Non-clinical safety evaluation of repeated intramuscular administration of the AS15 immunostimulant combined with various antigens in rabbits and cynomolgus monkeys.

Combination of tumor antigens with immunostimulants is a promising approach in cancer immunotherapy. We assessed animal model toxicity of AS15 combined with various tumor antigens: WT1 (rabbits), or p501, dHER2 and recPRAME (cynomolgus monkeys), administered in seven or 20 dose regimens versus a saline control. Clinical and ophthalmological examinations, followed by extensive post-mortem pathological examinations, were performed on all animals. Blood hematology and biochemistry parameters were also assessed. Antigen-specific antibody titers were determined by enzyme-linked immunosorbent assay. Additional assessments in monkeys included electrocardiography and immunohistochemical evaluations of the p501 expression pattern. Transient increases in body temperature were observed 4 h or 24 h after injections of recPRAME + AS15 and dHER2 + AS15. Edema and erythema were observed up to 1 week after most injections of recPRAME + AS15 and all injections of dHER2 + AS15. No treatment-related effects were observed for electrocardiography parameters. Mean fibrinogen levels were significantly higher in all treated groups compared to controls, but no differences could be observed at the end of the treatment-free period. Transient but significant differences in biochemistry parameters were observed post-injection: lower albumin/globulin ratios (p501 + AS15), and higher bilirubin, urea and creatinine (dHER2 + AS15). Pathology examinations revealed significant increases in axillary lymph node mean weights (recPRAME + AS15) compared to controls. A 100% seroconversion rate was observed in all treated groups, but not in controls. p501 protein expression was observed in prostates of all monkeys from studies assessing p501 + AS15. These results suggest a favorable safety profile of the AS15-containing candidate vaccines, supporting the use of AS15 for clinical development of potential anticancer vaccines.

Nonclinical reproductive and developmental safety evaluation of the MAGE-A3 Cancer Immunotherapeutic, a therapeutic vaccine for cancer treatment.

We assessed potential toxic effects of the MAGE-A3 Cancer Immunotherapeutic on female fertility and embryo-fetal, pre- and post-natal development in rats and on male fertility in rats and monkeys. Three groups of 48 female (Study 1) or 22 male (Study 2) CD rats received 5 or 3 injections of 100µL of saline, AS15 immunostimulant, or MAGE-A3 Cancer Immunotherapeutic (MAGE-A3 recombinant protein combined with AS15) at various timepoints pre- or post-mating. Male Cynomolgus monkeys (Study 3) received 8 injections of 500µL of saline (n=2) or the MAGE-A3 Cancer Immunotherapeutic (n=6) every 2 weeks. Rats were sacrificed on gestation day 20 or lactation day 25 (Study 1) or 9 weeks after first injection (Study 2) and monkeys, 3 days or 8 weeks after last injection. Injections were well tolerated. Female rat mating performance or fertility, pre- and post-natal survival, offspring development up to 25 days of age, and male mating performance (rats) or fertility parameters (rats and monkeys) were unaffected.

Recombinant human papillomavirus type 16 E7 protein as a model antigen to study the vaccine potential in control and E7 transgenic mice.

The early genes E6 and E7 of human papillomavirus type 16 (HPV16) are consistently and exclusively expressed in HPV16-induced cancer lesions and play major roles in the development and maintenance of the malignant phenotype. Because this protein is a good example of a tumor-associated antigen, we have used E7 as a model antigen to test the potential of an experimental vaccine as an immunotherapeutic approach. In this study, we used a murine E7-expressing tumor model (TC1 cells) to assess effects of an E7-based vaccine on tumor growth. We show that vaccination with the E7 protein, formulated in the SmithKline Beecham Biologicals proprietary adjuvants (SBAS 1 and SBAS 2), leads to the rejection of pre-established tumors. Tumor rejection was associated with the induction of a strong systemic T helper 1 response, including CTLs, and the presence of an inflammatory infiltrate within the regressing tumor. Because most identified tumor-associated antigens are self antigens rather viral antigens, we used E7 transgenic mice to evaluate the E7-based vaccine in conditions where E7 is a self antigen. Transgenic mice, which constitutively and specifically express the E7 HPV16 gene in the thyroid epithelium, rapidly develop thyroid goiters and, after several months, thyroid carcinomas. We show that E7-specific antibodies and CD4 T helper responses can be obtained by vaccinating E7 transgenic mice, although a CTL response was not detected. Despite the absence of measurable CTL responses, vaccination still reduced the growth of pre-established TC1 tumors, although less efficiently than in nontransgenic animals, but was unable to suppress or delay the development of the spontaneous thyroid pathology.

Therapeutic potential of protein and adjuvant vaccinations on tumour growth.

Over 90% of cervical cancers are associated with HPV infection, the commonest being the HPV-16 subtype. Two early viral genes, E6 and 7, play major roles in the development and maintenance of the malignant phenotype. The vaccine potential of a recombinant HPV16 E7 protein was examined in two murine models of E7-expressing tumours. Formulations including the immunostimulants MPL and QS21 induced therapeutically active immune responses leading to regression of pre-established TC1 tumour lesions, associated with induction of IgG antibodies, lymphoproliferation and CTL. Our data provide a clear incentive to investigate the clinical application of this approach in cancer immunotherapy.

The role of cellular- and prodrug-associated factors in the bystander effect induced by the Varicella zoster and Herpes simplex viral thymidine kinases in suicide gene therapy.

To investigate the factors influencing the bystander effect--a key element in the efficacy of suicide gene therapy against cancer--we compared the effect triggered by four extremely efficient gene/prodrug combinations, i.e., VZVtk/BVDU, the thymidine kinase of Varicella zoster virus associated with (E)-5-(2-bromovinyl)-2'-deoxyuridine; VZVtk/BVaraU, the same enzyme associated with (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil; HSVtk/BVDU, the association of the Herpes simplex virus thymidine kinase with BVDU; and the classical HSVtk/GCV (ganciclovir) paradigm. The cells used, the human MDA-MB-435 breast cancer, and the rat 9L glioblastoma lines were equally sensitive in vitro to these four associations. In both cell types, the combinations involving pyrimidine analogues (BVDU, BVaraU) displayed a smaller bystander killing than the combination involving the purine analogue (GCV). In addition, the bystander effect induced by all the tk/prodrug systems was reduced in MDA-MB-435 cells in comparison to 9L cells; albeit, the viral kinases were produced at a higher level in the breast cancer cells. All systems induced apoptotic death in the two cell types, but the MDA-MB-435 cells, deprived of connexin 43, were noncommunicating in striking contrast with the 9L cells. That functional gap junctions have to be increased in order to improve the breast cancer cell response to suicide gene therapy was demonstrated by transducing the Cx43 gene: this modification enhanced the bystander effect associated in vitro with GCV treatment and, by itself, decreased the tumorigenicity of the untreated cells. However, the noncommunicating MDA-MB-435 cells triggered a significant bystander effect both in vitro and in vivo with the HSVtk/GCV system, showing that communication through gap junctions is not the only mechanism involved.

Role and expression of CD40 on human retinal pigment epithelial cells.

PURPOSE: To examine the CD40 costimulatory molecule expression on normal resting or activated adult human retinal pigment epithelium (hRPE) cells and to evaluate its role as an activation molecule considering the potential antigen presentation functions of hRPE cells. METHODS: Expression of HLA-DR and costimulatory (CD40, B7.1, B7.2, CD54, and CD58) molecules on hRPE cells was analyzed by flow cytometry. CD40 triggering was performed using soluble CD40L or cocultures with CD40L transfected fibroblasts. Interleukin (IL)-6, -8, -10, and -12 secretions were measured by enzyme-linked immunosorbent assay. Antigen presentation function of hRPE cells was assessed by coculturing hRPE cells with allogeneic T cells. T-cell proliferation was measured by [(3)H]-thymidine incorporation, and T-cell apoptosis by measurement of caspase-3 activity. RESULTS: Interferon (IFN)gamma-activated hRPE cells expressed CD40, but not B7.1 or B7.2. Although interferongamma enhanced IL-6 and IL-8 production, CD40 triggering of IFNgamma-activated hRPE cells did not induce IL-12 secretion. hRPE cells did not stimulate allogeneic resting T cells and downregulated phytohemagglutinin-activated allogeneic T cells via a cell-to-cell contact-dependent mechanism. Some induction of apoptosis was detected. CONCLUSIONS: CD40 is expressed on IFNgamma-activated hRPE cells. Its ligation leads to an increased production of IL-6 and IL-8 but fails to induce B7.1 or B7. 2 expression, or to induce IL-12 secretion. Accordingly, hRPE cells do not activate allogenic T cells but inhibit T-cell proliferation, partly through induction of apoptosis. These results suggest that hRPE cells could be implicated more in a deviant antigen presentation. If the exact molecular mechanisms are unclear, it is likely that CD40-CD40L interaction could play a role in this process.

Comparative in vitro and in vivo cytotoxic activity of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and its arabinosyl derivative, (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil (BVaraU), against tumor cells expressing either the Varicella zoster or the Herpes simplex virus thymidine kinase.

The inhibitory effects of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and its arabinosyl derivative (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil (BVaraU) on the growth of both MDA-MB-435 human breast carcinoma and 9L rat gliosarcoma cells expressing the thymidine kinase (tk)-encoding gene of the Varicella zoster virus (VZV) or the Herpes simplex virus (HSV) were evaluated. In vitro, BVDU and BVaraU effectively killed both cell types expressing VZVtk, with 50% inhibitory concentration values ranging from 0.06 to 0.4 microM, whereas ganciclovir (GCV) lacked activity. On HSVtk+ cells, BVDU had high cytotoxic activity, with 50% inhibitory concentration values that were similar to those of GCV, whereas BVaraU was inactive. In vivo, BVDU applied intraperitoneally caused a 50% tumor growth inhibition in nude mice inoculated subcutaneously with VZVtk+ as well as HSVtk+ mammary tumor cells. In mice and at variance with the in vitro results, BVaraU had very little activity against the VZVtk+ mammary cells; GCV had the highest activity on the HSVtk+ cells, resulting in a 50% eradication of the tumors. With the 9L rat gliosarcoma model, the VZVtk/BVDU system completely failed to inhibit the development of VZVtk+ glioma tumors induced subcutaneously in syngeneic rats, although BVDU had a similar 45-minute half-life in both rats and mice. Factors other than degradation of the prodrug and related to the mode of action of these analogs are possibly involved in the observed discrepancies between the in vitro and in vivo results.

Curative potential of herpes simplex virus thymidine kinase gene transfer in rats with 9L gliosarcoma.

The transfer of the gene coding for the thymidine kinase of the herpes simplex virus (HSV-tk), followed by ganciclovir (GCV) administration, has been described for the treatment of several types of cancer, especially brain tumors. We further studied the efficacy of this approach by using the 9L rat gliosarcoma model, and cells producing 5 x 10(3), 9 x 10(4), 3 x 10(5) HSV-tk retroviral particles per milliliter. Their stereotactic injection in 9L brain tumors and GCV treatment did not result in any increase of survival. To study a model of optimal in vivo transduction, we examined the survival of rats with tumors growing from 9L cells that had been previously transduced in vitro with the HSV-tk vectors (9LTk cells). We observed that GCV administration cured 26% (n = 42) of the animals with 9LTk brain tumors, with most of the relapsing tumors remaining HSV-tk positive. The increase of either the dose or the duration of GCV treatment did not improve the survival rate. But the cerebral localization of the tumor played an important role, because this survival rate reached 67% (n = 12) when similar tumors were growing subcutaneously. No or only marginal antitumoral responses were induced by the presence of a selectable marker gene in the HSV-tk vectors. These results demonstrate that in vitro HSV-tk gene transfer in 9L tumor cells, but not in vivo gene transfer, followed by GCV treatment, is able to cure rats at a rate that is higher for subcutaneous than for intracerebral tumors.

Loss of tumorigenicity and increased immunogenicity induced by interleukin-10 gene transfer in B16 melanoma cells.

Because interleukin-10 (IL-10) has potent immunosuppressive and anti-inflammatory properties and is produced by some cancers, we hypothesized that its production might play a role in carcinogenesis by inhibiting adequate antitumoral immune responses. To test this hypothesis, retroviral vectors containing the IL-10 cDNA were generated and used to infect B16F1 melanoma cells that were injected subcutaneously in syngeneic mice. Surprisingly, IL-10 gene transfer resulted in a loss of tumorigenicity that was proportional to the amount of IL-10 secreted. Histological analysis showed massive area of necrosis of these tumor cells, with infiltration of polymorphic inflammatory cells. Parental cells simultaneously implanted had decreased tumorigenicity only when mixed with IL10-producing cells, but not when injected contralaterally, suggesting that their eradication is mediated mostly by a local phenomenon. Host T lymphocytes and natural killer (NK) cells were involved in this eradication because IL-10-producing cells grew in nude mice and in CD8+ or NK-depleted mice. Finally, mice injected with IL-10-secreting cells developed an antitumoral systemic immune response able to protect them against a subsequent challenge with parental cells. These results demonstrate that, in some settings, IL10 may have in vivo immunostimulating and proinflammatory properties that need to be considered in its therapeutic development.