Virotherapy with a type 2 herpes simplex virus-derived oncolytic virus induces potent antitumor immunity against neuroblastoma.

PURPOSE: We recently constructed an oncolytic virus from type 2 herpes simplex virus (HSV-2) that selectively targets and kills tumor cells with an activated Ras signaling pathway. Designated FusOn-H2, this virus has shown several discrete killing mechanisms. Here, we evaluated the antitumor immune responses after FusOn-H2-mediated virotherapy in a syngeneic murine neuroblastoma model. EXPERIMENTAL DESIGN: We directly injected FusOn-H2 into established tumors and then measured its antitumor effect and the accompanying tumor-specific immune responses. Several oncolytic HSVs constructed from HSV-1 were included in the same experiments for comparisons. RESULTS: Our data show that tumor destruction by FusOn-H2 in vivo induces potent antitumor immune responses in this syngeneic neuroblastoma model. The elicited cellular immunity not only eradicated neuroblastoma cells in vitro but also inhibited the growth of tumors at sites distant from the virus injection site. Moreover, adoptive transfer of splenocytes from mice receiving virotherapy to na?ve mice resulted in a measurable antitumor effect. CONCLUSION: We conclude that the ability of FusOn-H2 to induce tumor-specific cellular immunity expands the oncolytic repertoire of this virus and increases the likelihood that its use in patients would produce significant therapeutic benefits.     

Oncolytic virus and anti-4-1BB combination therapy elicits strong antitumor immunity against established cancer

Oncolytic virotherapy using vaccinia virus (Vv) has shown some encouraging antitumor responses in mouse models and patients, but the breadth of efficacy in clinical trials has been somewhat limited. Given that antitumor effects have correlated with increased host immune responses, we hypothesized that improved therapeutic outcomes may be achieved by using oncolytic virus (OV) in combination with a potent immune agonist reagent. In this study, we carried out a preclinical evaluation of a genetically engineered strain of oncolytic vaccinia virus (Vvdd) for its capacity to induce antitumor responses when combined with an agonist antibody (Ab) specific for the costimulatory molecule 4-1BB (CD137). In immune-competent syngeneic mouse models of cancer, this combination therapy significantly reduced the growth of established subcutaneous tumors relative to either treatment alone. Importantly, the development of pulmonary metastatic lesions was also reduced. Tumor growth inhibition was associated with increased numbers of CD11b(+) and CD11c(+) myeloid cells in the tumor draining lymph nodes, greater infiltration of CD8(+) effector T and natural killer (NK) cells, and a more sustained presence of neutrophils at the tumor site. Depletion of T or NK cells or neutrophils reduced efficacy, confirming their contribution to an effective therapeutic response. We further extended this conclusion through results from IFNγ-deficient mice. In summary, our findings offered a proof-of-concept for a combinatorial approach to enhance the antitumor efficacy of an OV, suggesting a strategy to improve their use as an immunotherapeutic treatment for cancer.     

Co-administration of carcinoembryonic antigen and HIV TAT fusion protein with CpG-oligodeoxynucleotide induces potent antitumor immunity

Although dendritic cells (DC) have been well demonstrated as a strong cellular adjuvant for a tumor vaccine, there are several limitations for clinical application. A protein-based vaccine using a potent adjuvant is an appealing approach for tumor antigen-specific immunotherapy because of their simplicity, safety, efficacy and capacity for repeated administration. CpG-oligodeoxynucleotides (ODN) have been used as adjuvants to stimulate innate and adaptive immune responses for cancer treatment. The authors evaluated the adjuvant effects of CpG-ODN in a vaccine incorporating recombinant fusion protein of the HIV TAT PTD domain and carcinoembryonic antigen (TAT-CEA). Mice vaccinated with TAT-CEA and CpG-ODN (TAT-CEA + CpG) showed enhanced CEA-specific immunity, including cytotoxic T-lymphocytes (CTL) activity and interferon (IFN)-γ secreting T cells compared with CEA and CpG-ODN (CEA + CpG) or TAT-CEA vaccination alone. Vaccination with TAT-CEA + CpG elicited Th1-based responses, as indicated by the higher ratio of immunoglobulin (Ig)G2a antibody/IgG1 antibodies specific for CEA. The survival rate was significantly increased after vaccination with TAT-CEA + CpG in a tumor model using MC38/CEA2. Furthermore, the TAT-CEA ± CpG vaccine groups showed similar antitumor immunity to the CEA peptide-pulsed DC (CEA peptide/DC) vaccine groups. These data suggest that coadministration of TAT fusion protein with CpG-ODN may serve as a potential formulation for enhancing antitumor activity. ( Cancer Sci 2008; 99: 1034–1039)     

Carbon‐ion beam treatment induces systemic antitumor immunity against murine squamous cell carcinoma

BACKGROUND:

Carbon‐ion beam (CIB) treatment is a powerful tool for controlling primary tumors in the clinical setting. However, to date, few clinical or experimental studies have investigated the effects of CIB treatment on tumor recurrence and antitumor immunity.

METHODS:

A multiple challenge test was performed using syngenic and nude mouse models of a poorly immunogenic squamous cell carcinoma cell line (SCCVII) after CIB treatment at a clinically available dose (77 kiloelectron volts [keV]/μm) to primary tumors. To further examine changes in antitumor immunity in this model, the authors used dendritic cell (DC)‐based immunotherapy.

RESULTS:

In a syngenic model, CIB treatment itself resulted not only in efficient elimination of the primary tumor but also in a dramatic reduction of tumor formation after secondary tumor challenge at a contralateral site (P < .0001). Conversely, CIB treatment eliminated neither the primary nor the secondary tumor in nude mice. This antitumor effect produced by CIB treatment was enhanced significantly by combining it with DC immunotherapy (P = .0007). Combined CIB and DC treatment induced more intense cytolytic activity than CIB in a chromium‐release assay. The third challenge tests, which included challenge with a third‐party tumor cell line (FM3A) and effector depletion, revealed that the antitumor effects were the results of tumor‐specific, long‐lasting antitumor immunity through CD8‐positive T lymphocytes.

CONCLUSIONS:

To the authors' knowledge, this is the first demonstration of strong antitumor immunity induced by CIB treatment in a dermal tumor, and this effect was enhanced by combining it with DC‐based immunotherapy. The authors concluded that this combination warrants further investigation as a promising modality for the prevention of tumor recurrence. Cancer 2010. © 2010 American Cancer Society.     

Artesunate induces oncosis-like cell death in vitro and has antitumor activity against pancreatic cancer xenografts in vivo

Pancreatic cancer is highly resistant to the currently available chemotherapeutic agents. Less than 5% of patients diagnosed with this disease could survive beyond 5 years. Thus, there is an urgent need for the development of novel, efficacious drugs that can treat pancreatic cancer. Herein we report the identification of artesunate (ART), a derivative of artemisinin, as a potent and selective antitumor agent against human pancreatic cancer cells in vitro and in vivo. ART exhibits selective cytotoxic activity against Panc-1, BxPC-3 and CFPAC-1 pancreatic cancer cells with IC₅₀ values that are 2.3- to 24-fold less than that of the normal human hepatic cells (HL-7702). The pan caspase inhibitor zVAD-fmk did not inhibit the cytotoxic activity of ART. Electron microscopy of ART-treated cells revealed severe cytoplasmic swelling and vacuolization, swollen and internally disorganized mitochondria, dilation (but not fragmentation) of the nuclei without chromatin condensation, and cell lysis, yielding a morphotype that is typical of oncosis. The ART-treated cells exhibited a loss of mitochondrial membrane potential (ΔΨm) and ART-induced cell death was inhibited in the presence of the reactive oxygen species (ROS) scavenger N-acetyl-cysteine (NAC). Importantly, ART produced a dose-dependent tumor regression in an in vivo pancreatic cancer xenografts model. The in vivo antitumor activity of ART was similar to that of gemcitabine. Taken together, our study suggests that ART exhibits antitumor activity against human pancreatic cancer via a novel form of oncosis-like cell death, and that ART should be considered a potential therapeutic candidate for treating pancreatic cancer.     

Posttransplant administration of cyclophosphamide and donor lymphocyte infusion induces potent antitumor immunity to solid tumor

PURPOSE: Nonmyeloablative allogeneic stem cell transplantation (SCT) has been increasingly used for the treatment of hematologic and solid malignancies, and mature donor T cells are considered to be the main effectors of the graft-versus-tumor (GVT) activity. However, the association between degree of donor chimerism and intensity of GVT effects has not been fully elucidated. We recently proposed a unique nonmyeloablative cell therapy using posttransplant cyclophosphamide and donor lymphocyte infusion, by which a significant antitumor effect against murine renal cell carcinoma, RENCA, was induced, although the level of mixed chimerism was relatively low. In this study, we attempted to clarify a role of chimerism for in vivo antitumor effects on GVT effects in radiation-associated nonmyeloablative SCT. EXPERIMENTAL DESIGN: We assessed antitumor effects on RENCA tumors and the degree of donor chimerism after several doses of irradiation followed by allogeneic SCT and compared the results with those of cyclophosphamide-based cell therapy. RESULTS: Allogeneic SCT following sublethal irradiation (6 Gy) induced almost complete donor chimerism, whereas cyclophosphamide-based cell therapy produced low levels of donor chimerism. Nonetheless, GVT activity was much more potent in cyclophosphamide-based cell therapy than irradiation-conditioned SCT. Furthermore, cyclophosphamide-conditioned SCT induced more potent immune reconstitution with less severe graft-versus-host disease than irradiation-conditioned SCT. CONCLUSIONS: Our results indicate that a high level of chimerism is not essential for the in vivo antitumor effect of nonmyeloablative allogeneic cell therapy against solid tumor and that the recovery of peripheral lymphocytes after the initial immunosuppression might be a critical event for the elicitation of in vivo antitumor effects of that treatment modality.     

Peripheral immunization against malignant rat glioma can induce effective antitumor immunity in the brain

Using two malignant rat glioma cell lines, we tested to what extent peripheral immunization could affect tumor growth in the brain of syngeneic rats. Peripheral subcutaneous (s.c.) immunization was performed with autologous Newcastle-disease-virus (NDV)-infected or non-infected live tumor cells. Thus immunized rats or non-immunized controls were intracerebrally implanted with increasing numbers of the respective malignant glioma cells. Without immunization the mean survival time after intracerebral implantation of 1x10(4) TZ363 or RG2 glioma cells was 9 and 29 days respectively. After s.c. immunization with either NDV-infected or non-infected TZ363 cells only 25% or less of challenged animals developed tumors in the brain. Immunization with NDV only had no effect. In RG2 glioma, s.c. immunization had no effect on tumor growth in the central nervous system and on survival time, no matter what kind of vaccine was used. These results clearly show, that in principle the efferent arm of the anti-tumor response can be effective accross the blood-brain barrier and extend into the microenvironment of the central nervous system. Whether or not glioma lines can induce this immunity and respond to it, seems to depend on their individual immunobiological characteristics.     

Natural killer cells play a key role in the antitumor immunity generated by chaperone-rich cell lysate vaccination

Tumor derived chaperone-rich cell lysate (CRCL) when isolated from tumor tissues is a potent vaccine that contains at least 4 of the highly immunogenic heat shock proteins (HSP) such as HSP70, HSP90, glucose related protein 94 and calreticulin. We have previously documented that CRCL provides both a source of tumor antigens and danger signals triggering dendritic cell (DC) activation. Immunization with tumor derived CRCL elicits tumor-specific T cell responses leading to tumor regression. In the current study, we further dissect the mechanisms by which CRCL simulates the immune system, and demonstrate that natural killer (NK) cells are required for effective antitumor effects to take place. Our results illustrate that CRCL directly stimulates proinflammatory cytokine and chemokine production by NK cells, which may lead to activation and recruitment of macrophages at the tumor site. Thus, this report provides further insight into the function of CRCL as an immunostimulant against cancer.     

Trichostatin A is a histone deacetylase inhibitor with potent antitumor activity against breast cancer in vivo.

PURPOSE: Trichostatin A (TSA), an antifungal antibiotic with cytostatic and differentiating properties in mammalian cell culture, is a potent and specific inhibitor of histone deacetylase (HDAC) activity. The purpose of this study was to evaluate the antiproliferative and HDAC inhibitory activity of TSA in vitro in human breast cancer cell lines and to assess its antitumor efficacy and toxicity in vivo in a carcinogen-induced rat mammary cancer model. EXPERIMENTAL DESIGN AND RESULTS: TSA inhibited proliferation of eight breast carcinoma cell lines with mean +/- SD IC(50) of 124.4 +/- 120.4 nM (range, 26.4-308.1 nM). HDAC inhibitory activity of TSA was similar in all cell lines with mean +/- SD IC(50) of 2.4 +/- 0.5 nM (range, 1.5-2.9 nM), and TSA treatment resulted in pronounced histone H4 hyperacetylation. In randomized controlled efficacy studies using the N-methyl-N-nitrosourea carcinogen-induced rat mammary carcinoma model, TSA had pronounced antitumor activity in vivo when administered to 16 animals at a dose of 500 microg/kg by s.c. injection daily for 4 weeks compared with 14 control animals. Furthermore, TSA did not cause any measurable toxicity in doses of up to 5 mg/kg by s.c. injection. Forty-one tumors from 26 animals were examined by histology. Six tumors from 3 rats treated with TSA and 14 tumors from 9 control animals were adenocarcinomas. In contrast, 19 tumors from 12 TSA-treated rats had a benign phenotype, either fibroadenoma or tubular adenoma, suggesting that the antitumor activity of TSA may be attributable to induction of differentiation. Two control rats each had tumors with benign histology. CONCLUSIONS: The present studies confirm the potent dose-dependent antitumor activity of TSA against breast cancer in vitro and in vivo, strongly supporting HDAC as a molecular target for anticancer therapy in breast cancer.     

Oncolytic treatment and cure of neuroblastoma by a novel attenuated poliovirus in a novel poliovirus-susceptible animal model

Neuroblastoma is one of the most common solid tumors in children. Treatment is of limited utility for high-risk neuroblastoma and prognosis is poor. Resistance of neuroblastoma to conventional therapies has prompted us to search for a novel therapeutic approach based on genetically modified polioviruses. Poliovirus targets motor neurons leading to irreversible paralysis. Neurovirulence can be attenuated by point mutations or by exchange of genetic elements between different picornaviruses. We have developed a novel and stable attenuated poliovirus, replicating in neuroblastoma cells, by engineering an indigenous replication element (cre), copied from a genome-internal site, into the 5'-nontranslated genomic region (mono-crePV). An additional host range mutation (A(133)G) conferred replication in mouse neuroblastoma cells (Neuro-2a(CD155)) expressing CD155, the poliovirus receptor. Crossing immunocompetent transgenic mice susceptible to poliovirus (CD155 tg mice) with A/J mice generated CD155 tgA/J mice, which we immunized against poliovirus. Neuro-2a(CD155) cells were then transplanted into these animals, leading to lethal tumors. Despite preexisting high titers of anti-poliovirus antibodies, established lethal s.c. Neuro-2a(CD155) tumors in CD155 tgA/J mice were eliminated by intratumoral administrations of A(133)Gmono-crePV. No signs of paralysis were observed. Interestingly, no tumor growth was observed in mice cured of neuroblastoma that were reinoculated s.c. with Neuro-2a(CD155). This result indicates that the destruction of neuroblastoma cells by A(133)Gmono-crePV may lead to a robust antitumor immune response. We suggest that our novel attenuated oncolytic poliovirus is a promising candidate for effective oncolytic treatment of human neuroblastoma or other cancer even in the presence of present or induced antipolio immunity.     

Combined suicide and granulocyte-macrophage colony-stimulating factor gene therapy induces complete tumor regression and generates antitumor immunity

The use of prodrug-activated ("suicide") gene therapy has been shown to be effective in inducing tumor regression when only a small proportion of tumor cells contains the suicide gene. These experiments were designed to test whether additional therapeutic benefit may be obtained by stimulating the immune response. Murine MC26 colon carcinoma cells, either untransduced or transduced with genes for herpes simplex virus-1 thymidine kinase (HSV1-TK) or human GM-CSF, were injected subcutaneously into syngeneic BALB/c mice in various combinations. Inoculation of equal numbers of untransduced and HSV1-TK-containing cells followed by ganciclovir (GCV) treatment resulted in almost complete tumor regression, but by 7 weeks, tumors had recurred in all mice. A similar initial regression was obtained using equal numbers of cells containing HSV1-TK and GM-CSF genes, but >80% of these mice remained tumor-free after 3 months. Groups of tumor-free mice that had received GM-CSF-containing cells were left for different periods of time and rechallenged with unmodified MC26 cells on the opposite flank. Of the mice rechallenged 14, 28, and 108 days later, 100%, 88%, and 57%, respectively, showed complete resistance to unmodified tumor cells. In mice that showed tumor regrowth, tumor volume was much less than in control mice. Adoptive transfer of spleen cells from resistant mice to na?ve syngeneic mice resulted in partial resistance to challenge with unmodified tumor cells. Specific cytotoxicity against MC26 cells was only demonstrable in mice receiving GM-CSF- and HSV1-TK-containing tumor cells. These experiments show that the presence of cells secreting GM-CSF in HSV1-TK-containing, regressing tumor is able to induce complete or partial resistance to tumor rechallenge. This indicates the potential usefulness of GM-CSF in enhancing other antitumor therapies.     

Anti-cancer effects of bortezomib against chemoresistant neuroblastoma cell lines in vitro and in vivo

The proteasome inhibitor bortezomib (Velcade) was recently approved for the treatment of therapy-refractive multiple myeloma and is under investigation for numerous other types of cancer. A phase I clinical trial in paediatric patients resulted in tolerable toxicity. Since the emergence of chemoresistance represents one of the major drawbacks in cancer therapy, we investigated the influence of bortezomib on multi-drug resistant human neuroblastoma cell lines characterised by P-glycoprotein expression and p53 mutation. Nanomolar concentrations of bortezomib inhibited the cell cycle and induced apoptosis in chemosensitive as well as in chemoresistant cell lines. In vivo growth of chemosensitive and chemoresistant neuroblastoma cell lines was inhibited to a similar extent. In addition, bortezomib inhibited vessel formation in neuroblastoma xenografts. These findings and the favourable toxicity profile of bortezomib in children make it reasonable to further pursue additional development of the drug for the treatment of neuroblastoma and other paediatric solid tumours.     

Vaccination with human HER-2/neu (435-443) CTL peptide induces effective antitumor immunity against HER-2/neu-expressing tumor cells in vivo

HER-2/neu is a self-antigen expressed by tumors and nonmalignant epithelial tissues. The possibility of self-tolerance to HER-2/neu-derived epitopes has raised questions concerning their utility in antitumor immunotherapy. Altered HER-2/neu peptide ligands capable of eliciting enhanced immunity to tumor-associated HER-2/neu epitopes may circumvent this problem. The human CTL peptide HER-2/neu (435-443) [hHER-2(9(435))] represents a xenogeneic altered peptide ligand of its mouse homologue, differing by one amino acid residue at position 4. In contrast to mHER-2(9(435)), vaccination of HLA-A*0201 transgenic (HHD) mice with hHER-2(9(435)) significantly increased the frequency of mHER-2(9(435))-specific CTL and also induced strong protective and therapeutic immunity against the transplantable ALC tumor cell line transfected to coexpress HLA-A*0201 and hHER-2/neu or rHER-2/neu. Similar results were also obtained with wild-type C57BL/6 mice inoculated with HER-2/neu transfectants of ALC. Adoptive transfer of CD8(+) CTL from mice immunized with hHER-2(9(435)) efficiently protected naive syngeneic mice inoculated with ALC tumors. In conclusion, our results show that HER-2(9(435)) serves as a tumor rejection molecule. They also propose a novel approach for generating enhanced immunity against a self-HER-2/neu CTL epitope by vaccinating with xenogeneic altered peptide ligands and provide useful insights for the design of improved peptide-based vaccines for the treatment of patients with HER-2/neu-overexpressing tumors.     

All-trans retinoic acid downregulates ALK in neuroblastoma cell lines and induces apoptosis in neuroblastoma cell lines with activated ALK

Recently, gene amplification and gain-of-function mutations of ALK have been found in some neuroblastoma cell lines and clinical tumor samples. We have previously reported that knockdown of ALK by RNAi induced apoptosis in neuroblastoma cells with gene amplification of ALK. We report that all-trans retinoic acid (ATRA) downregulates ALK in neuroblastoma cell lines. Downregulation of ALK protein by ATRA was accompanied by apoptosis in neuroblastoma cells with gene amplification or gain-of-function mutation of ALK but not in neuroblastoma cells without these genetic alterations. These results suggest that ALK downregulation by ATRA might lead to apoptosis in neuroblastoma cells with activated ALK.     

Selective antibody-mediated targeting of class I MHC to EGFR-expressing tumor cells induces potent antitumor CTL activity in vitro and in vivo

Epidermal growth factor receptor (EGFR) is highly overexpressed in many tumor types. We present a new fusion molecule that can target solid tumors that express EGFR. The fusion molecule combines the advantage(s) of the well-established tumor targeting capabilities of high affinity recombinant fragments of antibodies with the known efficient, specific and potent killing ability of CD8 T lymphocytes directed against highly antigenic MHC/peptide complexes. A recombinant chimeric molecule was created by the genetic fusion of the scFv antibody fragment derived from the anti-EGFR monoclonal antibody C225, to monomeric single-chain HLA-A2 complexes containing immunodominant tumor or viral-specific peptides. The fusion protein can induce very efficiently CTL-dependent lysis of EGFR-expressing tumor cells regardless of the expression of self peptide-MHC complexes. Moreover, the molecule exhibited very potent antitumor activity in vivo in nude mice bearing preestablished human tumor xenografts. These in vitro and in vivo results indicate that recombinant scFv-MHC-peptide fusion molecules might represent a novel and powerful approach to immunotherapy of solid tumors, bridging antibody and T lymphocyte attack on cancer cells.     

Chimeric, divalent and tetravalent anti-CD19 monoclonal antibodies with potent in vitro and in vivo antitumor activity against human B-cell lymphoma and pre-B acute lymphoblastic leukemia cell lines

CD19 is an attractive therapeutic target for treating human B-cell tumors. In our study, chimeric (c) divalent (cHD37) and tetravalent (cHD37-DcVV) anti-CD19 monoclonal antibodies (MAbs) were constructed, expressed and evaluated for their binding to human 19-positive (CD19(+)) tumor cell lines. They were also tested for proapoptotic activity and the ability to mediate effector functions. The antitumor activity of these MAbs was further tested in mice xenografted with the CD19(+) Burkitt's lymphoma cell line, Daudi or the pre-B acute lymphoblastic leukemia (ALL) cell line, NALM-6. The cHD37 and cHD37-DcVV MAbs exhibited specific binding and comparable proapoptotic activity on CD19(+) tumor cell lines in vitro. In addition, the cHD37 and cHD37-DcVV MAbs were similar in their ability to mediate antibody-dependent cell-mediated phagocytosis (ADCP). However, the tetravalent cHD37-DcVV MAb bound more avidly, had a slower dissociation rate, and did not internalize as well. It also had enhanced antibody-dependent cellular cytotoxicity (ADCC) with human but not murine effector cells. The cHD37 and cHD37-DcVV MAbs exhibited comparable affinity for the human neonatal Fc receptor (FcRn) and similar pharmacokinetics (PKs) in mice. Moreover, all the HD37 constructs were similar in extending the survival of mice xenografted with Daudi or NALM-6 tumor cells. Therefore, the cHD37 and cHD37-DcVV MAbs have potent antitumor activity and should be further developed for use in humans. Although not evident in mice, due to its increased ability to mediate ADCC with human but not mouse effector cells, the cHD37-DcVV MAb should have superior therapeutic efficacy in humans.     

Mebendazole elicits a potent antitumor effect on human cancer cell lines both in vitro and in vivo.

We have found that mebendazole (MZ), a derivative of benzimidazole, induces a dose- and time-dependent apoptotic response in human lung cancer cell lines. In this study, MZ arrested cells at the G(2)-M phase before the onset of apoptosis, as detected by using fluorescence-activated cell sorter analysis. MZ treatment also resulted in mitochondrial cytochrome c release, followed by apoptotic cell death. Additionally, MZ appeared to be a potent inhibitor of tumor cell growth with little toxicity to normal WI38 and human umbilical vein endothelial cells. When administered p.o. to nu/nu mice, MZ strongly inhibited the growth of human tumor xenografts and significantly reduced the number and size of tumors in an experimental model of lung metastasis. In assessing angiogenesis, we found significantly reduced vessel densities in MZ-treated mice compared with those in control mice. These results suggest that MZ is effective in the treatment of cancer and other angiogenesis-dependent diseases.     

In situ expression of soluble B7-1 in the context of oncolytic herpes simplex virus induces potent antitumor immunity

In vivo delivery of immunomodulatory genes is a promising strategy for solid tumor vaccination. A drawback is that it necessitates induction of a large effect from transgene expression in a small percentage of tumor cells. Although the B7 family is known to be the most potent of the costimulatory molecules, gene transduction of B7 alone has not been effective in inducing antitumor immunity in nonimmunogenic tumors by ex vivo methods, much less in vivo. We have developed a novel approach where a gene encoding soluble B7-1, a fusion protein of the extracellular domain of murine B7-1 and the Fc portion of human IgG1, is delivered to tumor cells in vivo in the context of an oncolytic replication-competent herpes simplex virus, and the gene product is secreted by tumor cells rather than expressed on the cell surface. Defective herpes simplex virus vectors containing the B7-1-immunoglobulin (B7-1-Ig) fusion transgene (dvB7Ig) were generated using G207 as a helper virus and tested in the poorly immunogenic murine neuroblastoma, Neuro2a, in syngeneic A/J mice. Intraneoplastic inoculation of dvB7Ig/G207 at a low titer successfully inhibited the growth of established s.c. tumors, despite the expression of B7-1-Ig being detected in only 1% or fewer of tumor cells at the inoculation site, and prolonged the survival of mice bearing intracerebral tumors. Immunohistochemistry of dvB7Ig/G207-inoculated tumors revealed a significant increase in CD4+ and CD8+ T-cell infiltration compared with control tumors inoculated with defective vector expressing alkaline phosphatase (dvAP/G207). The antitumor effect of dvB7Ig/G207 was not manifested in athymic mice. In vivo depletion of immune cell subsets in A/J mice further revealed that CD8+ T cells, but not CD4+ T cells, were required. Animals cured of their tumors by dvB7Ig/G207 treatment were protected against rechallenge with a lethal dose of Neuro2a cells but not SaI/N cells. The results demonstrate that the use of soluble B7-1 for immune gene therapy is a potent and clinically applicable means of in situ cancer vaccination.     

CHS 828, a novel pyridyl cyanoguanidine with potent antitumor activity in vitro and in vivo

A new class of recently discovered antineoplastic agents, the pyridyl cyanoguanidines, exert a potent antitumor activity in rodents after oral administration. Optimization in vitro and in vivo has resulted in the selection of the lead candidate CHS 828 (N-(6-chlorophenoxyhexyl)-N'cyano-N"-4-pyridylguanidine). CHS 828 was found to exert potent cytotoxic effects in human breast and lung cancer cell lines, with lesser effects on normal fibroblasts and endothelial cells. In a study using a panel of cell lines with different resistance patterns, the effects of CHS 828 showed a low correlation with the activity patterns of known anticancer agents, and no sensitivity to known mechanisms of multidrug resistance was observed. In nude mice bearing human tumor xenografts, CHS 828, at doses from 20 to 50 mg/kg/day p.o., inhibited the growth of MCF-7 breast cancer tumors and caused regression of NYH small cell lung cancer tumors. Oral administration of CHS 828 once weekly improved efficacy without increasing toxicity. CHS 828 was found to compare favorably with established chemotherapeutic agents such as cyclophosphamide, etoposide, methotrexate, and paclitaxel. In mice with NYH tumors, long-term survival (>6 months) was observed after treatment with CHS 828 was stopped. In conclusion, CHS 828 is an effective new antitumor agent, with a potentially new mechanism of action. CHS 828 is presently being tested in Phase I clinical trials in collaboration with the European Organization for Research and Treatment of Cancer.