In a previous study we demonstrated that vesicular stomatitis virus (VSV) can be used as a vector to express a soluble protein in mammalian cells. Here we have generated VSV recombinants that express four different membrane proteins: the cellular CD4 protein, a CD4-G hybrid protein containing the ectodomain of CD4 and the transmembrane and cytoplasmic tail of the VSV glycoprotein (G) We reported recently that VSV can be used as a genetically stable, high-level expression vector (2). Here we have investigated VSVs ability to express cellular and viral membrane proteins and we have also examined the incorporation of these proteins, into the vital membrane. Incorporation of foreign viral spike glycoproteins into the envelope of recombinant VSV particles might allow development of a new generation of killed virus vaccines or retargeting of virus particles to specific cell types. Also, expression of other membrane proteins from the VSV genome and their subsequent incorporation into the virions could yield large quantities of the expressed protein in highly purified form for structural or functional studies. Because of these potential applications, we are especially interested in understanding the factors governing membrane protein incorporation into VSV particles. By generating VSV recombinants expressing foreign envelope proteins, we are able to achieve high-level expression of foreign membrane proteins at the same time that the VSV proteins are being made. This system allows us to examine the structural requirements for foreign protein incorporation into VSV particles.Previous studies have shown that the envelope proteins of several other viruses can be incorporated into the envelope of VSV particles when VSV and a second virus are propagated in the same cells. This phenomenon of pseudotype formation is well known (reviewed in ref.3), but the extent of foreign envelope protein incorporation into VSV has not been examined in detail. Also, the cellular CD4 protein expressed from a vaccinia vector was found to be incorporated into VSV particles at a low level of about 60 molecules per virion, and no preference was seen for a chimeric CD4 carrying the transmembrane and cytoplasmic domains of VSV G protein (4). In contrast, a study of HIV envelope protein incorporation into VSV particles lacking G protein indicated a requirement for the cytoplasmic tail of G on the HIV envelope protein (5). These studies complemented earlier studies showing a lower efficiency of incorporation of G proteins with truncated tails into the VSV G ts mutant lacking the G protein (6).Here, using recombinant VSVs expressing very high levels of foreign membrane proteins, we have been able to quantitate both the expression levels of the foreign proteins and their incorporation into VSV particles. We conclude that there is significant extra space in the VSV envelope that can, in many cases, accommodate large amounts of foreign membrane proteins. MATERIALS AND METHODSPlasmid Construction. A plasmid expressing the positive strand RNA complem...

The incidence of and 5-year survival after diagnosis of colorectal adenocarcinoma in transplant recipients is markedly different than the general population. Patients are often diagnosed at a younger age. With current screening guidelines, over 25% of at-risk patients would not be screened. We propose modifying these guidelines to allow earlier detection of colorectal cancer in this population.

Immunocytokines (IC), consisting of tumor-specific monoclonal antibodies fused to the immunostimulatory cytokine interleukin 2 (IL2), exert significant antitumor effects in several murine tumor models. We investigated whether intratumoral (IT) administration of IC provided enhanced antitumor effects against subcutaneous tumors. Three unique ICs (huKS-IL2, hu14.18-IL2, and GcT84.66-IL2) were administered systemically or IT to evaluate their antitumor effects against tumors expressing the appropriate IC-targeted tumor antigens. The effect of IT injection of the primary tumor on a distant tumor was also evaluated. Here, we show that IT injection of IC resulted in enhanced antitumor effects against B16-KSA melanoma, NXS2 neuroblastoma, and human M21 melanoma xenografts when compared to intravenous (IV) IC injection. Resolution of both primary and distant subcutaneous tumors, and a tumor-specific memory response were demonstrated following IT treatment in immunocompetent mice bearing NXS2 tumors. The IT effect of huKS-IL2 IC was antigen-specific, enhanced compared to IL2 alone, and dose-dependent. Hu14.18-IL2 also showed greater IT effects than IL2 alone. The antitumor effect of IT IC did not always require T cells since IT IC induced antitumor effects against tumors in both SCID and nude mice. Localization studies using radiolabeled 111 In-GcT84.66-IL2 IC confirmed that IT injection resulted in a higher concentration of IC at the tumor site than IV administration. In conclusion, we suggest that IT IC is more effective than IV administration against palpable tumors. Further testing is required to determine how to potentially incorporate IT administration of IC into an antitumor regimen that optimizes local and systemic anticancer therapy.

Although great advances have been made in the treatment of low- and intermediate-risk neuroblastoma in recent years, the prognosis for advanced disease remains poor. Therapies based on monoclonal antibodies that specifically target tumour cells have shown promise for treatment of high-risk neuroblastoma. This article reviews the use of monoclonal antibodies either as monotherapy or as part of a multifaceted treatment approach for advanced neuroblastoma, and explains how toxins, cytokines, radioactive isotopes or chemotherapeutic drugs can be conjugated to antibodies to enhance their effects. Tumour resistance, the development of blocking antibodies, and other problems hindering the effectiveness of monoclonal antibodies are also discussed. Future therapies under investigation in the area of immunotherapy for neuroblastoma are considered.

Purpose: Radiofrequency ablation (RFA) is a common treatment modality for surgically unresectable tumors. However, there is a high rate of both local and systemic recurrence. Experimental Design: In this preclinical study, we sought to enhance the antitumor effect of RFA by combining it with huKS-IL2 immunocytokine [tumor-specific monoclonal antibody fused to interleukin-2 (IL2)] in mice bearing CT26-KS colon adenocarcinoma. Mice were treated with RFA, huKS-IL2 via intratumoral injection, or combination therapy. Results: Treatment of mice bearing s.c. tumors with RFA and huKS-IL2 resulted in significantly greater tumor growth suppression and enhanced survival compared with mice treated with RFA or huKS-IL2 alone. When subtherapeutic regimens of RFA or huKS-IL2 were used, tumors progressed in all treated mice. In contrast, the combination of RFA and immunocytokine resulted in complete tumor resolution in 50% of mice. Treatment of a tumor with RFA and intratumoral huKS-IL2 also showed antitumor effects against a distant untreated tumor. Tumor-free mice after treatment with RFA and huKS-IL2 showed immunologic memory based on their ability to reject subsequent challenges of CT26-KS and the more aggressive parental CT26 tumors. Flow cytometry analysis of tumor-reactive T cells from mice with complete tumor resolution showed that treatment with RFA and huKS-IL2 resulted in a greater proportion of cytokineproducing CD4 T cells and CD8 T cells compared with mice treated with RFA or huKS-IL2 alone. Conclusions: These results show that the addition of huKS-IL2 to RFA significantly enhances the antitumor response in this murine model, resulting in complete tumor resolution and induction of immunologic memory.