antibody-drug conjugate described herein, or a pharmaceutical composition comprising the anti-gp350 antibody or an immunogenic fragment thereof, one or more immunogenic peptides, or the EBV antibody-drug conjugate.
In certain embodiments, disclosed herein is a method of treating or preventing EBV infection in a subject, comprising administering a therapeutically effective amount of an anti-gp350 antibody disclosed herein or an immunogenic fragment thereof, an immunogenic peptide described herein, an EBV antibody-drug conjugate described herein, or a pharmaceutical composition comprising the anti-gp350 antibody or an immunogenic fragment thereof, one or more immunogenic peptides, or the EBV antibody-drug conjugate.
In certain embodiments, disclosed herein is a method of treating or preventing EBV-associated PTLD in a subject, comprising administering a therapeutically effective amount of an anti-gp350 antibody disclosed herein or an immunogenic fragment thereof, one or more immunogenic peptides described herein, an EBV antibody-drug conjugate described herein, or a pharmaceutical composition comprising the anti-gp350 antibody or an immunogenic fragment thereof, one or more immunogenic peptides, or the EBV antibody-drug conjugate, before or after the transplant in the subject.
As shown in the working examples, 15 novel EBV gp350-specific mAbs were generated, their binding to gp350 was characterized, their neutralization activity against EBV infection in vitro was determined, and their epitopes were mapped. The newly developed mAbs have many uses in vaccine development, diagnosis of viral infection, and therapeutic/prophylactic management of post-transplant lymphoproliferative diseases, either individually, in combination with nAb-72A1, or with other mAbs such as anti-gH/gL (E1D1).
To overcome the existing challenges facing PTLD treatment, novel EBV antibodies and EBV antibody-drug conjugates (ADCs) are developed. The EBV neutralizing antibodies (nAbs) that specifically block new or reactivated EBV infection are conjugated with small molecules that specifically target latent viral protein, EBV nuclear antigen 1 expressed in all EBV+ malignancies. The recent identification and isolation of nAbs against the highly variable viruses HIV-1 (10, 11), influenza (12-14), and respiratory syncytial virus (15) has direct implications for successful EBV protection. Indeed, in 2012, an international, multidisciplinary expert panel recommended use of intravenous (IV) anti-viral nAbs for preventing or treating EBV+ PTLD (16). EBV uses multiple surface glycoproteins (gps), including the major gp350, to infect host cells (17, 18). These gps are expressed on EBV virions and in EBV+ cells (19, 20), and stimulate immune responses in humans and in animal models (21-23), making them attractive targets for an EBV vaccine (24). Multiple lines of evidence suggest that use of anti-gp350 nAbs to protect against EBV-PTLDs is feasible (16): (A) Maternal Abs protect against EBV infection in neonates (25, 26); (B) gp350-expressing EBV+ cells activate complement (27) and mediate Ab-dependent cellular cytotoxicity (28); (C) gp350 vaccines reduce EBV load and protect against EBV+ lymphomas in marmosets (29-32) and protect EBV-naïve adults from EBV-induced mononucleosis (32-34); (D) Compared to control mice, SCID mice injected with peripheral blood mononuclear cells from EBV-naïve donors and immunized with anti-gp350 (72A1) mouse nAb are completely protected against EBV and development of EBV+ tumors or PTLD-like lesions (35); and (E) 72A1 also conferred short-term protection against acquiring EBV after transplantation in 3 out of 4 pediatric patients in a small phase 1 clinical trial (35). However, there was a major drawback: all 4 patients who received 72A1 developed human anti-mouse Abs (HAMA), which can cause side effects and limit treatment efficacy, with one developing a hypersensitivity reaction. This suggests that 72A1 in its native form is not a safe treatment for humans (35). Thus, chimeric (human/mouse), humanized, or human nAbs, which are safe and effective in the treatment of various cancers, are needed (7, 36-38).
Pre-existing antibodies provide the primary defense against viral infection. Prophylactic prevention of EBV primary infection has mainly focused on blocking the first step of viral entry by generating neutralizing antibodies (nAbs) that target EBV envelope glycoproteins. Five glycoproteins, in particular, gp350/220 (gp350), gp42, gH, gL, and gB, are required for efficient infection of permissible host cells and have emerged as potential prophylactic targets (23, 24, 61, 64).
Several studies have indicated that the EBV gp350 as the major immunodominant glycoprotein is an ideal target for EBV nAbs production. Although the ectodomain of EBV gp350 (AA 1-841) has been shown to contains at least seven unique CD21 binding epitopes located in the ectodomain of gp350 (58), at least one of these epitopes (AA 142-161) is capable of eliciting nAbs (57-58). The AA residues 142-161 are also one of the binding epitopes for nAb 72A1 (59, 68). The AA residues that constitute the other epitopes and their role in generating nAb has not been elucidated, as this information would be valuable in the precise design of effective EBV peptide vaccine. To date, nAb-72A1 remains the only EBV antibody with proven clinical prophylactic efficacy, as its been shown to confer short-term protection by reducing and delaying EBV infection onset in immunized pediatric transplant patients (35).
EBV predominantly infects epithelial cells and B cells, reflecting the viral tropism and the cellular ontogeny for EBV-associated malignancies (17). There are two schools of thought on how the initial EBV transmission into the human host occurs. In the first infection model, the incoming virus engages with ephrin receptor A2 via heterodimeric gH/gL, which triggers gB fusion with the epithelial cell membrane and entry of the virus into the cytoplasm (17). This interaction is thought to occur in the oral mucosa, where the virus undergoes lytic replication to release virions that subsequently infect B cells. In the alternative model, the incoming virus binds to the host cell via complement receptor type 1 (CR1)/CD35 (45) and/or CR2/CD21 through its major immunodominant glycoprotein, gp350 (65). The interaction between gp350 and CD35 and/or CD21 triggers viral adsorption, capping, and endocytosis into the B cell (66), which subsequently leads to interaction between heterotrimeric viral glycoproteins complex, gp42/gH/gL, binding to HLA class II molecules to activate gB membrane fusion and entry. Because these two models are not necessarily mutually exclusive, and given that both gp350 and gH/gL complex are important in initiating the first viral contact with host cells, use of nAbs that target either gp350 or gH/gL complex or both may potently block incoming virus at the oral mucosa.
Nearly all EBV-infected individuals develop nAbs directed to the ectodomains of these glycoproteins (52, 67). These antibodies can prevent neonatal infection, and can protect against acute infectious mononucleosis in adolescents and several human lymphoid and epithelial malignancies associated with EBV infection (30, 32-34). Although numerous monoclonal antibodies (mAbs) have been generated against EBV gp350 (53, 68, 69), only two murine mAbs, the non-neutralizing 2L10 and the neutralizing 72A1, have been extensively characterized and made commercially available (68, 69). Importantly, nAb-72A1 conferred short-term clinical protection against EBV transmission after transplantation in pediatric patients in a small phase I clinical trial (35).
EBV gp350 is the most immunogenic envelope glycoproteins on the virions. It is a type 1 membrane protein that encodes for 907 amino acid residues. A single splice of the primary transcript deletes 197 codons between codons 501 and 699, and joins two fragments of gp350 codons in frame to generate the gp220 messenger RNA. Both gp350 and gp220 are comprised of the same 18-AA residue at the C terminus that is located within the viral membrane, a 25-AA residue at the transmembrane-spanning domain, and a large highly glycosylated N-terminal ectodomain, AA 1-841 (57). The first 470 AA of gp350 are sufficient for binding CD21 in B cells, as demonstrated by a truncated gp350 (AA 1-470) blocking the binding of EBV to B cells and reducing viral infectivity (17). The gp350-binding domain on CD21 is mapped to N-terminal short consensus repeats (SCRs) 1 and 2, which also bind to a bioactive fragment of complement protein 3 (C3d) (34, 68). A soluble truncated EBV gp350 fragment (AA 1-470) and soluble CD21 SCR1 and SCR2 can block EBV infection and immortalization of primary B cells (57). However, gp350 binding to CD35 is not restricted to N-terminal SCRs; it binds long homologous repeat regions as well as SCRs 29-30 (57).
The gp350 ectodomain is heavily glycosylated, with both N- and O-linked sugars, which accounts for over half of the molecular mass of the protein. Currently there is only one crystal structure available for gp350, comprised of a truncated structure between 4-443 AA, with at least 14 glycosylated asparagine residues coating the protein with sugars, with the exception of a single glycan-free patch (59). Mutational studies of several residues in the glycan-free patch resulted in the loss of CD21 binding (59), suggesting that binding of CD35 and CD21 by gp350 is mediated within this region.
Using gp350 synthetic peptides binding to CD21 on the surface of a B cell line, additional gp350 epitope was identified in the C-terminal region of gp350 (AA 822-841), suggesting that this region is involved in EBV infection of B cells (58). The role of other epitopes in eliciting nAbs has not been fully investigated. Furthermore, the exact AA residues that comprise the core binding epitopes capable of eliciting neutralization and non-neutralization antibodies have not been determined. Mapping the EBV gp350 protein residues defining immunodominant epitopes, identifying the critical AA residues of the epitopes, and defining their roles in generating nAbs and non-nAbs will guide rational design and construction of an efficacious EBV gp350-based vaccine that would focus the B-cell responses to the protective epitopes.
As demonstrated in the working examples, 23 hybridomas producing antibodies against EBV gp350 were generated. To assess their clinical and diagnostic potential and utility in informing future prophylactic and therapeutic vaccine design: (1) the ability of the antibodies produced by the new hybridomas to detect gp350 protein was tested by enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunoblot; (2) the unique CDRs of the heavy and light chains of all 23 hybridomas were sequenced to identify novel mAbs; (3) the efficacy of each mAb to neutralize EBV infection in vitro was measured; and (4) competitive cell and/or linear peptide binding assays were used to identify gp350 regions recognized by neutralizing and non-neutralizing mAbs; and (5) peptide binding assays were used to identify gp350 core linear AA residues recognized by neutralizing and non-neutralizing mAbs.
Out of the 23 hybridomas, 15 were monoclonal and novel, based on their VH and VL CDR sequences, compared to the reported sequence of m72A1 (50). Following confirmation that the new 15 mAbs recognized gp350 antigen and contained unique VH-VL sequences, further characterization revealed that mAbs HB1, HB5, HB11, and HB20 inhibited EBV infection of a human B-cell line in a dose-dependent manner, with HB5 being the best neutralizer, comparable to m72A1 and h72A1. Thus, provided herein are four new nAbs against EBV infection of B cells with potential clinical utility in blocking viral infection in immunosuppression settings. The amino acid sequences of the heavy chain and light chain of the mAbs are provided in Table 2 below.

TABLE 2

Sequences of Heavy Chain and Light Chain

	Heavy Chain	Light Chain

72A1	PELVKPGTSMKISCKASGSSFTDY	QAVLTQESALTTSPGETVTLTCRSST
	TMNWMKQSHGKNLEWIGLINPYN	GAVTTSNYANWVQEKPDHLFTGLIG
	GGTRYNQKFKGKATLTLDKSSSTA	GTNNRVPGVPARFSGSLIGDKAALTI
	YMEVLSLTSEDSAVYYCAGGLRRV	TGAQTEDEAIYFCVLWHSNHWVFGG
	NWFAYWGQGTLVSVSAAKTTPPS	GTKLTVL (SEQ ID NO: 101)
	VYPLAPGSAAQTNSMVTLG (SEQ
	ID NO: 100)

HB1	PGLVAPSQSLSITCTVSGFLLTTYG	KFMSTSVGDRVSVTCKASQNVGTNV
	VHWVRQPPGKGLEWLGVIWAGGS	AWYQQKPGQSPKALIYSTSSRYTGV
	TNYNSALMSRLSINKDISKSQVFLK	PDRFAGSGSGTDYTLTISNVQSEDLA
	MNSLQTDDTAMYYCTRDRGYGYL	EYFCQQYNTYPYTFGGGTRLDIKRAD
	YAMDYWGQGTSVTVSSAKTTPPS	AAPTV (SEQ ID NO: 103)
	VYPLAPGSAAQTNSMVTLG (SEQ
	ID NO: 102)

HB2	PELKKPGETVKISCKASGYTFTAYS	AILSASPGEKVTMTCRATSSVNYMH
	MHWVKLTPGKGLKWMGWINTKTG	WYQQKPGSSPKPWIYATSNLASGVP
	EPTYADDFKGRFAFSLETSASTAY	ARFSGSGSGTSYSLTISRVEAEDAAT
	LQINNLKNEDTATYFCAPYGYALDY	YYCQQWSSNPPTFGAGTKLELKRAD
	WGQGTSVTVSSAKTTPPSVYPLAP	AAPTV (SEQ ID NO: 105)
	GSAAQTNSMVTLG (SEQ ID
	NO: 104)

HB3	AELVRPGASVKLSCKASGYTFASY	SSLSASLGDRVTISCRASQDIGNYLN
	WMQWVKQWPGQGLEWIGEINPN	WYQQKPDGTIKLLIYYTSRLHSGVPS
	NGHTNYNERFKNKASLTVDKSSST	RFSGSGSGTDYSLTISNLEEEDIATYF
	AYMQLSSLTSEDSAVYYCARNLYY	CQQGNTLPPTFGGGTKLEIKRADAAP
	YGRPDYWGQGTSVTVSSAKTTAP	TV (SEQ ID NO: 107)
	SVYPLAPVCGDTTGSSVTLG (SEQ
	ID NO: 106)

HB5	AELVRPGASVKISCKAFGYTFTNH	AILSVSPGERVSFSCRASQSIGTSIHW
	NINWVKQRPGQGLDWIGYINPYND	YQQRTNDSPRLLIKYASESISGIPPRF
	YTSYNQKFKGKATLTVDKSSNTAY	SGSGSGTDFTLSINSVESEDIADYHC
	MELSSLTSEDSAVYYCARSEGWL	QQSNSWPMLTFGAGTKLELKRADAA
	RRGAWFAYWGQGTLVTVSAAKTT	PTV (SEQ ID NO: 109)
	APSVYPLAPVCGDTTGSSVTLG
	(SEQ ID NO: 108)

HB6	PELRKPGETVKISCKASGYTFTDYS	AILSASPGEKVTMTCRATSSVNYMH
	MHWVKQTPGKGLKWMGWINTRT	WYQQKPGSSPKPWIYATSNLASGVP
	GEPRYADDFKGRFAFSLETSASTA	ARFSGSGSGTSYSLTISRVEAEDAAT
	YLQINNLKNEDTATYFCAPYGYALD	YYCQQWSSNPPTFGAGTKLELKRAD
	YWGQGTSVTVSSAKTTPPSVYPLA	AAPTV (SEQ ID NO: 111)
	PGSAAQTNSMVTLG (SEQ ID
	NO: 110)

HB7	AELVRPGASVKLSCKALGYTFTDY	KFMSTSVGDRVNITCKASQSVGNAV
	EMHWVKQTPVHGLEWIGTISPGRS	AWFQQKPGQSPKLLIYSASNRYTGIP
	GTAYNQKFKGKATLTADKSSRTAY	DRFTGSGSGTDFTLTCNNMQSEDLA
	MELNSLTSEDSAVYYCSRYGHPSY	DYFCQQYSSYPLTFGAGTKLELKRAD
	LDVWGAGTTVTVSSAKTTPPSVYP	AAPTV (SEQ ID NO: 113)
	LAPGSAAQTNSMVTLG (SEQ ID
	NO: 112)

HB8	PELKKPGETVKISCKASGYSFTNY	LSLPVSLGDQASISCRSSQSIVHSNG
	GMNWVKQAPGKGLKWMGWINTY	NTYLEWYLQKAGQSPKLLIYKVSNRF
	TGEPTYADDFKGRFAFSLETSAST	SGVPDRFGGSGSGTDFTLKISRVEAE
	AFLQINNLKNEDTATYLCARYYYGS	DLGVYYCFQGSHVPYTFGGGTKLEIK
	VYSAWFAYWGQGTLVTVSAAKTT	RADAAPTV (SEQ ID NO: 115)
	PPSVYPLAPGSAAQTNSMVTLG
	(SEQ ID NO: 114)

HB9	GGLVKPGGSLKLSCAASGFTFSSY	LSLPVSLGDQASISCRSSQSIVHSNG
	TMSWVRQTPEKRLEWVATISSGG	NTYLEWYLQKAGQSPKLLIYKVSNRF
	SYIYYPDSVKGRFTISRDNAKNTLY	SGVPDRFGGSGSGTDFTLKISRVEAE
	LQMSSLKSEDTAIYYCTREDFYYG	DLGVYYCFQGSHVPYTFGGGTKLEIK
	SSYGFFDVWGAGTTVTVSSAKTTA	RADAAPTV (SEQ ID NO: 117)
	PSVYPLAPVCGDTTGSSVTLG
	(SEQ ID NO: 116)

HB10	AELVRPGASVKLSCKASGYTFTSY	SSLSASLGDRVTISCRASQDIGNYLN
	WMHWVKQWPGQGPEWIGEINPS	WYQQKPDGTVKLLIYYTSRLHSGVPS
	NGHTNYNERFKNKATLTVDKSSST	RFSGSGSGTDYSLTISNLEEEDIATYF
	AYMQLSSLTSEDSAVYYCARNLYY	CQQGNTLPPTFGGGTKLEIKRADAAP
	YGRPDYWGQGTSVTVSSAKTTPP	TV (SEQ ID NO: 119)
	SVYPLAPGSAAQTNSMVTLG (SEQ
	ID NO: 118)

HB11	PSLVKPSQTLSLTCSVTGDSITSGF	AIMSASLGEKVTMSCRASSSVNFMN
	WNWIRKFPGNKLEYMGYISYSGST	WYQQKSDDSPKLLIYYISNLAPGVPA
	YYNPSLKSRISITRDTSKNQYYLQL	RFSGSGSGNSYSLTISGMEGEDAAT
	NSVTTEDTATYYCARGNGGNYDW	YYCQQFTSSPSVVTFGGGTKLEIKRA
	YFDVWGAGTTVTVSSAKTTPPSVY	DAAPTV (SEQ ID NO: 121)
	PLAPGSAAQTNSMVTLG (SEQ ID
	NO: 120)

HB12	AELVRPGASVKLSCKASGYTFTNY	LSLPVSLGDQASISCRSSQSLVHSNG
	WIH2VKQWPGQGLEWIGEINPNN	NTYLHWYLQKPGQSPKLLIYKVSNRF
	GHTNYNERFKNKASLTVDKSSSTA	SGVPDRFSGSGSGTDFTLKISRVEAE
	YMQLSSLTSEDSAVYYCARNLYYY	DLGVYFCSQSTHVPLTFGSGTKLEIK
	GRPDYWGQGTSVTVSS (SEQ ID	(SEQ ID NO: 123)
	NO: 122)

HB14	SGAELVRPGASVNLSCKALGYTFT	LSLPVSLGDQASISCRSSQSIVHDNG
	DYEMHWVKQTPVYGLEWIGTIHPR	NTYLEWYLQKPGQSPKLLIYKVSNRF
	RGGTAYNQRFKGKAALTADKSSST	SGVLDKFSGSGSGTDFTLKISRVEAE
	AYMELSSLTSEDSAVYYCARYGYP	DLGIYYCFQGSHVPPTFGGGTKLEIK
	WYFDVWGAGTTVTVSS (SEQ ID	(SEQ ID NO: 125)
	NO: 124)

HB17	AELVIPGASVKVSCKASGYTFTSY	SSLSASLGDRVTISCRASQDIGNYLN
	WIHWVKQWPGQGLEWIGEINPNN	WYQQKPDGTIKLLIYYTSRLHSGVPS
	GHTNYNEKFKSKATLTVDKSSSTA	RFSGSGSGTDYSLTISNLEEEDIATYF
	YMQLSSLTSEDSAVYYCARNLFYY	CQQGNTLPPTFGGGTKLEIKRADAAP
	SRPDYWGQGTSVTVSSAKTTPPS	TV (SEQ ID NO: 127)
	VYPLAPGCGDTTGSSVTLG (SEQ
	ID NO: 126)

HB20	AELVKPGASVKLSCKASGYTFTSY	SSLSASLGDRVTISCRASQDIGNYLN
	WIQWVKQRPGQGLEWIGEINPTN	WYQQKPDGTVKLLIYYTSRLHSGVPS
	GHTNYNEKFKTKATLTVDKSSSTA	RFSGSGSGTDYSLTISNLEQEDIATYF
	YMRLSSLTSEDSAVYYCARNLYYY	CQQGNALPPTFGGGTKLEIKRADAAP
	GRPDYWGQGTSVTVSSAKTTAPS	TV (SEQ ID NO: 129)
	VYPLAPVCGDTTGSSVTLG (SEQ
	ID NO: 128)

HB22	PGLVAPSQSLSITCTVSGFSLTNYG	LSLPVSLGDQASISCRSSQSIVHSNG
	IHWVRQPPGKGLEWLVVIWSDGS	NTYLEWYLQKPGQSPKLLIYKVSNRF
	TIYNSALKSRLSISKDNSKSQVFLK	SGVPDRFSGSGSGTDFTLRISRVEAE
	MNSLQTDDTAMYYCARNYYGNSY	DLGVYYCFQGSHVPWTFGGGTKLEI
	PAWFAYWGQGTLVTVSAAKTTPP	KRADAAPTV (SEQ ID NO: 131)
	SVYPLAPGSAAQTNSMVTLG (SEQ
	ID NO: 130)

The identification of four new potent nAbs (HB1, HB5, HB11 and HB20) and sequencing of their CDR regions, as well as the humanization of m72A1 and E1D1, opens the possibility of using these nAbs for clinical applications, such as reducing or preventing EBV infection in transplant settings, with the consequent potential to reduce the incidence of EBV+ PTLDs. The h72A1 IgG1 antibody recognized both native gp350 as well as gp350 peptides that constitute the principal gp350 neutralizing epitope (142-161) and completely eliminated anti-murine IgG immunoreactivity. Importantly, h72A1—and the four newly generated nAbs (HB1, HB5, HB11, and HB20)—significantly blocked in vitro EBV infection of B cells to a degree comparable to or better than m72A1. Combining two or more nAbs that bind to different peptides on gp350 and gH/gL can significantly reduce infection in both B cells and epithelial cells.
In addition, both nAbs and non-nAbs can be used as research tools to provide insight into epitope targets important for vaccine development. In the past, various methods, including lectin/ricin immune-affinity assay, purified mAbs, purified soluble gp350 mutants, synthetic peptides, cell binding assays, and X-ray crystallography of partial gp350 protein (AA 4-443), have been used to identify the critical gp350 epitopes responsible for its interaction with the CD21 and CD35 cellular receptors (summarized in Table 3). Despite several attempts to identify gp350 epitopes important for eliciting nAbs, to date only a single epitope, AA 142-161, has been identified, which is also the binding epitope for nAb 72A1. Currently, the lack of a crystal structure of full-length gp350 protein and the unavailability of multiple nAbs hinder the opportunity to identify other gp350 epitopes that might elicit nAbs and inform design of effective vaccine strategies. To identify gp350 epitopes responsible for eliciting nAbs, the newly generated nAbs (HB1, HB5, HB11, and HB20) and non-nAbs (HB10, HB17, and HB22) were used to perform competitive cell binding and ELISA-based linear peptide binding assays. Although both approaches have various limitations, they offer useful information that when combined might inform and/or advance vaccine development efforts. Competitive cell binding assays can provide information on whether two antibodies bind overlapping or non-overlapping epitopes, although they are unable to indicate whether the competing antibodies bind the same or nearby epitopes, nor identify actual AA residues involved in the binding. On the other hand, the ELISA peptide binding assay is only reactive to linear epitopes and may or may not take into consideration post-translational protein modifications, depending on whether a full protein or peptides are used as the target antigen(s).
Using biotinylated antibodies, it was shown that the newly generated gp350 nAbs (HB1, HB5, HB11, and HB20) bound targets that overlapped with those of both m72A1 and h72A1, although HB20 showed only partial binding to the overlapping targets. The non-Ab, HB17 showed little to no competitive binding when compared to nAbs, suggesting that they bound different gp350 epitopes. These results strongly suggest that two distinct binding regions have been identified, one bound predominantly by nAbs and the other by non-nAbs, and that nAbs potentially bind targets within the same proximity, if not the same AA sequences. Thus, the current antibodies provide the first step toward generating reagents required for mapping neutralizing versus non-neutralizing epitopes on gp350, should the full-length crystal structure of the protein remain unavailable. Using linear peptide epitope mapping, three major mAb-binding regions, 1-101, 102-201, and 402-501 were identified; all three regions incorporable previously identified linear epitopes (58, 51, 56). Regions 1-101 and 402-501 were bound by both nAbs and non-nAbs, suggesting that these regions are immunodominant. However, the 102-201 region containing the nAb epitope 142-161 was only bound by nAbs (HB5, HB11, HB20, and both m72A1 and h72A1), with the exception of the nAb HB1. These results suggest that epitopes/regions capable of eliciting nAbs are located within the N-terminus of gp350.
Previous studies have generated and characterized several anti-gp350 mAbs, both neutralizing and non-neutralizing. Some of these have been effectively used to map the immunodominant or neutralizing epitopes present in the gp350 ectodomain, which has relevance for future strategies to design sterilizing prophylactic vaccines (Table 3).

TABLE 3

Summary of published gp350 epitope mapping using various methodologies

Method	mAbs/protein/peptides	Number of epitopes identified	Reference

Competitive binding assay	mAbs		7 epitopes - Sequence not	(53)
		defined
Binding studies:	mAbs	2 possible regions identified	(54)
Determine the effects of		by sequence alignment to C3d
anti-gp350 mAbs on gp350		sequence:
binding to CR2		1. aa 21-28
		2. aa 372-378
Peptide digest and	Truncated and mutant	Narrowed down to the first	(57)
immunoprecipitation	protein; mAbs (72A1 and	470 residues
	BOS-1)
Binding studies	Peptide and protein	2 sequences defined:	(55)
		1. aa 21-28
		2. N-terminus of gp350
Dot Blot immunoassay:	Protein - 8 clones overlapping	3 sequences defined:	(56)
Purified truncated protein	N- and C- terminal portions	1. aa 310-325
incubated with mAbs	of protein;	2. aa 326-439
	mAbs from Qualtiere et al.,	3. aa 733-841
	1987
Peptide cell binding assay	Synthesized peptides	7 regions, 3 identified:	(58)
to 2 CR2-positive (Raji and	covering gp350 (907 aa)	1. aa 142-161
Ramos) and 1 -negative		2. aa 282-301
(P3HR-1) cell lines		3. aa 822-841
Crystal structure and	Mutant proteins; mAbs 72A1	3 epitopes (based on 72A1	(59)
binding studies		binding and gp350 4-443)
		1. aa 16-29
		2. aa 142-161
		3. aa 282-301
Structural docking studies	gp350 and CR2 crystal	Single epitope (based on	(60)
and antigenicity mapping	structure alignment/docking	gp350 aa 1-470)
		1. aa 147-165
Structural alignment:	Peptides (used in	4 epitopes: identified	(51)
computer modeling of	immunization); mAb (72A1)	1. aa 14-20
gp350 and 72A1 and		2. aa 144-161
docking studies		3. aa 194-211
		4. aa 288-291

Virus-specific treatments are less likely to target basic metabolic mechanisms of healthy cells, making them more likely to efficiently kill virus-infected cells with fewer side effects. Until recently, few drug regimens have specifically targeted EBV+ lymphomas. However, in 2015, a few small molecules showed activity against EBV-transformed cells (39). Furthermore, in 2017, Jiang et al. described a novel small molecule (L₂P₄) that shows discriminating anti-proliferative activities against EBV-transformed B lymphoma cells (40).
Having described the invention with reference to the embodiments and illustrative examples, those in the art may appreciate modifications to the invention as described and illustrated that do not depart from the spirit and scope of the invention as disclosed in the specification. The examples are set forth to aid in understanding the invention but are not intended to, and should not be construed to limit its scope in any way. The examples do not include detailed descriptions of conventional methods. Such methods are well known to those of ordinary skill in the art and are described in numerous publications. Further, all references cited above and in the examples below are hereby incorporated by reference in their entirety, as if fully set forth herein.

EXAMPLES

Example 1: Materials and Methods

Cells and viruses. AGS-EBV-eGFP, a human gastric carcinoma cell line infected with a recombinant Akata virus expressing enhanced fluorescent green protein (eGFP) was a kind gift of Dr. Lisa Selin (University of Massachusetts Medical School). Anti-EBV gH/gL (E1D1) hybridoma cell line was a kind gift of Dr. Lindsey Hutt-Fletcher (Louisiana State University Health Sciences Center). Chinese hamster ovary cells (CHO); human embryonic kidney cells expressing SV-40 T antigen (HEK-293T); HEK-293 6E suspension cells; EBV-positive Burkitt lymphoma cells (Raji); myeloma cells (P3X63Ag8.653); and anti-EBV gp350 nAb-72A1 hybridoma cells (HB168) were purchased from American Type Culture Collection (ATCC). ExpiCHO cells were purchased from ThermoFisher Scientific.
AGS-EBV-eGFP cells were maintained in Ham's F-12 media supplemented with 500 μg/ml neomycin (G418, Gibco). Raji, P3X63Ag8.653, and HB168 hybridoma cells were maintained in RPMI 1640. CHO and HEK-293T cells were maintained in DMEM. HEK-293 6E cells and ExpiCHO cells were maintained in FreeStyle F17 Expression Medium supplemented with 0.1% Pluronic F-68 and Gibco ExpiCHO Expression Media, respectively. All culture media were supplemented with 10% fetal bovine serum (FBS) from Millipore Sigma, 2% penicillin-streptomycin, and 1% I-glutamine, with the exception of Freestyle F17 expression medium and Gibco ExpiCHO Expression Media. All media were purchased from ThermoFisher Scientific unless otherwise specified.
Antibodies and plasmids. Primary antibodies: EBV anti-gp350 nAb (m72A1) and anti-gH/gL (E1D1) were purified from the supernatant of HB168 and E1D1 hybridoma cell lines, respectively, using Capturem™ Protein A Maxiprep spin columns (Takara) or protein G affinity and size-exclusion chromatography. The non-nAb anti-gp350/220 mAb (2L10) was purchased from Millipore Sigma. Anti-KSHV gH/gL 54A1 mAb was generated and characterized as previously disclosed (79).
Secondary antibodies: Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG for immunoblot or ELISA were purchased from Bio-Rad. HRP-conjugated goat anti-human IgG for ELISA was purchased from ThermoFisher Scientific. Alexa Fluor (AF) 488-conjugated goat anti-mouse IgG (H+L) for flow cytometry was purchased from Life Sciences Tech. Goat anti-mouse IgG (H+L) secondary antibody and DyLight 650 for epitope mapping were purchased from Thermo Fisher Scientific. Anti-biotin monoclonal antibody conjugated to AF488 for competitive binding assay was purchased from ThermoFisher Scientific.
The construction of the pCI-puro vector and pCAGGS-gp350/220-F has been described (23, 45).
Virus production and purification. eGFP-tagged EBV was produced from the EBV-infected AGS cell line as described (46). Briefly, AGS-EBV-eGFP cells were seeded to 90% confluency in T-75 flasks in Ham's F-12 medium containing G418 antibiotic. After the cells reached confluency, G418 media was replaced with Ham's F-12 medium containing 33 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA) and 3 mM sodium butyrate (NaB) to induce lytic replication of the virus. Twenty-four hours post-induction, the media was replaced with complete Ham's F-12 media without G418, TPA, or NaB and cells were incubated for 4 days at 37° C. in a 50% CO₂incubator. The cell supernatant was collected, centrifuged, and filtered using 0.8 μm filter to remove cell debris. The filtered supernatant was ultra-centrifuged using a Beckman-Coulter type 19 rotor for 70 min at 10,000 rpm to pellet the virus. EBV-eGFP virus was titrated in both HEK-293T cells and Raji cells, and stocks were stored at −80° C. for subsequent experiments.
Generation and purification of gp350 virus-like particles. To generate gp350 VLPs, equal amounts (8 μg/plasmid) of the relevant plasmids (pCAGGS-Newcastle disease virus (NDV) matrix (M), and nucleocapsid proteins (NP), and gp350 ectodomain fused to NDV fusion (F) protein cytoplasmic and transmembrane domains) were co-transfected into 80% confluent CHO cells seeded in T-175 cm²flasks; supernatant from transfected cells containing VLPs was collected and VLPs were purified and composition characterized as previously described (47).
Production of hybridoma cell lines. Seven days prior to immunization, two eight-week-old BALB/c mice were bled for collection of pre-immune serum. The mice were immunized with purified UV-inactivated EBV three times (Day 0, 21, and 35), and then boosted every 7 days three times (Day 42, 49, and 56) with VLPs incorporating gp350 on the surface after Day 35. The mice were sacrificed, and their splenocytes were isolated, purified, and used to fuse with P3X63Ag8.653 myeloma cells at a ratio of 3:1 in the presence of polyethylene glycol (PEG, Sigma). Hybridoma cells were seeded in flat-bottom 96-well plates and selected in specialized hybridoma growth media with HAT (Sigma) and 10% FBS as previously described (80).
Indirect ELISA. Hybridoma cell culture supernatant from wells that had colony-forming cells were tested for antibody production by indirect ELISA. Briefly, immunoplates (Costar 3590; Corning Incorporated) were coated with 50 μl of 0.5 μg/ml recombinant EBV gp350 ectodomain (Immune Technology Corporation) diluted in 1× phosphate buffered saline (PBS, pH 7.4) and incubated overnight at 4° C. After washing three times with 1× PBS containing 0.05% (v/v) Tween 20 (washing buffer), plates were blocked with 100 μl washing buffer containing 2% (w/v) bovine serum albumin (BSA) (blocking buffer) then incubated for 1 h at room temperature and washed as above. 100 μl of hybridoma supernatant or 50 μl of 10 μg/ml purified mAbs was added to each well (in triplicate) and incubated for 2 h at room temperature. Anti-KSHV gH/gL 54A1 and m72A1 mAbs were added as negative and positive controls, respectively. The plates were washed as described above, followed by incubation with 50 μl of goat anti-mouse IgG HRP-conjugated secondary antibody (1:2,000 diluted in 1× PBS) at room temperature for 1 h. The plates were washed again and 100 μl of chromogenic substrate 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS, Life Science Technologies) was added. The reaction was stopped using 100 μl of ABTS peroxidase stop solution containing 5% sodium dodecyl sulfate (SDS) in water. The absorbance was read at an optical density of 405 nm using an ELISA reader (Molecular Devices). All experiments were performed in triplicate and confirmed in three independent experiments.
Antibody purification, quantification, and isotyping. Hybridoma cells from selected individual positive clones were expanded stepwise from 96-well plates to T-75 flasks. At confluence in T-75 flasks, supernatant from individual clones was collected, clarified by centrifugation (4,000 g, 10 min, 4° C.), and filtered through a 0.22-μm membrane filter (Millipore). Antibodies were further purified by Capturem™ Protein A Maxiprep (Takara) and stored in 1× PBS (pH 7.4) at 4° C. Alternatively, antibodies were purified using protein G affinity chromatography followed by size-exclusion chromatography at the Beckman Institute of City of Hope X-ray Crystallography Core facility. Antibodies were analyzed by SDS-PAGE to determine purity. Bicinchoninic acid assay (BCA assay; Thermo Fisher Scientific) was conducted to determine the concentration of purified antibodies. Isotype identification was performed with the Rapid ELISA mouse mAb isotyping kit (Thermo Fisher Scientific). Two independent experiments were performed.
RNA extraction, cDNA synthesis, and sequencing and analysis of the variable region of the mAbs. Total RNA was extracted from 1×10⁶hybridoma cells using the RNeasy Mini Kit (Qiagen). Each hybridoma clone cDNA was synthesized in a total volume of 20 μl using Tetro Reverse Transcriptase (200 u), RiboSafe RNase Inhibitor, Oligo(dT)18 primer, dNTP mix (10 mM each nucleotide), and 100-200 ng RNA. Reverse transcription was performed at 45° C. for 30 min, and terminated at 85° C. for 5 min. The cDNA was stored at −20° C. Immunoglobulin (Ig) VH and VL were amplified using the mouse Ig-specific primer set purchased from Novagen (48). The VH and VL genes were amplified in separate reactions and PCR products were visualized on 1% agarose gel.
The VH and VL amplicons were sequenced using an Illumina MiSeq platform: duplicate 50 μl PCR reactions were performed, each containing 50 ng of purified cDNA, 0.2 mM dNTPs, 1.5 mM MgCl2, 1.25 U Platinum Taq DNA polymerase, 2.5 μl of 10× PCR buffer, and 0.5 μM of each primer designed to amplify the VH and VL. The amplicons were purified using an AxyPrep Mag PCR Clean-up kit (Thermo Fisher Scientific). The Illumina primer PCR PE1.0 and index primers were used to allow multiplexing of samples. The library was quantified using ViiA™ 7 Real-Time PCR System (Life Technologies) and visualized for size validation on an Agilent 2100 Bioanalyzer (Agilent Technologies) using a high-sensitivity cDNA assay. The sequencing library pool was diluted to 4 nM and run on a MiSeq desktop sequencer (Illumina). The 600-cycle MiSeq Reagent Kit (Illumina) was used to run the 6 μM library with 20% PhiX (Illumina), and FASTQ files were used for data analysis (81). The determination of immunoglobulin families was analyzed using the IMGT/V-QUEST tool 210 (www.imgt.org/IMGT_vquest/vquest) (82).
Chimeric mAb construct generation. To generate chimeric mAbs, the VH and VL sequences were cloned into the dual-vector system such as pFUSE CHIg/pFUSE CLIg (InvivoGen), which express the constant region of the heavy and light chains of human immunoglobulins, respectively (Genewiz). The constructs were transiently transfected into HEK-293 6E cells. The supernatants were collected at 72 h post-transfection and IgG was purified using protein A/G affinity chromatography.
Humanization of 72A1. To generate humanized mAbs, the BioLuminate interface (Schrödinger) was used to identify the human VH and VL framework using 72A1. The resulting model was visually inspected to ensure appropriate packing at the base of the CDR. The sequence was meditope-enabled to add functionality for generating bispecific antibodies in the future (83). The resulting sequences were codon-optimized, synthesized, and cloned into the PD2610 vector (Atum). The constructs were transiently transfected into ExpiCHO cells following the manufacturer's protocol. Supernatant was collected at 10 days post-transfection and IgG was purified using protein G affinity chromatography, followed by size-exclusion chromatography.
Immunoblot analysis. CHO cells were cultured and stably co-transfected with full-length pCAGGS-gp350/220 and pCl-puro vector containing a puromycin resistance gene. Forty-eight hours post-transfection, DMEM media containing 10 μg/ml of puromycin was added to enrich for cells expressing gp350 protein. Puromycin-resistant clones were expanded, followed by flow cytometry sorting using m72A1 to a purity >90%. EBV gp350-positive CHO cells were harvested and lysed in radioimmunoprecipitation assay buffer (RIPA) followed by centrifugation at 15,000 g for 15 min on a benchtop centrifuge. The lysate was collected and heated at 95° C. for 10 min in SDS sample buffer containing β-mercaptoethanol, then separated using SDS-PAGE. Proteins were transferred onto a nitrocellulose membrane using an iBlot™ Transfer System (Thermo Fisher Scientific) followed by incubation in blocking buffer (1% BSA; 20 mM Tris-HCl, pH 7.5; 137 mM NaCl; and 0.1% Tween-20 [TBST]) for 1 hour. The blots were incubated in TBST containing purified anti-gp350 antibodies (1:50) overnight at 4° C. After three washes with TBST, the blots were incubated with HRP-conjugated goat anti-mouse (1:2000) in TBST for 1 hour. After three washes, the antibody-protein complexes were detected using the Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare). All experiments were independently repeated three times.
Flow cytometry. To assess the ability of purified anti-gp350 mAb to detect surface expression of EBV gp350 protein by flow cytometry, CHO cells that stably express EBV gp350 were harvested and stained with purified anti-gp350 (10 μg/ml), followed by AF488 goat anti-mouse IgG secondary antibody. Flow cytometric analysis was performed on a C-6 FC (BD Biosciences) and data was analyzed using FlowJo Cytometry Analysis software (FlowJo, LLC) as described (47). All the experiments were independently repeated three times.
EBV neutralization assay. EBV neutralization assay was performed in Raji cells as previously described (47). Briefly, purified individual anti-gp350 mAbs were incubated with purified AGS-EBV-eGFP (titer calculated to infect at least 8% of HEK293 cells seeded in 100 μl of serum-free DMEM) for 2 hours at 37° C. To represent EBV infection of B cells, the pre-incubated anti-gp350 mAbs/AGS-EBV-eGFP were used to infect 5×10⁵Raji cells seeded in a 96-well plate for 2 hours at 37° C. Neutralizing 72A1 and non-neutralizing 2L10 anti-gp350 mAbs served as positive and negative controls, respectively. Infected cells were washed three times with PBS to remove any unbound viruses and antibodies. Washed, infected cells were incubated in 96-well plates at 37° C. for 48 hours post-infection and the number of eGFP+ (infected) cells was determined using flow cytometry. All dilutions were performed in quintuplicate and the assays were repeated three times. Antibody EBV neutralization activity was calculated as: % neutralization=(EBValone−EBVmAb)/(EBValone)×100.
Epitope mapping by competitive cell binding assay. To evaluate conformation epitope mapping of the selected mAbs, competitive binding assays were conducted using biotinylated mAbs. A one-step antibody biotinylation kit (MACS Miltenyi Biotec) was used to biotinylate the mAbs. Approximately 1×10⁵CHO cells that stably expressed EBV gp350 were incubated for 2 hours with serially diluted (500, 250, 125, and 67.5 μg/ml) unlabeled competitor mAbs and non-specific anti-KSHV gH/gL 54A1 mAb. After being washed with PBS, the cells were incubated for 2 hours in the presence of 1 μg/ml biotinylated mAbs. To determine maximum binding, cells in which the biotinylated mAb was added in the absence of unlabeled mAbs were included in the assay. Cells were washed with PBS, followed by incubation for 1 hour with anti-biotin AF488 at a dilution of 1:500. After the final wash in PBS, cells were resuspended in 1 paraformaldehyde and analyzed by flow cytometry as described above. Percentage of inhibition was calculated as: 100−[(% fluorescent cells with competitor mAb/% fluorescent cells without competitor mAb)×100]. The complete prevention of binding of a biotinylated mAb by its unlabeled counterpart was used as a validation of the assay, as previously described (84).
Synthesis of 20-mer linear peptides of gp350 proteins. Forty-five sequential 20-mer linear peptides, covering the whole sequence of gp350 (GenBank: NC 007605.1), with an exception of aa 862-881, were synthesized using a solid phase method and cleaved using a low-high hydrogen fluoride method by the GenicBio, as previously described (58). Synthesis of aa 862-881 (pep-44) was not possible due to multiple hydrophobic aa.
Linear epitope mapping by peptide-mAb binding assay. The binding of anti-gp350 mAbs to 45 synthesized 20-mer sequential peptides covering the total length of gp350 was analyzed using indirect ELISA as described (58). Briefly, immunoplates were coated with 50 μl of 10 μg/ml EBV gp350 peptides (forty-five 20-mers) diluted in PBS and incubated overnight at 4° C.; 0.5 μg/ml recombinant EBV gp350 ectodomain protein was used as a positive control. After washing three times with washing buffer (PBS containing 0.05% (v/v) Tween 20), plates were blocked with 100 μl washing buffer containing 3% BSA (blocking buffer), incubated for 1 hour at room temperature, and washed as above. Ten μg/ml purified mAbs were added to each well in triplicate and incubated for 2 hours at room temperature. Anti-gp350 antibodies m72A1 and h72A1 were added as positive controls and anti-KSHV-gH/gL 54A1 mAb was used as negative control. The plates were washed as described above, followed by incubation with goat anti-mouse IgG or goat anti-human IgG HRP-conjugated secondary antibody (1:2,000 diluted in PBS) at room temperature for 1 hour. The plates were washed again and the chromogenic substrate ABTS was added. The reaction was stopped using ABTS peroxidase stop solution. The absorbance was read at an optical density of 405 nm using an ELISA plate reader.
Statistical Analysis. Unpaired Mann-Whitney U test was used to assess statistical differences between two independent groups. Statistical calculations were performed in Graphpad Prism. Data was considered statistically significant at p<0.05.

Example 2: Novel Anti-Gp350 mAbs Target Linear and Conformational Epitopes

New EBV gp350-specific mAbs were generated and biochemically characterized, and their ability to neutralize EBV infection was evaluated. In addition, the antibodies were used to map immunodominant epitopes on the EBV gp350 protein. 23 novel monoclonal antibodies specific against EBV gp350 were developed. To generate hybridomas, BALB/c mice were immunized three times with purified UV-inactivated EBV and boosted three times with virus-like particles (VLPs) that incorporate EBV gp350 ectodomain (1-841) on the surface to improve antibody affinity maturation and avidity. Then the splenocytes were isolated from the immunized mice and fused with myeloma cells to generate hybridomas. Specifically, five eight-week-old BALB/c mice were immunized with virus-like particles incorporating gp350/220 on the surface, four times (day 0, 14, 28, and 56) via intraperitoneal injection without adjuvants. At day 64, immunized mice were boosted once intravenously. Animals were sacrificed at Day 70 to harvest splenocytes for fusion with the mouse myeloma P3X63Ag8 cell line.
Indirect ELISA was used to screen supernatants from the hybridomas for specificity against purified EBV gp350 ectodomain protein (AA 4-863) and 23 hybridomas producing gp350 specific antibodies were identified. To further characterize the biochemical properties of the 23 antibodies generated, the antibodies were purified from the hybridoma supernatants using protein A spin columns, followed by SDS-PAGE to confirm the purity of all antibodies (FIG. 1A).
These novel antibodies were analyzed by flow cytometric analysis for surface expression of gp350 protein on 10⁶CHO cells transfected with 1 μg of pCAGGS-gp350. gp350 expressing cells were resuspended in PBS, stained with anti-gp350 mAb, which detects the gp350 ED, followed by secondary Ab goat anti-mouse conjugated to AF488. Additionally, western blot analysis was conducted on untransfected and pCAGGS-gp350 transfected CHO lysate. Anti-gp350 mAb 72A1 was used as a positive control (1:100) and anti-gp350 hybridoma clone supernatants were used at 1:50, and anti-mouse secondary antibody was used at 1:2000.
It was found that all 23 hybridoma producing antibodies, designated HB1-23, recognized the gp350 antigen in an initial ELISA screening using unfractionated and unpurified hybridoma supernatants (data not shown). When the quantified amount of the purified antibodies (10 μg/ml) was reevaluated using indirect ELISA, all of the 23 antibodies and m72A1 (anti-gp350 positive control) had ELISA signals greater than two times those of anti-KSHV gH/gL mAb 54A1 (negative control), and were considered positive/specific to gp350 (FIG. 1B). Of the 23 gp350-positive hybridoma producing antibodies identified, HB4, HB5, HB7, HB13, and HB14 demonstrated binding strength equal to or greater than that of the positive control, m72A1. This difference in binding of the antibodies could be due to differential exposure of cognate epitopes on gp350 in the assay performed.
Determining the nature of the binding between an antibody and its target antigen is an important consideration for the performance and specificity of an antibody, as it can involve the recognition of a linear or conformational epitope (49). The ability of the purified antibodies to bind linear epitopes was evaluated by performing immunoblot analysis of denatured gp350 antigen expressed on Chinese hamster ovary (CHO) cells, and 16 of the 23 antibodies detected both the 350 kDa and the 220 kDa splice variant. In contrast, HB2, HB3, HB6, HB7, HB13, HB20, and HB21, as well as the negative control 54A1, failed to recognize either of the denatured isoforms of gp350 (FIG. 1C). The antibodies' ability to bind conformational epitopes was further characterized by flow cytometric analysis of CHO cells stably expressing gp350 on the cell surface. HB1, HB2, HB3, HB5, HB6, HB9, HB11, HB12, HB15, HB17, HB19, HB20, HB21, and m72A1 antibodies readily recognized gp350 (FIGS. 1D and 1E). The fact that HB2, HB3, HB20, and HB21 detected gp350 by flow cytometry, but not by immunoblot, suggests that these four antibodies only recognized conformational epitopes (i.e., native) on gp350, whereas HB5, HB9, HB11, HB15, HB17, and HB19 recognized both linear and conformational epitopes (FIGS. 1C-1E). The observation that all 23 anti-gp350 antibodies recognized the gp350 antigen either by indirect ELISA, flow cytometry, or immunoblot assay suggests that antibodies that are specific to EBV gp350 protein were successfully produced. In addition, the isotypes of the newly generated antibodies were determined to be IgG1 (n=14), IgG2a (n=5), IgG2b (n=1), a mixture of IgG1 and IgG2b (n=1), and a mixture of IgG1 and IgM (n=2) (Table 4).

TABLE 4

Summarized biochemical and functional characterization
of anti-gp350 antibodies

			ELISA binding	Flow
Anti-	IgG	Light	to purified EBV	cytometry	Western
body	sub-class	chain	gp350/220	(CHO Cells)	blot

HB1	IgG1	κ	+	+	+
HB2	IgG2a	κ	+	+	−
HB3	IgG2a	κ	+	+	−
HB4	IgG1	κ	+	−	+
HB5	IgG2a	κ	+	+	+
HB6	IgG1	κ	+	+	−
HB7	IgG1	κ	+	−	−
HB8	IgG1	κ	+	−	+
HB9	IgG2a	κ	+	+	+
HB10	IgG1	κ	+	−	+
HB11	IgG1	κ	+	+	+
HB12	IgG1	κ	+	+	+
HB13	IgG1	κ	+	−	−
HB14	IgG1	κ	+	−	+
HB15	IgG1	κ	+	+	+
HB16	IgG1/IgM	κ	+	−	+
HB17	IgG2b	κ	+	+	+
HB19	IgG1/IgM	κ	+	+	+
HB20	IgG2a	κ	+	+	−
HB21	IgG1/IgG2b	κ	+	+	−
HB22	IgG1	κ	+	−	+
HB23	IgG1	κ	+	+	+
m72A1	IgG1	κ/λ	+	+	+

Example 3: Identification and Characterization of 15 Novel Anti-350 Monoclonal Antibodies

To determine whether the generated hybridomas were monoclonal or a mixture of antibodies, the VH and VL variable region genes of the 23 new anti-gp350 antibodies, as well as m72A1 (positive control), were sequenced using Illumina MiSeq. The sequence of the CDR of m72A1 antibody was recently determined and published (50, 51). PCR was used to amplify the genes encoding the VH and VL chain regions in cDNAs generated from the 23 hybridoma cells, as well as from m72A1. The PCR products presented distinct bands for VH (˜350-450 bp) and VL (˜450-500 bp). FIG. 2 is a representation of identified bands for only a few selected hybridomas which did not yield additional multiple non-specific bands from PCR amplifications that required extra-purification steps. The purified fragments were sequenced, followed by in silico analysis, and CDRs for both VH and VL were identified (Table 1). As previously reported, two unique IgG1 VH and two unique VL chains, one kappa and one lambda sequence of m72A1, were identified using the light chain kappa degenerative primers and specific primers for the lambda light chain (50). These sequences were >94% identical to the previously published sequences, suggesting that m72A1 exists as a mixture of antibodies, instead of the reported mAb (51). Similar to m72A1, HB4, HB13, HB15, and HB23 hybridomas each produced a mixture of two antibodies, with two unique sequences of the VH chain showing at >5% frequencies, suggesting that they are not mAbs (Table 5). Coding sequences for VL chains for HB7, HB9, and HB17 were unable to be identified, unless the frequencies were lowered to >1% (Table 5); in this case, the identified coding VL chain sequences were identical.

TABLE 5

Summary of Illumina Dual Demultiplex of V_Hand V_LRegions

								>5%
			PEAR	Length	Primer		>5%	Unique
		Starting	Merged	Filtered	Matched	3x	Unique	Non-
Sample	Chain	Pairs	Reads	Reads	Reads	Reads	Coding	Coding

HB1	HEAVY	51,641	51,210	46,655	32,482	22,725	1	0
	LIGHT	280,048	279,012	100,725	68,041	58,570	1	1
HB2	HEAVY	22,793	22,621	16,475	11,415	7,429	1	0
	LIGHT	167,230	166,496	161,764	132,752	115,886	1	1
HB3	HEAVY	26,382	26,162	25,542	16,910	11,709	1	0
	LIGHT	12,681	12,609	11,753	9,809	8,023	1	1
HB4	HEAVY	38,811	38,238	17,151	11,957	7,217	2	0	§
	LIGHT	179,249	129,752	111,996	78,392	66,419	1	1
HB.5	HEAVY	42,951	42,173	35,793	25,842	17,173	1	0
	LIGHT	176,073	175,267	168,806	141,712	127,045	1	1
HB6	HEAVY	26,142	25,981	22,245	15,658	10,453	1	0
	LIGHT	171,996	171,370	167,730	138,348	122,397	1	1
HB7	HEAVY	32,443	32,094	25,449	17,615	11,836	1	0
	LIGHT	67,031	63,924	37,344	26,271	22,378	1	1	*
HB8	HEAVY	140,091	103,349	92,744	58,292	31,583	1	0
	LIGHT	151,244	115,527	102,439	82,154	70,803	1	1
HB9	HEAVY	37,057	36,473	19,544	11,585	7,358	1	0
	LIGHT	409,432	310,529	136,074	106,820	90,063	1	2	*
HB10	HEAVY	38,181	37,981	26,043	17,104	11,391	1	0
	LIGHT	114,255	112,498	106,914	84,368	75,370	1	1
HB11	HEAVY	22,225	21,841	6,956	4,408	2,465	1	0
	LIGHT	106,465	102,278	65,332	50,232	44,527	1	0
HB12	HEAVY	83,044	82,355	46,350	30,276	20,886	1	0
	LIGHT	53,098	47,336	15,823	7,560	5,845	1	1
HB13	HEAVY	81,451	80,372	47,995	32,216	20,139	2	0	§
	LIGHT	27,314	24,774	8,987	5,457	4,104	2	1
HB14	HEAVY	76,299	75,357	28,309	19,104	12,939	1	0
	LIGHT	153,011	149,264	48,474	29,710	25,133	1	1
HB.15	HEAVY	26,551	26,410	16,387	11,434	7,002	2	0	§
	LIGHT	78,525	77,778	43,509	29,504	24,731	1	1
HB16	HEAVY	54,249	53,943	9,517	7,128	4,179	1	0
	LIGHT	42,048	40,351	30,602	22,758	18,251	2	1	§
HB17	HEAVY	111,614	110,882	81,428	50,844	35,949	1	0
	LIGHT	102,490	100,488	83,925	65,925	57,727	1	1	*
HB18	HEAVY	211,215	155,410	146,256	91,009	50,308	1	0
	LIGHT	212,261	161,879	155,235	123,096	105,959	1	1
HB19	HEAVY	109,692	82,221	20,546	12,587	7,274	1	1
	LIGHT	70,828	69,744	62,572	48,354	42,051	1	1
HB20	HEAVY	15,781	15,632	12,789	7,757	4,852	1	0
	LIGHT	135,527	133,208	118,513	90,717	78,701	1	1
HB21	HEAVY	15,312	15,202	8,577	5,645	3,420	1	0
	LIGHT	102,450	100,171	89,059	68,552	60,500	1	1
HB22	HEAVY	217,959	156,488	154,008	95,755	50,245	1	0
	LIGHT	205,334	156,986	143,386	108,728	85,136	1	0
HB23	HEAVY	196,390	143,929	123,028	71,076	39,358	2	0	§
	LIGHT	158,594	120,140	115,476	90,004	78,787	2	0
72A1	HEAVY	213,480	158,199	156,215	107,395	68,486	2	0	§
	LIGHT	187,216	140,964	132,945	105,783	91,208	1	1

* Hybridoma with VL chain sequences identified with >1% frequency,
§ Hybridoma with more than one unique, plausible-coding VH chain sequence with >5% frequency.
The term “unique” refers to unique sequence counts (so, identical sequences found in a substantial frequency of merged reads, not necessarily unique compared to other samples).

The analysis and comparison of the VH and VL chain gene sequences of the 23 hybridomas compared to m72A1 showed unique sequences within the CDR 1-3 region (Table 1). Only HB8 and HB18 had identical VH and VL chain gene sequences, suggesting that the two are the same clone isolated separately; therefore, HB18 was excluded from subsequent experiments. One of the two paired sequences from HB15 hybridoma mixture was confirmed to have identical VH and VL gene sequences to that of mAb HB10; however, based on the previous characterization, the presence of the additional paired sequenced in the HB15 hybridoma was sufficient to confer subtle differences in biochemical interactions with gp350 between the HB10 and HB15 purified antibodies. In addition, the germline genes for the VH and VL chains of the new 15 anti-gp350 mAbs and m72A1 were determined (Table 6).

TABLE 6

Identification of the germline genes for
VH and VL of 15 new mAbs and m72A1

Anti-	V-GENE	J-GENE	V-GENE	J-GENE
bodies	and allele	and allele	and allele	and allele

HB1	IGHV2-9*02	IGHJ4*01	IGKV6-15*01	IGKJ2*01
HB2	IGHV9-2-1*01	IGHJ4*01	IGKV4-72*01	IGKJ5*01
HB3	IGHV1S81*02	IGHJ4*01	IGKV10-96*01	IGKJ1*01 or
				IGKJ1*02
HB5	IGHV1S45*01	IGHJ3*01	IGKV5-48*01	IGKJ5*01
HB6	IGHV9-2-1*01	IGHJ4*01	IGKV4-72*01	IGKJ5*01
HB7	IGHV1-15*01 or	IGHJ1*01	IGKV6-13*01	IGKJ5*01
	IGHV1-23*01
HB8	IGHV9-3-1*01	IGHJ3*01	IGKV1-117*01	IGKJ2*01
HB9	IGHV5-6-4*01	IGHJ1*01	IGKV1-117*01	IGKJ2*01
HB10	IGHV1S81*02	IGHJ4*01	IGKV10-96*01	IGKJ1*01 or
				IGKJ1*02
HB11	IGHV3-8*02	IGHJ1*01	IGKV4-50*01	IGKJ1*01
HB12	IGHV1S81*02	IGHJ4*01	IGKV1-110*01 F	IGKJ4*01
HB14	IGHV1-15*01	IGHJ1*01	GKV1-110*01 F	IGKJ4*01
HB17	IGHV1S81*02	IGHJ4*01	IGKV10-96*01	IGKJ1*01 or
				IGKJ1*02
HB20	IGHV1S81*02	IGHJ4*01	IGKV10-96*01	IGKJ1*01 or
				IGKJ1*02
HB22	IGHV2-6*02	IGHJ3*01	IGKV1-117*01	IGKJ1*01
m72A1	IGHV1-18*01 or	IGHJ3*01	IGLV1*01	IGLJ1*01
	IGHV1-26*01

These results show that although only two mice were used in the generation of the antibodies, germline diversity was still present to some extent, and few mAbs shared the same germline gene rearrangement and evolution. Thus, based on the sequence analysis (FIG. 3), 15 unique anti-gp350 mAbs were generated, with distinct sequence identities from commercially available m72A1. The sequence of the widely used non-neutralizing antibody 2L10 (originally from G. Pearson's laboratory) was not available and therefore, 2L10 was not used in the sequence comparative studies.

Example 4: Humanization of m72A1 and HAMA Testing

The m72A1 VH and VL chain sequences identified in this study were identical to the ones published by Herrman et al., and they were used to generate a humanized 72A1 352 (h72A1) as a strategy to reduce and/or eliminate HAMA (FIG. 4A). The disclosed h72A1 bound gp350 with similar strength to m72A1 in both ELISA (FIG. 4B). The levels of anti-mouse and anti-human activity retained in the h72A1 nAb were determined using ELISA. As shown in FIG. 4C, mouse 72A1 mAb reacted strongly to goat-anti-mouse IgG as compared to goat anti-human IgG (9-fold, p<0.0001) and 28-fold above the background (1× PBS) (p<0.0001). In contrast, h72A1 mAb did not react at all to goat anti-mouse, but specifically reacted strongly to goat anti-human IgG (2100-fold, p<0.0001) over the background. To determine whether h72A1 still recognized gp350 in its native conformation, flow cytometric analysis was performed. h72A1 recognized native epitopes of gp350 expressed on CHO cells surface, comparable to m72A1 360 (FIG. 4D). These results indicate that humanization of m72A1 did not affect its ability to recognize native gp350, but it abrogated anti-mouse reactivity and increased anti-human reactivity.

Example 5: Neutralization Assay

Currently, m72A1 is the only commercially available anti-gp350 nAb (68). However, this antibody was recently reported to be a mixture of both functional and non-functional antibodies (50). The ability of the 15 new mAbs (10 μg/ml or 50 μg/ml) to neutralize purified eGFP-tagged-EBV infection of a B cell line (Raji) in vitro was evaluated and compared to that of m72A1 (mixture) and the newly cloned and biochemically characterized h72A1, following standardized procedures (47, 52). The percentage of eGFP+ cells (percent infection) was determined using flow cytometry as described (47). The nAbs 72A1 and E1D1 were used as positive controls, whereas the anti-gp350 non-neutralizing mAb 2L10 and KSHV gH/gL mAb 54A1 were used as negative controls. Because HB4, HB13, HB15, HB16, HB19, HB21, and HB23 were confirmed to be polyclonal based on isotyping and/or sequence data, they were eliminated from further consideration in the neutralization assay. HB18 was not used in neutralization experiments because it was identical to HB8.
The purified eGFP-tagged-EBV was titered in Raji cells to determine percent EBV infection using a range of volumes (50-250 μl) (FIG. 5A). Then initial neutralization of EBV was conducted in Raji cells using purified mAbs at various concentrations (12.5, 25, 50, and 100 μg/ml). Only HB1, HB5, HB11, 375 HB22 and m72A1 showed a dose-dependent neutralization of EBV in Raji cells; the neutralization capability of HB5 (60-80%) was comparable to that of m72A1 (35-80%) (FIG. 5B). In contrast, HB2, HB3, HB6, HB7, HB8, HB9, HB10, HB12, HB14, HB17, and HB20 mAbs failed to neutralize EBV infection, even at the highest concentration. As expected, neither 2L10 nor 54A1 neutralized EBV infection, even at the highest mAb concentration of 100 μg/ml (FIG. 5B).
Subsequently, seven representative novel nAb and non-nAb anti-gp350 mAbs (HB1, HB5, HB10, HB11, HB17, HB20 and HB22) were purified as well as controls (m72A1, h72A1 and 54A1) using protein G affinity chromatography and size-exclusion chromatography in order to eliminate any potential impurities, then their potency in blocking EBV infection of Raji cells was reevaluated. Chromatography-purified HB1, HB5, HB11, and HB20, blocked EBV infection in a dose-dependent manner (FIG. 5C). HB5 was the most effective nAb, efficiently blocking EBV infection (90%) at percentages comparable to both m72A1 (93%) and h72A1 (98%), even at the lowest concentration of 12.5 μg/ml, with 97% nAb activity at 100 μg/ml (FIG. 5C). HB1, HB11, and HB20 neutralized EBV infection between 57-73% at the lowest concentration (12.5 μg/ml) and 90% at 100 μg/ml. Neither HB17, HB22, nor 54A1 blocked EBV infection; although HB10 blocked some EBV infection, nAb activity did not reach 50% even at the highest concentration of antibody used, thus it was classified as a non-nAb.

Example 6: Four Novel Gp350 nAbs Bind Antigenic Epitopes that Overlap with Those of 72A1

At least seven unique CD21 binding epitopes on EBV gp350 have been predicted (Table 3). One of these epitopes (AA 142-161) has been identified as the primary epitope recognized by m72A1 (59) and mice immunized with the 142-161 peptide elicit nAbs against EBV infection (51). To evaluate whether the selected novel nAbs (HB1, HB5, HB11, and HB20) and non-nAbs (HB10, HB17, and HB22) bind overlapping or non-overlapping target epitopes to those of 72A1, their ability to compete for binding to gp350 expressed stably on transfected CHO cells were determined. Antigen binding competition was observed between biotinylated m72A1 (1 μg/ml) and serially diluted (500, 250, 125, and 67.5 μg/ml) unlabeled gp350 nAbs (HB1, HB5, HB11, HB20, and h72A1), but not the gp350 non-nAbs (HB10, HB17, or HB22) or anti-KSHV gH/gL antibody 54A1 (negative control) (Table 7).

TABLE 7

Cell binding mAbs competition assay with
EBV gp350 using biotinylated m72A1

% inhibition of biotinylated nAbs

	500	250	125	67.5
Unlabeled mAbs	μg/ml	μg/ml	μg/ml	μg/ml

HB1	91	89	81	81
HB5	95	93	92	94
HB10	12	14	16	13
HB11	95	95	89	87
HB17	32	32	6	0
HB20	96	91	87	89
HB22	10	3	16	9
m72A1	91	93	94	97
h72A1	98	95	93	89
54A1	18	7	10	4

However, previously non-nAbs HB10 and HB22, were shown not to bind native gp350 expressed on CHO cells using FACS (FIG. 1D), suggesting that the observed lack of competitive binding could be attributed to these two Abs not binding the native gp350 expressed on the CHO cells. These results indicate that nAbs HB1, HB5, HB11, and HB20, as well as h72A1, bind overlapping target epitopes with that of m72A1, while non-nAbs HB17, the only non-nAbs able to recognize gp350 in conformational form, binds different target epitopes. Similar results were obtained when cross-competition binding assays between 1 μg/ml biotinylated and 500 μg/ml unlabeled gp350 nAbs HB1, HB5, HB11, HB20, m72A1, and h72A1 and non-nAb HB17 were performed (Table 8), confirming that the newly developed gp350 nAbs bind overlapping epitopes to 72A1.

TABLE 8

Cross-competitive binding of EBV gp350
% inhibition of biotinylated nAbs

Unlabeled mAbs	HB1	HB5	HB10	HB11	HB17	HB20	HB22	m72A1	h72A1	54A1

HB1	92	97	ND	96	16	61	ND	98	95	4
HB5	92	98	ND	93	9	73	ND	97	97	13
HB10	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
HB11	93	96	ND	96	17	3	ND	97	97	9
HB17	23	32	ND	11	86	17	ND	24	16	0
HB20	88	97	ND	89	13	59	ND	98	96	0
HB22	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
m72A1	82	97	ND	90	37	31	ND	98	98	1
h72A1	87	97	ND	79	31	26	ND	98	98	0
54A1	0	0	ND	0	0	0	ND	0	3	0

ND—Not determined

Because of inability of non-nAbs, HB10 and HB22 to recognize conformational gp350, they were excluded from the cross competitive cell binding assays. Importantly, even though HB1, HB5, HB11, and HB20 competed with 72A1 for the same antigenic epitope, each of these nAbs had unique VH and VL sequences from 72A1 (Table 1, Table 2).

Example 7: Novel Anti-Gp350 nAbs and Non-nAbs Bind Three Major Immunodominant Regions on Gp350

To identify linear epitopes on gp350, anti-gp350 nAbs (HB1, HB5, HB11 and HB20) and non-nAbs (HB10, HB17, and HB22) were scanned in an ELISA-based assay using a peptide library consisting of sequential peptides (FIGS. 6A-6I). The peptide library, consisting of 20-mer peptides, covered the entire gp350 protein, with the exception of AA 862-881 (Table 9).

TABLE 9

Sequence, length and position of EBV gp350
peptides

Peptide
name	Peptide	gp350		Pep-
length	position	region	Peptide sequence	tide

Pep-1	1-20	1	MEAALLVCQYTIQSLIHL	20
			TG (SEQ ID NO: 132)

Pep-2	21-40	1	EDPGFFNVEIPEFPFYPT	20
			CN (SEQ ID NO: 133)

Pep-3	41-61	1	VCTADVNVTINFDVGGK	21
			KHQL (SEQ ID NO: 134)

Pep-4	62-81	1	DLDFGQLTPHTKAVYQP	20
			RGA (SEQ ID NO: 135)

Pep-5	82-101	1	FGGSENATNLFLLELLG	20
			AGE (SEQ ID NO: 136)

Pep-6	102-121	2	LALTMRSKKLPINVTTGE	20
			EQ (SEQ ID NO: 137)

Pep-7	122-141	2	QVSLESVDVYFQDVFGT	20
			MWC (SEQ ID NO: 138)

Pep-8	142-161	2	HHAEMQNPVYLIPETVP	20
			YIK (SEQ ID NO: 139)

Pep-9	162-181	2	WDNCNSTNITAVVRAQ	20
			GLDV (SEQ ID NO: 140)

Pep-10	182-201	2	TLPLSLPTSAQDSNFSV	20
			KTE (SEQ ID NO: 141)

Pep-11	202-221	3	MLGNEIDIECIMEDGEIS	20
			QV (SEQ ID NO: 142)

Pep-12	222-241	3	LPGDNKFNITCSGYESH	20
			VPS (SEQ ID NO: 143)

Pep-13	242-261	3	GGILTSTSPVATPIPGTG	20
			YA (SEQ ID NO: 144)

Pep-14	262-281	3	YSLRLTPRPVSRFLGNN	20
			SIL (SEQ ID NO: 145)

Pep-15	282-301	3	YVFYSGNGPKASGGDY	20
			CIQS (SEQ ID NO: 146)

Pep-16	302-321	4	NIVFSDEIPASQDMPTNT	20
			TD (SEQ ID NO: 147)

Pep-17	322-341	4	ITYVGDNATYSVPMVTS	20
			EDA (SEQ ID NO: 148)

Pep-18	342-361	4	NSPNVTVTAFWAWPNN	20
			TETD (SEQ ID NO: 149)

Pep-19	362-381	4	FKCKWTLTSGTPSGCE	20
			NISG (SEQ ID NO: 150)

Pep-20	382-401	4	AFASNRTFDITVSGLGTA	20
			PK (SEQ ID NO: 151)

Pep-21	402-421	5	TLIITRTATNATTTTHKVI	20
			F (SEQ ID NO: 152)

Pep-22	422-441	5	SKAPESTTTSPTLNTTG	20
			FAD (SEQ ID NO: 153)

Pep-23	442-461	5	PNTTTGLPSSTHVPTNL	20
			TAP (SEQ ID NO: 154)

Pep-24	462-481	5	ASTGPTVSTADVTSPTP	20
			AGT (SEQ ID NO: 155)

Pep-25	482-501	5	TSGASPVTPSPSPWDN	20
			GTES (SEQ ID NO: 156)

Pep-26	502-521	6	KAPDMTSSTSPVTTPTP	20
			NAT (SEQ ID NO: 157)

Pep-27	522-541	6	SPTPAVTTPTPNATSPT	20
			PAV (SEQ ID NO: 158)

Pep-28	542-561	6	TTPTPNATSPTLGKTSP	20
			TSA (SEQ ID NO: 159)

Pep-29	562-581	6	VTTPTPNATSPTLGKTS	20
			PTS (SEQ ID NO: 160)

Pep-30	582-601	6	AVTTPTPNATSPTLGKT	20
			SPT (SEQ ID NO: 161)

Pep-31	602-621	7	SAVTTPTPNATGPTVGE	20
			TSP (SEQ ID NO: 162)

Pep-32	622-641	7	QANATNHTLGGTSPTPV	20
			VTS (SEQ ID NO: 163)

Pep-33	642-661	7	QPKNATSAVTTGQHNIT	20
			SSS (SEQ ID NO: 164)

Pep-34	662-681	7	TSSMSLRPSSNPETLSP	20
			STS (SEQ ID NO: 165)

Pep-35	682-701	7	DNSTSHMPLLTSAHPTG	20
			GEN (SEQ ID NO: 166)

Pep-36	702-721	8	ITQVTPASISTHHVSTSS	20
			PA (SEQ ID NO: 167)

Pep-37	722-741	8	PRPGTTSQASGPGNSS	20
			TSTK (SEQ ID NO: 168)

Pep-38	742-761	8	PGEVNVTKGTPPQNATS	20
			PQA (SEQ ID NO: 169)

Pep-39	762-781	8	PSGQKTAVPTVTSTGGK	20
			ANS (SEQ ID NO: 170)

Pep-40	782-801	8	TTGGKHTTGHGARTSTE	20
			PTT (SEQ ID NO: 171)

Pep-41	802-821	9	DYGGDSTTPRPRYNATT	20
			YLP (SEQ ID NO: 172)

Pep-42	822-841	9	PSTSSKLRPRVVTFTSPP	20
			VTT (SEQ ID NO: 173)

Pep-43	842-861	9	AQATVPVPPTSQPRFSN	20
			LSM (SEQ ID NO: 174)

Pep-44	862-881	9	LVLQWASLAVLTLLLLLV	20
			MA (SEQ ID NO: 175)

Pep-45	882-901	9	DCAFRRNLSTSHTYTTP	20
			PYD (SEQ ID NO: 176)

Pep-46	888-907	9	NLSTSHTYTTPPYDDAE	20
			TYV (SEQ ID NO: 177)

This peptide could not be synthesized due to high hydrophobicity of AA residues in the sequence. Purified m72A1 and h72A1 nAbs were used as positive controls and anti-KSHV gH/gL 54A1 antibody as a negative control. Purified recombinant gp350 ectodomain was used as a control to validate the binding activity for all of the antibodies used. The overall gp350 sequence was divided into nine different regions consisting of ˜100 AA (FIG. 7). The three major regions that exhibited the greatest affinity to anti-gp350 mAbs were: 1-101, 102-201, and 402-501 (Table 10).

TABLE 10

Summarized analysis of anti-gp350 mAb linear
epitope binding to various regions of gp350

gp350 Regions

mAbs

	1	2	3	4	5	6	7	8	9

HB1	x	—	—	—	x	x	—	x	—
HB5	x	x	x	x	x	x	x	—	—
HB10	x	—	—	—	x	—	x	x	x
HB11	x	x	—	—	x	—	x	—	—
HB17	x	—	—	—	—	x	—	—	x
HB20	x	x	x	—	x	—	—	—	—
HB22	x	—	—	—	—	—	—	—	—
m72A1	x	x	—	—	x	—	—	—	—
h72A1	x	x	—	—	x	—	—	—	—

x represents positive binding of antibody to the region,
— represents no binding of antibody to the region

The AA 102-201 region was bound by only nAbs (HB5, HB11, HB20, m72A1 and h72A1), with the exception of HB1. Notably, this region (102-201) contains the epitope (AA 142-161) previously identified as a binding epitope for 72A1 and as a binding receptor for CR2 (Table 3), confirming that this is the main region that interacts with most gp350 nAbs. Because both nAbs and non-nAbs bound to AA 1-101 and 402-501, these two regions were considered to be immunodominant.

Example 8: Construction of Chimeric Gp350 nAbs

Chimeric gp350 nAbs were constructed according to the diagrams of FIG. 8. First, mouse antibodies against human gp350 were developed, and then the mouse antibody variable region is fused to human constant region, for example, to human IgG. The heavy chain and light chain variable regions of the mouse antibody were cloned into expression vectors such as pFUSE-CHIg and pFUSE2-CLIg vectors, respectively, followed by co-transfection of mammalian cells with recombinant pFUSE-CHIg and pFUSE2-CLIg vectors. The expression vectors were obtained from InvivoGen, and the expression was conducted in CHO cells or HEK293 cells, available from ATCC. The construction schemes and expression of heavy and light chains of clone 19 are shown in FIG. 8.
Analysis of VH-VL sequence from the HB168 (nAb-72A1) hybridoma revealed that the hybridoma produced two antibodies: one that is gp350-specific and another that recognizes mineral oil-induced plasmacytoma (MOPC) (57). To further investigate gp350 for additional neutralizing epitopes, the gp350-specific nAb-72A1 VH-VL sequence was used to generate chimeric (mouse/human) recombinant antibodies. Similarly, the VH-VL sequence for the HB20 antibody, which the neutralization analysis above showed to be one of the best nAb, was used to generate chimeric antibody. A negative control chimeric recombinant antibody was generated using VH-VL sequences from the gp350-specific but non-neutralizing HB5 antibody.

Example 9: Development of Antibody-Small Molecule Conjugates (ADCs)

Using small molecule L₂P₄as an example, antibody-small molecule conjugates can be developed as illustrated in FIG. 9. One or more small molecules can be conjugated to the antibody heavy chain or light chain via a reactive donor group and a C-terminal acceptor group. Small molecule L₂P₄was disclosed in the publication by Jiang et al.⁴⁰After validating the function of the purified chimeric gp350 nAbs using ELISA, flow cytometry (FC), and surface plasmon resonance, the nAbs can be conjugated to L₂P₄in a site-specific manner via a Val-Cit dipeptide linker, which releases the active agent upon internalization of the ADC.^{41, 42}

Example 10: Efficacy Test of ADCs

To screen for optimal dose and identify the best nAb clone for pre-clinical studies, the ability of the ADC to neutralize EBV in vitro and to protect against PTLD in vivo is tested using a humanized mouse, as described^43,44. In brief, purified chimeric ADC (or controls: PBS, or isotype-matched non-nAbs) is injected by I.V. into humanized mice, followed by EBV-B95-8-eGFP challenge. Mice are monitored regularly and euthanized upon signs of illness or after a preset limit of 100 days. Routine histology and necropsy are conducted to assess the efficacy of the ADC to protect against EBV infection and PTLD development.

Example 11: Humanization of E1D1

Similar to Example 4, humanized E1D1 antibody was produced and its specificity was compared to mouse E1D1 by flow cytometric analysis (FIG. 10). Humanization of E1D1 did not affect its binding specificity to EBV gH/gL.

Example 12: Biochemical Characterization of Chimeric and Humanized Antibodies

As demonstrated by FIG. 11, murine, chimeric and humanized antibodies purified by affinity and size-exclusion chromatography were analyzed by SDS-PAGE under reducing conditions. Protein band sizes of 50 kDa and 25 kDa corresponding to the heavy and light chain, respectively were observed. ELISA was performed to determine the reactivity of chimeric and humanized antibodies to murine IgG. Soluble EBV gp350 or gH/gL protein was used as the target antigen at 0.5 μg/ml. Plates were incubated with 0.5 μg/ml of primary antibody, followed by three washes. Bound antibodies were detected using HRP-conjugated anti-mouse IgG or anti-human IgG (1:2,000). The chimeric and humanized antibodies did not react to murine IgG. To determine whether chimeric and humanized anti-gp350 retained the ability to recognize linear epitopes on gp350, lysate from CHO WT cells and CHO cell stably expressing gp350 were analyzed by western blot analysis. The chimeric and humanization process of anti-gp350 did not affect the binding affinity of the antibodies to gp350 linear epitopes, as indicated by the protein band observed at ˜350 kDa.
ELISA binding of anti-gp350 and anti-gH/gL to soluble gp350 and gH/gL proteins was performed. Soluble EBV gp350 and gH/gL proteins were used as the target antigen at 0.5 μg/ml. 1× phosphate buffered saline (PBS) was used as negative (not shown) control. Bound antibodies were detected using HRP-conjugated anti-mouse or anti-human IgG (1:2,000). The chimeric and humanization process did not affect the ability of anti-gp350 and anti-gH/gL to bind to purified recombinant gp350 and gH/gL proteins, respectively.
Flow cytometric analysis of gp350 and gH/gL specificity was performed. CHO wild type cells and gp350 or gH/gL-expressing CHO cells were stained with primary antibodies, followed by secondary goat anti-mouse or anti-human conjugated to AF488. Unstained cells and cells stained with secondary goat anti-mouse or anti-human conjugated to AF488 alone, were used as negative controls. Binding of chimeric and humanized anti-gp350 or gH/gL to conformational epitopes was comparable to that of the parental murine antibodies.

Example 13: EBV Inhibitory Effects of Anti-Gp350 and Anti-gH/gL nAbs

As shown in FIG. 12, EBV-eGFP was pre-incubated with serial diluted (10-0.01 μg/ml) purified murine, chimeric and humanized anti-gp350 (HB5 and 72A1) and anti-gH/gL (E1D1) followed by incubation for 24 hours with 2.5×10⁵HEK 293 (12A) or SVKCR2 cell (12B) or 5×10⁵Raji cells (12C). EBV-eGFP+ cells were enumerated using flow cytometry. Murine nAb (mHB5, m72A1 and mE1D1) served as positive controls. h72A1 and chHB5 were able to neutralize EBV infection in both B cells (Raji cells) and CR2+ epithelial cells (HEK 293 and SVKCR2 cells) similar to or better than the murine versions, m72A1 and mHB5.

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The references listed below, and all references cited in the specification are hereby incorporated by reference in their entireties, as if fully set forth herein.

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Claims

1. An antibody or an immunogenic fragment thereof that specifically binds to Epstein-Barr virus (EBV) glycoprotein (gp) 350 or glycoprotein 220.

2. The antibody or the immunogenic fragment thereof of claim 1, wherein the antibody or the immunogenic fragment thereof specifically binds to a fragment of gp350 comprising the amino acid sequence of AA1-101, 102-201, or 402-501.

3. The antibody or the immunogenic fragment thereof of claim 1, comprising a VH region comprising CDR1, CDR2 and CDR3 having an amino acid sequence of SEQ ID NOs: 5-19, 21-35, and 37-51.

4. The antibody or the immunogenic fragment thereof of claim 1, comprising a VL region comprising CDR1, CDR2 and CDR3 having an amino acid sequence of SEQ ID NOs: 53-67, 69-83, and 85-99.

5. The antibody of claim 1, wherein the antibody is a monoclonal antibody.

6. The antibody of claim 1, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

7. The antibody of claim 6, wherein the antibody is humanized 72A1 or humanized E1D1.

8. An immunogenic peptide comprising one or more fragments of gp350, wherein each fragment has an amino acid sequence identical to or sharing at least 60% similarity to AA1-101, AA102-201 or AA402-501.

9. The immunogenic peptide of claim 8, further comprising a known immunogenic peptide such as keyhole limpet hemocyanin (KLH) peptide.

10. An antibody-small molecule conjugate comprising:

the antibody or the immunogenic fragment thereof of claim 1; and

a small molecule having an anti-proliferative activity against EBV-transformed cells, wherein the small molecule is conjugated to the antibody.

11. The conjugate of claim 10, wherein the antibody is a monoclonal antibody.

12. The conjugate of claim 11, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

13. The conjugate of claim 10, wherein the small molecule is a growth inhibitor of EBV infected B cells.

14. The conjugate of claim 10, wherein the small molecule is L₂P₄, 2-butynediamide, or a derivative thereof.

15. The conjugate of claim 10, wherein the small molecule is conjugated to the antibody via a linker or an adaptor.

16. The conjugate of claim 10, wherein the small molecule is conjugated to the constant region of the heavy chain or the light chain of the antibody.

17. A pharmaceutical composition comprising the antibody or the immunogenic fragment thereof of claim 1.

18. A method of neutralizing EBV infection, preventing EBV infection, preventing a post-transplant lymphoproliferative disease (PTLD), or treating an EBV-associated cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or the immunogenic fragment thereof of claim 1.

19. (canceled)

20. (canceled)

21. The method of claim 18, wherein the subject is a pediatric transplant recipient who is EBV naïve or an adult transplant recipient.

22. The method of claim 21, wherein the administration is before, during, and/or after the transplant.

23. (canceled)

24. The method of claim 18, wherein the EBV-associated cancer is selected from the group consisting of Hodgkin lymphoma, Burkitt lymphoma, gastric cancer, and nasopharyngeal carcinoma.

25-29. (canceled)