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

We developed a murine model of CNS disease to obtain a better understanding of the pathogenesis of CNS involvement in pre-B-cell acute lymphoblastic leukemia (ALL). Semiquantitative proteomic discovery-based approaches identified unique expression of asparaginyl endopeptidase (AEP), intercellular adhesion molecule 1 (ICAM1), and ras-related C3 botulinum toxin substrate 2 (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 cell lines with low/absent expression of AEP and ICAM1 did not result in an invasive phenotype or murine CNS disease. Flow cytometric analysis identified an enriched population of blast cells expressing ICAM1/lymphocyte function associated antigen-1 (LFA-1)/CD70 in the CD10(+)/CD19(+) fraction of bone marrow aspirates obtained from relapsed compared with 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 suggest that combinations of processes are involved in the pathogenesis of CNS disease in pre-B-cell ALL, support a model in which CNS disease occurs as a result of external invasion, and suggest 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.

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 tumor 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.

Immunotherapy success in prophylaxis cannot predict therapy: prime-boost vaccination against the 5T4 oncofoetal antigen.

We have investigated the tumour therapeutic efficacy of homologous and heterologous prime-boost vaccine strategies against the 5T4 oncofoetal antigen, using both replication defective adenovirus expressing human 5T4 (Ad5T4), and retrovirally transduced DC lines (DCh5T4) in a subcutaneous B16 melanoma model (B16h5T4). In naïve mice we show that all vaccine combinations tested can provide significant tumour growth delay. While DCh5T4/Adh5T4 sequence is the best prophylactic regimen (P > 0.0001), it does not demonstrate any therapeutic efficacy in mice with established tumours. In active therapy the Adh5T4/DCh5T4 vaccination sequence is the best treatment regimen (P = 0.0045). In active therapy, we demonstrate that B16h5T4 tumour growth per se induces Th2 polarising immune responses against 5T4, and the success of subsequent vaccination is dependant on altering the polarizing immune responses from Th2 to Th1. We show that the first immunization with Adh5T4 can condition the mice to induce 5T4 specific Th1 immune responses, which can be sustained and subsequently boosted with DCh5T4. In contrast immunisation with DCh5T4 augments Th2 immune responses, such that a subsequent vaccination with Adh5T4 cannot rescue tumour growth. In this case the depletion of CD25(+) regulatory cells after tumour challenge but before immunization can restore therapeutic efficacy. This study highlights that all vaccine vectors are not equal at generating TAA immune responses; in tumour bearing mice the capability of different vaccines to activate the most appropriate anti-tumour immune responses is greatly altered compared to what is found in naïve mice.

Combination of vaccination and chimeric receptor expressing T cells provides improved active therapy of tumors.

We have generated murine T cells expressing chimeric immune receptors (CR) against human 5T4 oncofetal Ag (h5T4) and evaluated their tumor therapeutic efficacy alone and in combination with immunization using a replication-defective adenovirus encoding h5T4 (Rad.h5T4) and bone marrow-derived dendritic cells (BMDC). The h5T4-specific engineered T cells demonstrated Ag-specific, non-MHC-restricted cytolysis of h5T4-positive B16 and CT26 tumor cells in vitro by cytotoxicity assay and antitumor activity in vivo using a Winn assay. In the s.c. injected B16h5T4 melanoma model, early local but not systemic i.v. administration of syngeneic h5T4-specific CR T cells significantly increased mice survival. This improvement was further enhanced when combined with immunization with Rad.h5T4, followed by post-CR T cell treatment with BMDC in the active therapy model, possibly through mechanisms of enhancing Ag-specific cellular immune responses. This synergistic effect was lost without delivery of the BMDC. Our findings suggest that combining engineered T cells with specific vaccination strategies can improve the active tumor therapy.

Adoptive transfer of anti-idiotypic T cells cure mice of disseminated B cell lymphoma.

There is extensive interest in idiotypic vaccination as a treatment of lymphoma. An alternative approach is the adoptive transfer of in vitro generated T cells. This strategy has been used to treat posttransplantation EBV-related diseases. The ability to generate in vitro T cells to peptides derived from immunoglobulin idiotypes raises the possibility of directly using such cells as a treatment of lymphoma. Investigating the adoptive transfer of specific T cells to idiotype derived peptides in a murine lymphoma model is therefore an important part of the clinical translation of this alternative approach. We have generated an idiotype-specific T cell line, able to recognise a defined, naturally processed idiotype-derived epitope. This line has been used to successfully treat mice with disseminated lymphoma supporting the clinical use of idiotype specific T cells.

Immunization with a recombinant adenovirus encoding a lymphoma idiotype: induction of tumor-protective immunity and identification of an idiotype-specific T cell epitope.

The Ig Id of a B cell lymphoma is a tumor-specific Ag, although as a self-Ag it is likely to be a weak immunogen. Provision of a foreign gene may enhance the immunogenicity of the idiotype. Viral vectors allow highly efficient transfer of genetic material and are themselves innately immunogenic. We have investigated the ability of recombinant adenoviral vectors, encoding the idiotypic gene with or without fusion to the human Fc region, to produce anti-idiotypic Ab- and T cell-mediated responses in a syngeneic BALB/c A20 murine lymphoma model. The idiotypic V(H) and V(L) sequences were assembled as a single chain variable fragment (scFv) and adenoviral vectors encoding the A20 scFv (Ad.A20) and A20 scFv linked to the Fc fragment of human IgG1 (Ad.A20hFc) were constructed. A single immunization of BALB/c mice with Ad.A20hFc but not Ad.A20 induced a specific anti-idiotypic Ab response. T cell lines generated from mice vaccinated with either vector displayed specific cytotoxicity, proliferation, and IFN-gamma release against a syngeneic dendritic cell line transduced using a retroviral vector to express the A20 scFv idiotype (XS52.A1.A20). Importantly, both T cell lines lysed the A20 lymphoma cells. An immunodominant H-2K(d)-restricted CD8(+) T cell peptide, DYWGQGTEL (A20[106-114]), was identified as a naturally occurring A20 scFv epitope. A single immunization with Ad.A20hFc but not Ad.A20 provided protection in >40% of animals challenged with a lethal dose of the A20 tumor line and was more effective, in this model, than a previously optimized plasmid vaccine.