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. 2006;117:55–74.

Theodore E. Woodward Award: Development of Novel, EBV-Targeted Therapies for EBV-Positive Tumors

Shannon Kenney 1,✉
PMCID: PMC1500921  PMID: 18528464

Abstract

The near universal presence of EBV in certain tumors suggests that new EBV-based therapies could be developed for these malignancies. We have explored one EBV-based therapy that involves the purposeful induction of lytic EBV infection in tumors. Induction of lytic EBV infection in tumors activates expression of EBV-encoded kinases that convert the prodrug, ganciclovir, to its active cytotoxic form. In mouse models for EBV-positive tumors, the combination of lytic-inducing chemotherapy and ganciclovir is much more effective than either agent alone for treating tumors. Another potential EBV-based target is the cellular protein, CD70. EBV-positive tumors commonly express CD70, while CD70 expression in normal cells is restricted to a few highly activated B cells and T cells. Anti-CD70 monoclonal antibody inhibits the growth of CD70-positive (but not CD70-negative) Burkitt’s lymphomas in SCID mice. Finally, while completely lytic EBV infection is clearly incompatible with tumor cell growth, we recently discovered that small numbers of lytically-infected cells actually promote the growth of EBV-immortalized lymphocytes in SCID mice, through the release of paracrine growth factors as well as angiogenic factors. Thus, agents that prevent the earliest stage of lytic EBV infection (such as fatty acid synthase inhibitors), rather than the later stage of viral replication, might also be useful in the treatment of early-stage EBV-positive tumors.

Introduction

Epstein-Barr virus (EBV) is a ubiquitous human herpes virus that causes infectious mononucleosis and is strongly associated with certain B-cell and epithelial-cell malignancies (1). Certain malignancies almost always contain the EBV genome, including transplant-associated lymphoproliferative disease (LPD), AIDS-related CNS lymphomas, African Burkitt’s lymphomas, and nasopharyngeal carcinomas (Table 1). Other malignancies sometimes (but not always) contain the EBV genome, including Hodgkin’s Disease (EBV present in up to 50% of cases) and gastric cancer (EBV present in up to 10% of cases).

TABLE 1.

Human Tumors Associated with Epstein-Barr Virus Infection

  • EBV Genome Almost Always Present
    • —Post-transplant lymphoproliferative disease
    • —AIDS-related CNS lymphoma
    • —AIDS-related leiomyosarcoma
    • —African Burkitt’s lymphomas
    • —Nasopharyngeal carcinoma
  • EBV Genome Commonly Present
    • —Hodgkin’s Disease
    • —Gastric Carcinoma
    • —AIDS-associated peripheral lymphomas
    • —AIDS-associated primary effusion lymphomas
    • —Asian T-cell lymphomas
  • Possibly EBV Associated
    • —Breast Cancer
    • —Liver cancer (Asia)
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EBV infection efficiently immortalizes primary human B cells in vitro. Furthermore, EBV-immortalized B-cell lines (“lymphoblastoid cell lines” (LCLs)) induce lymphoproliferative disease (LPD) when injected into immunodeficient (SCID) mice. Several different viral proteins are known to play important roles during EBV immortalization of primary B cells in vitro, particularly LMP-1 and EBNA-2 (1), and presumably also contribute to the development of EBV-positive tumors in patients. Independent of the exact role that EBV may play in the development of various different human malignancies, the very fact that EBV is essentially universally present in certain human tumors suggests that novel EBV-based therapies could be developed for these malignancies.

Viral Pathogenesis

Like all herpesviruses, EBV can infect cells in either a latent or lytic state (Figure 1). Unlike herpes simplex virus and cytomegalovirus, however, most illnesses attributable to EBV infection are associated with the latent forms of infection. During primary infection, EBV probably initially infects oropharyngeal epithelial cells in a lytic form, and then subsequently infects B cells, where the virus usually assumes one of the latent forms of infection. Primary EBV infection in some individuals, particularly adolescents, results in the clinical syndrome, infectious mononucleosis, approximately 1 month after infection (2,3). The EBV-positive B cells in patients with infectious mononucleosis primarily contain the latent form(s) of infection, and the symptoms associated with this illness are caused by the onset of a vigorous cytotoxic T cell response against the virally-infected B cells (2–5).

Fig. 1.

Fig. 1

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Differences between latent and lytic EBV infection. A. Latent EBV infection. In the latent forms of EBV infection, the virus persists as an episome in the nucleus and is replicated once per cell cycle by the cellular DNA polymerase and the viral protein, EBNA-1, using an origin of replication called oriP. The host cell is not killed by latent EBV infection. EBV proteins expressed during some forms of latent viral infection (especially LMP-1) have transforming functions. Drugs that inhibit the latent form of EBV replication are not currently available. Viral proteins expressed during type III latent infection are shown. B. Lytic EBV infection. Expression of the two viral immediate-early proteins, BZLF1 (Z) and BRLF1 (R), results in activation of the lytic form of viral replication. The virus is replicated by the virally-encoded DNA polymerase, using the oriLyt origin of replication. Infectious virion particles are released and the host cell is generally killed. Acyclovir and ganciclovir inhibit this form of viral replication.

Following recovery from infectious mononucleosis, it is essentially impossible to find any lytically-infected cells in the peripheral blood of immunocompetent individuals (6). Lytic viral infection is most commonly found in tonsillar B cells, as well as tonsillar epithelial cells. The fact that immunocompetent individuals persistently shed EBV asymptomatically in the saliva (7–11) explains why EBV is so highly prevalent (greater than 90% of adults seropositive) throughout the world. EBV is also sometimes present in both male and female genital secretions, suggesting that this virus could also be sexually transmitted (12,13).

Latent EBV infection

Latently-infected EBV-positive cells persist in the host for life but are usually confined to a small subset of memory B cells (1). In latently infected cells, EBV is replicated by the host cell DNA polymerase and the viral protein EBNA-1 (Figure 1A) (1). Drugs that prevent the latent form of EBV replication are not currently available. In the most stringent form of viral latency (type I), only one viral protein (EBNA-1) is expressed in the B cell, and since this particular viral protein is not transforming, this form of viral latent infection is generally harmless. In the less stringent forms of latent viral infection (type II and type III), up to nine viral proteins are expressed in the host cell, and some of these viral proteins (in particular LMP-1) can function as oncogenes (1). Fortunately, the viral proteins expressed during the potentially oncogenic types of EBV infection (type II and type III latency) are usually readily recognized by cytotoxic T cells in the immunocompetent host, and thus cells with these forms of EBV infection are normally rapidly eliminated (1). However, immunosuppressed patients, particularly transplant recipients and AIDS patients, do not efficiently eliminate B cells with type II or type III latent EBV infection and thus these individuals are at much higher risk of developing EBV-associated B-cell malignancies. Interestingly, EBV-associated epithelial cell tumors (nasopharyngeal carcinoma and gastric carcinoma), as well as EBV-positive Hodgkin’s Disease, generally occur in immunocompetent hosts. Exactly why some otherwise immunocompetent hosts cannot recognize and reject EBV-infected tumor cells is not currently understood, but is an area of intense study.

Lytic EBV infection

The lytic form of EBV infection is required for the production of progeny virus, and is thus essential for cell-to-cell spread of the virus, as well as transmission from host to host (Figure 1B). However, there is only one human disease that is unequivocally due to lytic EBV infection. This disease, oral hairy leukoplakia (OHL), occurs in epithelial cells on the lateral aspect of the tongue (14–18). OHL is a hyperproliferative lesion that is observed only in highly immunocompromised patients and is easily treated by antiviral agents such as acyclovir that inhibit the lytic form of EBV replication.

In the human host, B-cell receptor stimulation by antigen, differentiation of B-cells into plasma cells, and terminal differentiation of oral epithelial cells are all thought to activate the lytic form of EBV infection (19,20). In addition, certain cytokines, particularly TGF-beta, can induce lytic viral infection in a subset of Burkitt’s lymphoma lines in vitro, and could potentially reactivate EBV in vivo (21,22). The interaction between CD4 T cells and EBV-infected B cells has also been reported to induce lytic infection (23). Finally, there is increasing evidence that severe host cell stress in response to many different toxic stimuli (including chemotherapy and irradiation) can induce lytic EBV infection (24–27).

In the lytic form of EBV infection, the virus is replicated by the viral (rather than the cellular) DNA polymerase (Figure 1B). EBV lytic genes are expressed in a temporally regulated manner. Entry into the viral lytic cycle is triggered by expression of either of the two EBV immediate-early (IE) genes, BZLF1 and BRLF1 (reviewed in 28). The gene products of BZLF1 (referred to as Z, Zta, or Zebra) and BRLF1 (referred to as Rta or R) function as transcriptional activators and initiate an ordered cascade of viral lytic gene expression which culminates in release of infectious virus and usually death of the host cell (29–31). Once activated, the IE gene products together activate transcription of the early viral genes. Early viral genes encode the viral replication proteins, including the virally-encoded DNA polymerase. Following viral replication, the late viral genes are transcribed; many late genes encode structural proteins that make up the virion particle.

Treatment of lytic EBV infection

Lytic EBV replication can be inhibited by both acyclovir and ganciclovir in vitro (32–35). Acyclovir and ganciclovir are nucleoside analogues which must be phosphorylated prior to being incorporated by the viral (or cellular) DNA polymerase into DNA. Neither uninfected cells, nor cells that contain one of the latent forms of EBV infection, can efficiently phosphorylate either acyclovir or ganciclovir. However, cells infected with the lytic form of viral infection express two virally-encoded kinases (EBV thymidine kinase and the BGLF4 gene product, PK), thus allowing phosphorylation/activation of both acyclovir and ganciclovir in these cells (36–38). As acyclovir is less toxic than ganciclovir in patients, acyclovir is the preferred agent for treating oral hairy leukoplakia. There is no convincing evidence that acyclovir treatment of immunocompetent patients with infectious mononucleosis shortens or ameliorates this illness (39).

Lytic EBV infection, by enhancing the amount of horizontal transmission of EBV from cell to cell, may contribute to the development of EBV-associated tumors

The fact that all EBV-associated malignancies have a predominantly latent pattern of viral gene expression (1), in addition to studies demonstrating that a number of viral latency proteins are absolutely required for transformation of B cells in vitro (40–43), initially led investigators to believe that only the latent forms of viral infection are important for the development of EBV-associated malignancies. Nevertheless, small numbers of lytically-infected cells are frequently detected in biopsies of EBV-associated lymphoproliferative disease (LPD) (44), as well as in nasopharyngeal carcinomas. However, as lytically-infected cells are thought to die within a few days, such cells must be constantly regenerated within EBV-positive tumors.

There are several potential mechanisms by which EBV lytic gene expression could contribute to the growth of EBV-associated LPD in vivo. By increasing the horizontal transmission of the virus from cell to cell, lytic infection may increase the total number of latently-infected cells (Fig. 2A). However, whether acyclovir or ganciclovir are useful for treating early polyclonal EBV-associated lymphoproliferative disease in post-transplant patients is somewhat controversial (2,45). Nevertheless, there is increasingly convincing evidence suggesting that anti-viral prophylaxis reduces the subsequent development of EBV-associated lymphoproliferative disease in transplant recipients (46–51). In this case, inhibition of lytic EBV infection presumably reduces the pool of latently-infected B cells, thereby decreasing the probability that one or more of these cells eventually becomes malignant. Interestingly, we and our collaborators recently showed that low doses of the drug methotrexate can induce the lytic form of EBV replication in vitro, and that rheumatoid arthritis patients treated with methotrexate-containing regimens are more likely to have high EBV viral loads in the blood than patients treated with equally-immunosuppressive regimens not containing methotrexate (52). These results suggest that the previously noted association between methotrexate treatment and the development of EBV-positive lymphomas in rheumatoid arthritis patients may be partially due to enhanced lytic EBV replication induced by methotrexate.

Fig. 2.

Fig. 2

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Mechanisms by which lytic EBV infection in a small number of tumor cells could contribute to tumor development A. Infectious virus derived from lytically-infected cells can infect new B cells, thus increasing the total number of latently-infected EBV-positive B cells in the host. B. Lytically-infected cells produce the B-cell growth factors, IL-6, cellular IL-10 and viral IL-10, allowing B cells to grow more efficiently. Lytically-infected cells also produce VEGF and thus contribute to angiogenesis in both B-cell and epithelial-cell malignancies.

Lytic EBV infection promotes lymphoproliferative disease by enhancing expression of the paracrine B-cell growth factor, IL-6

In addition to enhancing horizontal viral transmission, the lytic form of EBV infection might also potentially stimulate tumor growth by inducing the expression of paracrine factors that promote tumor growth (Fig. 2B). For example, the Kaposi’s Sarcoma herpesvirus (KSHV) lytic protein ORF74 triggers paracrine secretion of the angiogenic factor VEGF (53), and thereby participates in the formation of Kaposi’s Sarcoma (54). Another KSHV lytic gene, vIL-6, encodes a homolog of IL-6 (55) and may function as a paracrine B-cell growth factor in KSHV-associated lymphomas (56).

Similarly, EBV-immortalized LCLs are dependent on paracrine/autocrine secretion of growth factors for optimal growth (57,58). Both cellular IL-6 and cellular IL-10 enhance the growth of LCLs in vitro and in mouse models of LPD (59–61). The immediate-early viral lytic protein, BZLF1, induces expression of cellular IL-10 (cIL-10) (62–64), and a late lytic EBV-encoded protein (vIL-10) that is a homologue of cellular IL-10 (65) enhances the growth of B cells in vitro (66).

To determine whether lytic infection contributes to EBV-induced LPD in SCID mice, we examined the phenotype of LCLs derived with wild-type (WT), BZLF1-deleted (Z-KO), or BRLF1-deleted (R-KO) viruses (67), using peripheral blood leukocytes from several different donors to generate LCLs. The Z-KO and R-KO viruses cannot express most viral lytic genes or replicate lytically unless the deleted gene product is supplied in trans (68). Somewhat surprisingly, we found that early-passage LCLs generated from the Z-KO and R-KO viruses (Z-KO and R-KO LCLs) were significantly impaired in comparison to the corresponding WT LCLs in terms of their ability to form tumors in SCID mice (69). In contrast, acyclovir treatment (which prevents lytic viral replication but not early lytic viral gene expression) did not inhibit the growth of WT LCLs in SCID mice, showing that release of infectious viral particles is not required for efficient tumor formation in this particular SCID mouse model. Instead, we found that early-passage lytic-defective LCLs express substantially lower levels of IL-6 than the corresponding wild-type LCLs, suggesting a potential paracrine mechanism for the growth defect of these lines in SCID mice. Interestingly, neutralizing antibodies against IL-6 were recently shown to inhibit transplant-associated LPD in patients (70).

Our results unexpectedly suggest that most of the cellular IL-6 in LPD lesions is derived from the small population (approximately 1%) of lytically-infected cells. It is likely that one or both of the two EBV IE proteins (BZLF1 and BRLF1) is responsible for activating IL-6 transcription in lytically-infected B cells. If so, therapies that prevent expression of the two EBV immediate-early proteins might be useful for inhibiting the growth of early-stage lymphoproliferative disease. In this regard, it is important to note that acyclovir does not prevent the expression of either BZLF1 or BRLF1. However, we recently discovered that agents which inhibit fatty acid synthase activity (such as C75 and cerulenin) also reduce the expression of two EBV immediate-early proteins (71). Thus, fatty acid synthase inhibitors could potentially be useful for preventing all stages of lytic EBV infection in tumors.

Lytic EBV infection in tumor cells may also promote malignancy by enhancing angiogenesis. Angiogenesis is critical for the efficient growth of both B-cell and epithelial cell malignancies. Virus-induced upregulation of angiogenesis factors in virally-mediated malignancies may play a significant role in tumor progression. For example, in the case of KSHV, multiple viral lytic proteins are capable of inducing upregulation of VEGF. The capacity of VEGF to induce angiogenesis contributes to the development of Kaposi’s Sarcoma (KS), a highly vascularized lesion. Angiogenesis is also thought to be critically important for the growth of the EBV-associated malignancies, nasopharyngeal carcinoma (NPC) and Burkitt’s’s lymphoma (BL), as angiogenesis inhibitors restrict tumor growth in mouse models of either malignancy.

To determine if lytically-infected cells contribute to angiogenesis in the context of EBV-associated tumors, we examined whether lytic-defective LCLs are impaired in mediating angiogenesis in comparison to WT LCLs. Considering that at most a few percent of WT LCLs have the lytic form of viral infection, we somewhat surprisingly found that the supernatants from lytic-defective LCLs contain significantly less angiogenic activity than the supernatants from WT LCLs (72). The supernatants of the lytic-defective LCLs also contained much less VEGF than the supernatants of the WT LCLs. These results suggest that in early passage LCLs, angiogenic factors are primarily derived from the small number of lytically-infected cells.

Lytic induction as a strategy for treating EBV-positive tumors

As discussed above, a small number of lytically-infected cells may actually help EBV-positive tumors to grow. Nevertheless, it is also clear that too many lytically-infected cells is detrimental for tumor growth. Therefore, we and others have been exploring the idea that the purposeful induction of lytic EBV infection in EBV-positive tumor cells could be a potentially novel way to selectively kill EBV-infected tumor cells (73–75).

Theoretically, EBV-positive tumors containing the latent forms of viral infection could be switched to the lytic form of infection by either inducing expression of the EBV immediate-early genes from the endogenous viral genome in tumors, or by using gene delivery methods to express either of the two EBV immediate-early proteins in tumor cells (Fig. 3A). Our laboratory has pursued both types of approaches. As a gene delivery strategy, we constructed both BZLF1 and BRLF1 expressing adenovirus vectors. When we injected the BZLF1 or BRLF1 adenovirus vectors directly into EBV-positive nasopharyngeal carcinoma tumors grown in nude mice, tumor growth was inhibited, whereas a control adenovirus vector had no effect (76).

Fig. 3.

Fig. 3

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Therapeutic lytic induction strategies for treating EBV-positive tumors. A. Gene delivery techniques. Expression of either EBV immediate-early protein (BZLF1 or BRLF1) under a strong consitutively active promoter in gene delivery vectors (such as adenovirus) can induce lytic infection in EBV+ tumors and cell lysis B. Induction of lytic viral gene expression by chemotherapy and radiation therapy. Treatment of latently infected tumors cells with chemotherapy or radiation leads to activation of signal transduction pathways (including MAPK, p38, and PI3K). Activation of these kinases enhances the transcriptional activity of cellular transcription factors that bind to and activate the two EBV immediate-early promoters, BZLF1 and BRLF1, as shown. Expression of the BZLF1 and BRLF1 viral proteins (which are transcriptional activators) is then sufficient to activate the entire EBV lytic gene cascade. C. Ganciclovir (GCV) enhances the cytotoxic effect of chemotherapy and radiation in an EBV-dependent manner. Certain chemotherapy agents, as well as irradiation, activate lytic EBV gene transcription in a portion of EBV+ tumors cells, allowing these cells to express virally-encoded kinases that can phosphorylate (and activate) the nucleoside analogue, ganciclovir. Phosphorylated ganciclovir inhibits the host cell DNA polymerase and kills the tumor cell. In addition, phosphorylated ganciclovir can be spread to nearby cells, inducing “bystander” killing. Latently infected tumor cells cannot phosphorylate ganciclovir.

In addition, we have pursued methods for activating lytic EBV gene transcription in latently infected tumor cells using drug-based strategies. We discovered that certain cytotoxic therapies, including some chemotherapy agents and gamma irradiation, can induce lytic EBV gene transcription in at least a portion of tumor cells (24–27). This induction is mediated through transcriptional activation of the two EBV immediate-early promoters and requires the PI3 kinase, p38 kinase, and MAP kinase pathways (24,25) (Fig. 3B). Specific transcription factor binding sites in both the BZLF1 promoter (MEF2 and CRE), and the BRLF1 promoter (EGR-1) are also required (25). Agents that inhibit histone deacetylases (such as butyrate compounds) have also been shown to enhance the amount of lytic EBV infection in some mouse tumor models (26).

Lytic induction strategies are most effective for inhibiting tumor growth when combined with the antiviral drug, ganciclovir (24–27,77,78). In cells containing the lytic (but not latent) type of EBV infection, virally encoded kinases (BGLF4 and the viral thymidine kinase) are expressed which phosphorylate the nucleoside analogue, ganciclovir, converting it to its active cytotoxic form (37) (Fig. 3C). Phosphorylated ganciclovir inhibits not only viral DNA replication, but also inhibits the host cell DNA replication, and is cytotoxic. Furthermore, phosphorylated ganciclovir can be transferred into nearby cells that are unable to phosphorylate ganciclovir (i.e., tumor cells with latent EBV infection), and thus induce “bystander” killing. As chemotherapy and irradiation induce lytic infection in only a portion of tumor cells, the combination of these agents with ganciclovir is much more effective than either agent alone for treating EBV-positive tumors in mouse models (24,25). Whether ganciclovir will be effective in combination with lytic induction strategies for treating EBV-positive human tumors is currently being investigated (78,79).

Development of Therapeutic monoclonal antibodies targeted against EBV-induced proteins

The development of therapeutic monoclonal antibodies directed against proteins that are preferentially expressed in EBV-positive malignancies is another potentially promising avenue of research. A monoclonal antibody directed against the B-cell specific protein, CD20 (Rituximab), is already widely used to treat early lymphoproliferative disease in transplant recipients. However, CD20 is expressed on almost all B cells, not just the malignant B cells. Ideally, a monoclonal antibody that specifically recognizes a virally-encoded cell surface protein (such as LMP-1 or LMP-2) could be developed that mediates efficient cell killing in a completely EBV-dependent manner. As an alternative strategy, we recently investigated whether monoclonal antibodies directed against the cellular protein, CD70, could mediate cell killing in an at least partially EBV-dependent manner (80). We chose CD70 as a target because CD70 is expressed on the surface of all cells containing types II or III latent EBV infection (including lymphoproliferative Disease, Hodgkin’s Disease, and nasopharyngeal carcinoma) (81,82) but is otherwise only expressed on very rare, highly activated B cells and T cells (Fig. 4). Our in vitro studies demonstrated that two different monoclonal antibodies directed against CD70 induced complement-dependent killing of EBV+ Burkitt’s lymphomas expressing CD70 (due to type III latent viral infection), and that this killing effect was comparable to that achieved with Rituximab plus complement. As expected, the CD70-directed antibodies did not induce complement-mediated killing of Burkitt’s lymphoma cells with type I latent EBV infection, since these cells do not express CD70. Most importantly, a monoclonal CD70 antibody was shown to inhibit the growth of CD70-positive (but not CD70-negative) Burkitt’s lymphoma cells in a SCID mouse model. These results suggest that it should be possible to develop monoclonal antibodies that would more specifically target EBV-positive tumor cells for destruction than is the case with the currently available therapeutic monoclonal antibodies.

Fig. 4.

Fig. 4

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Expression of cellular CD70 and EBV proteins in different types of latent EBV infection. The tumors associated with each type of EBV latency, the viral proteins expressed, and cellular CD70 expression are indicated.

Conclusions

Epstein-Barr virus has evolved in its human host in a manner that allows the virus to persist in each particular host forever, while at the same time be efficiently spread from host to host. Normally, the interaction between EBV and its host is relatively benign and causes little or no disease. However, when individuals become immunosuppressed, the propensity of the virus to induce B cell proliferation (an attempt by the virus to ensure the lifelong persistence of latently-infected B cells) is not adequately controlled by cytotoxic T cell killing of EBV-positive cells and can sometimes eventually lead to lymphoproliferative disease. In other EBV-associated malignancies (such as Burkitt’s lymphoma, Hodgkin’s Disease, nasopharyngeal carcinoma and gastric carcinoma), viral infection appears to collaborate with additional genetic mishaps (such as c-myc translocation, inactivation of tumor suppressor genes, and/or activation of various oncogenes) to enhance the likelihood of tumor development in immunocompetent hosts.

As we continue to learn more about how various EBV proteins contribute to different forms of EBV-associated malignancies, we will no doubt identify additional viral targets that could be used to prevent tumor cell growth. Our recent findings that lytic EBV proteins actually contribute to tumor angiogenesis, and induce the expression of the B-cell growth factor, IL-6, suggest that new treatment strategies aimed at completely suppressing the expression of all lytic viral proteins might be useful in controlling early EBV-associated malignancies. Furthermore, lytic induction strategies, whereby the lytic form of viral infection is intentionally induced in tumors containing the latent form of infection, appear increasingly promising in mouse tumor models and are being actively tested in humans with EBV-positive tumors. Finally, there is every reason to believe that EBV-positive tumor cells should express many viral-dependent targets that could be utilized in the development of tumor-specific therapeutic monoclonal antibodies.

ACKNOWLEDGMENTS

NIH grants R01-CA66519, R01-CA58853, and the many past and present members of the Kenney laboratory. Particular thanks to Mark McDermott for help with figures.

DISCUSSION

Tweardy: Houston: I enjoyed that a lot. The question that I have relates to the possibility of additional roles for IL-6 and IL-10 cytokine production that you are suggesting act as growth factors for the lymphoma. The other thing that IL-6 can be doing is actually reducing apoptosis secondary to the stress of radiation and chemotherapy in the second pathway that you have outlined. I’m wondering if you’ve pre-treated in vitro or in vivo the cells with IL-6 to see whether or not it can reduce the effect of the radiation therapy and chemotherapy combination?

Kenney: Chapel Hill: We recently found that neutralizing anti-IL-6 antibodies dramatically inhibit the growth of EBV-immortalized B cells in SCID mice. In addition, there was actually a small study reported a few years ago in which patients with post-transplant lymphoproliferative disease were treated with anti-IL-6 antibodies. In this study, anti-IL-6 antibodies improved the lymphoproliferative disease in 8 of the 12 patients in the absence of any other therapy.

Howley: Boston: The IL-6 and the angiogenic factors—are they direct targets of transcriptional activation of the Z and R proteins or is there another pathway involved? And have you identified activated transcription of IL-6 or VEGF.

Kenney: We are actually in the process of doing that. I can tell you that the IL-6 effect is, indeed, transcriptional, and that we just cloned the IL-6 promoter upstream of a reporter gene to determine which EBV protein turns it on. Our preliminary results suggest that the EBV R protein efficiently activates the IL-6 promoter. The VEGF effect is not transcriptional, but post-transcriptional, and we’re in the process of determining exactly how that occurs and which viral protein is responsible.

Jordan: Los Angeles: Enjoyed your presentation quite a bit. I deal with transplant patients on a daily basis and we do see a lot of EBV infection, especially in pediatric liver transplant patients, that can persist for long periods of time. We really worry about these kids. We do maintain them on ganciclovir and when we see tumors or we see situations that look like PTLD, we treat them with Rituxibam or IVIG. This usually results in rapid tumor regression. Can Rituximab induce lytic EBV infection? If so, are the tumors more susceptible to ganciclovir at that point?

Kenney: I think, first of all, that rituximab is a wonderful agent. And second of all, it has recently been shown by another group (although we could not show this) that rituximab treatment also induces lytic EBV infection in at least some EBV-positive B cell lines in vitro. Based on our work, these investigators wanted to find out if rituximab induces lytic EBV infection. If so, it would make sense that it could induce synergistic killing of EBV-positive cells when combined with ganciclovir.

Jordan: Another question—any work with the interleukin-6 receptor humanized monoclonal and PTLD, not anti-interleukin-6, but the receptor blocker?

Kenney: I’m unfamiliar with that, but what I did not have time to present was work we have done with a monoclonal antibody directed against CD70, which is a cellular protein induced by EBV infection. We find that this antibody works well in inhibiting the growth of EBV-positive CD70 expressing lymphomas in mouse models. I think there’s going to be a lot more done in the next ten years in developing additional novel EBV-targeted therapies.

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