Diffusible VP22-E2 protein kills bystander cells and offers a route for cervical cancer gene therapy

Human papillomaviruses (HPVs) are a causative agent of cervical cancer and are implicated in several other types of malignant disease including cancer of the vulva, oral cancer, and skin cancer. In HPV-transformed cells, expression of the viral E6 and E7 oncogenes increases cell proliferation and inhibits apoptosis. Expression of the viral E2 protein in HPV-transformed cells represses transcription of E6 and E7 and induces apoptosis and/or growth arrest. We have shown previously that herpes simplex virus type 1 (HSV-1) VP22–HPV E2 fusion proteins can traffic between cells and induce apoptosis. Here we show that replication-defective adenoviruses can be used to deliver VP22–E2 fusion proteins to target cells. We show that the use of adenoviral vectors to deliver VP22–E2 proteins leads to high levels of apoptosis. Interestingly, VP22–E2 proteins produced in adenovirus-infected cells are able to enter uninfected cells and induce apoptosis. Trafficking between cells and the induction of apoptosis in bystander cells are detectable in a three-dimensional tumor model. These results suggest that adenoviral vectors expressing VP22–E2 fusion proteins could be used to treat cervical cancer and other HPV-associated diseases

Attenuated recombinant vaccinia virus expressing oncofetal antigen (tumor-associated antigen) 5T4 induces active therapy of established tumors

The human oncofetal antigen 5T4 (h5T4) is a transmembrane glycoprotein overexpressed by a wide spectrum of cancers, including colorectal, ovarian, and gastric, but with a limited normal tissue expression. Such properties make 5T4 an excellent putative target for cancer immunotherapy. The murine homologue of 5T4 (m5T4) has been cloned and characterized, which allows for the evaluation of immune intervention strategies in “self-antigen ” in vivo tumor models. We have constructed recombinant vaccinia viruses based on the highly attenuated and modified vaccinia virus ankara (MVA strain), expressing h5T4 (MVA-h5T4), m5T4 (MVA-m5T4), and Escherichia coli LacZ (MVA-LacZ). Immunization of BALB/c and C57BL/6 mice with MVA-h5T4 and MVA-m5T4 constructs induced antibody responses to human and mouse 5T4, respectively. C57BL/6 and BALB/c mice vaccinated with MVA-h5T4 were challenged with syngeneic tumor line transfectants, B16 melanoma, and CT26 colorectal cells that express h5T4. MVA-h5T4-vaccinated mice showed significant tumor retardation compared with mice vaccinated with MVA-LacZ or PBS. In active treatment studies, inoculation with MVA-h5T4 was able to treat established CT26-h5T4 lung tumors and to a lesser extent B16.h5T4 s.c. tumors. Additionally, when C57BL/6 mice vaccinated with MVA-m5T4 were challenged with B16 cells expressing m5T4, resulting growth of the tumors was significantly retarded compared with control animals. Furthermore, mice vaccinated with MVA-m5T4 showed no signs of autoimmune toxicity. These data support the use of MVA-5T4 for tumor immunotherapy

RAC2, AEP, and ICAM1 expression are associated with CNS disease in a mouse model of pre-B childhood acute lymphoblastic leukemia

To obtain a better understanding of the pathogenesis of central nervous system (CNS) involvement in pre-B acute lymphoblastic leukemia (ALL), we have developed a murine model of CNS disease. Semi quantitative proteomic discovery based approaches identified unique expression of AEP, ICAM1 and RAC2 among others in an invasive pre-B cell line that produced CNS leukemia in NOD-SCID mice. Targeting RAC2 significantly inhibited in vitro invasion and delayed disease onset in mice. Induced expression of RAC2 in cells lines with low/absent expression of AEP and ICAM1 did not result in an invasive phenotype or murine CNS disease. Flow cytometry analysis identified an enriched population of blast cells expressing ICAM1/LFA-1/CD70 in the CD10+/CD19+ fraction of bone marrow aspirates obtained from relapsed compared to normal controls and those with primary disease. CD10+/CD19+ fractions obtained from relapsed patients also express RAC2 and give rise to CNS disease in mice. Our data suggests combinations of processes are involved in the pathogenesis of CNS disease in pre-B ALL; supports a model in which CNS disease occurs as a result of external invasion and suggests that targeting the processes of adhesion and invasion unique to pre-B cells may prevent recurrences within the CNS

Regulation of autologous immunity to the mouse 5T4 oncofoetal antigen : implications for immunotherapy

Effective vaccination against tumour-associated antigens (TAA) such as the 5T4 oncofoetal glycoprotein may be limited by the nature of the T cell repertoire and the influence of immunomodulatory factors in particular T regulatory cells (Treg). Here, we identified mouse 5T4-specific T cell epitopes using a 5T4 knock out (5T4KO) mouse and evaluated corresponding wild-type (WT) responses as a model to refine and improve immunogenicity. We have shown that 5T4KO mice vaccinated by replication defective adenovirus encoding mouse 5T4 (Adm5T4) generate potent 5T4-specific IFN-γ CD8 and CD4 T cell responses which mediate significant protection against 5T4 positive tumour challenge. 5T4KO CD8 but not CD4 primed T cells also produced IL-17. By contrast, Adm5T4-immunized WT mice showed no tumour protection consistent with only low avidity CD8 IFN-γ, no IL-17 T cell responses and no detectable CD4 T cell effectors producing IFN-γ or IL-17. Treatment with anti-folate receptor 4 (FR4) antibody significantly reduced the frequency of Tregs in WT mice and enhanced 5T4-specific IFN-γ but reduced IL-10 T cell responses but did not reveal IL-17-producing effectors. This altered balance of effectors by treatment with FR4 antibody after Adm5T4 vaccination provided modest protection against autologous B16m5T4 melanoma challenge. The efficacy of 5T4 and some other TAA vaccines may be limited by the combination of TAA-specific T regs, the deletion and/or alternative differentiation of CD4 T cells as well as the absence of distinct subsets of CD8 T cells