MIG (CXCL9) chemokine gene therapy combines with antibody-cytokine fusion protein to suppress growth and dissemination of murine colon carcinoma.

The induction of a CTL response capable of eradicating disseminated tumor metastases and the establishment of a persistent tumor-protective immunity remain major goals of cancer immunotherapy. Here, we demonstrate for the first time that the combination of interleukin 2 (IL-2) targeted to the tumor microenvironment by a recombinant antibody-IL-2 fusion protein (huKS1/4-IL-2) with gene therapy by the murine chemokine MIG (CXCL9) markedly reduced s.c. tumor burden and decisively suppressed dissemination of experimental lung metastases of CT26-KSA colon carcinoma in syngeneic BALB/c mice. This combined therapy significantly prolonged the life span of these mice 3-4-fold by concurrently delivering MIG and IL-2 to the tumor site and thereby achieving chemoattraction of T cells together with their activation. The antitumor effect obtained was mediated predominantly by MHC class I antigen-restricted CD8(+) T cells with help from MHC class II antigen-restricted CD4(+) T lymphocytes. In addition, the MIG chemokine also induced angiostatic effects in the tumor vasculature. Taken together, this combination of MIG chemokine gene therapy with tumor-targeted cytokine IL-2 provides an approach for the rational design of novel cancer immunotherapy modalities.

In vivo activity in a catalytic antibody-prodrug system: Antibody catalyzed etoposide prodrug activation for selective chemotherapy.

Effective chemotherapy remains a key issue for successful cancer treatment in general and neuroblastoma in particular. Here we report a chemotherapeutic strategy based on catalytic antibody-mediated prodrug activation. To study this approach in an animal model of neuroblastoma, we have synthesized prodrugs of etoposide, a drug widely used to treat this cancer in humans. The prodrug incorporates a trigger portion designed to be released by sequential retro-aldol/retro-Michael reactions catalyzed by aldolase antibody 38C2. This unique prodrug was greater than 10(2)-fold less toxic than etoposide itself in in vitro assays against the NXS2 neuroblastoma cell line. Drug activity was restored after activation by antibody 38C2. Proof of principle for local antibody-catalyzed prodrug activation in vivo was established in a syngeneic model of murine neuroblastoma. Mice with established 100-mm3 s.c. tumors who received one intratumoral injection of antibody 38C2 followed by systemic i.p. injections with the etoposide prodrug showed a 75% reduction in s.c. tumor growth. In contrast, injection of either antibody or prodrug alone had no antitumor effect. Systemic injections of etoposide at the maximum tolerated dose were significantly less effective than the intratumoral antibody 38C2 and systemic etoposide prodrug combination. Significantly, mice treated with the prodrug at 30-fold the maximum tolerated dose of etoposide showed no signs of prodrug toxicity, indicating that the prodrug is not activated by endogenous enzymes. These results suggest that this strategy may provide a new and potentially nonimmunogenic approach for targeted cancer chemotherapy.

IFN-gamma-inducible protein-10 is essential for the generation of a protective tumor-specific CD8 T cell response induced by single-chain IL-12 gene therapy.

The successful induction of T cell-mediated protective immunity against poorly immunogenic malignancies remains a major challenge for cancer immunotherapy. Here, we demonstrate that the induction of tumor-protective immunity by IL-12 in a murine neuroblastoma model depends entirely on the CXC chemokine IFN-gamma-inducible protein 10 (IP-10). This was established by in vivo depletion of IP-10 with mAbs in mice vaccinated against NXS2 neuroblastoma by gene therapy with a linearized, single-chain (sc) version of the heterodimeric cytokine IL-12 (scIL-12). The efficacy of IP-10 depletion was indicated by the effective abrogation of scIL-12-mediated antiangiogenesis and T cell chemotaxis in mice receiving s.c. injections of scIL-12-producing NXS2 cells. These findings were extended by data demonstrating that IP-10 is directly involved in the generation of a tumor-protective CD8+ T cell-mediated immune response during the early immunization phase. Four lines of evidence support this contention: First, A/J mice vaccinated with NXS2 scIL-12 and depleted of IP-10 by two different anti-IP-10 mAbs revealed an abrogation of systemic-protective immunity against disseminated metastases. Second, CD8+ T cell-mediated MHC class I Ag-restricted tumor cell lysis was inhibited in such mice. Third, intracellular IFN-gamma expressed by proliferating CD8+ T cells was substantially inhibited in IP-10-depleted, scIL-12 NXS2-vaccinated mice. Fourth, systemic tumor protective immunity was completely abrogated in mice depleted of IP-10 in the early immunization phase, but not if IP-10 was depleted only in the effector phase. These findings suggest that IP-10 plays a crucial role during the early immunization phase in the induction of immunity against neuroblastoma by scIL-12 gene therapy.

Protective immunity against human carcinoembryonic antigen (CEA) induced by an oral DNA vaccine in CEA-transgenic mice.

Peripheral T-cell tolerance toward human carcinoembryonic self-antigen (CEA) was broken in CEA-transgenic C57BL/6J mice by an oral CEA-based DNA vaccine. This vaccine, delivered by the live, attenuated AroA- strain of Salmonella typhimurium (SL7207), induced tumor-protective immunity mediated by MHC class I-restricted CD8+ T cells. Activation of these T cells was indicated by increased secretion of proinflammatory cytokines IFN-gamma, interleukin (IL)-12 and granulocyte/macrophage-colony stimulating factor, as well as specific tumor rejection and growth suppression in vaccinated CEA-transgenic mice after a lethal challenge with murine MC38 colon carcinoma cells. These tumor cells were double transfected with CEA and the human epithelial cell adhesion molecule (Ep-CAM)/KSA and consequently served as a docking site for a recombinant antibody-IL2 fusion protein (KS1/4-IL2) recognizing KSA. Importantly, the efficacy of the tumor-protective immune response was markedly increased by boosts with this antibody-IL2 fusion protein, resulting in more effective tumor rejection coupled with increased expression of costimulatory molecules B7.2/B7.2 and intercellular adhesion molecule 1 (ICAM-1) on dendritic cells and intensified release of proinflammatory cytokines IFN-gamma, IL-12, and granulocyte/macrophage-colony stimulating factor from T cells of successfully vaccinated CEA-transgenic C57BL/6J mice. Increased T-cell activation mediated by boosts with KS1/4-IL2 fusion protein after tumor cell challenge was further indicated by expanded expression of T-cell activation markers CD25, CD28, CD69, and LFA-1. The application of such CEA-based DNA vaccines and its further improved versions may ultimately prove useful in combination therapies directed against human carcinomas expressing CEA self-antigens.

Tyrosine hydroxylase-based DNA-vaccination is effective against murine neuroblastoma.

BACKGROUND: The disruption of self-tolerance against neuroblastoma is the ultimate goal of an effective DNA-vaccine. PROCEDURE: Here we demonstrate the induction of a protective immunity against syngeneic murine NXS2 neuroblastoma in A/J mice, following vaccination with tyrosine hydroxylase (TH) derived antigens. Oral gene delivery was accomplished using an attenuated strain of Salmonella typhimurium as a carrier harboring vectors encoding for mTH antigens. RESULTS: Vaccination was effective in protecting animals from a lethal challenge with wild-type NXS2 tumor cells. CONCLUSIONS: These results provide the first evidence of the TH self antigen being recognized by T-cells and demonstrate that a TH-based DNA vaccine is a potentially useful immunotherapeutic strategy for neuroblastoma.

Histone H2A-mediated transient cytokine gene delivery induces efficient antitumor responses in murine neuroblastoma.

A major goal of cancer immunotherapy is the induction of a cell-mediated antitumor response in poorly immunogenic malignancies. We tested the hypothesis that this can be achieved by cytokine gene therapy with a novel histone H2A-based transient transfection procedure. This was tested by using cytokine genes encoding for IL-2 and a single chain IL-12 (scIL-12) fusion protein in a recently developed murine neuroblastoma model. Here, we demonstrate that cytokine gene transfer of IL-2 and scIL-12 with histone H2A results in the induction of an antitumor immune response that is superior in some respects to gene transfer with Superfect, a commercially available activated dendrimer commonly used to effect transfection with plasmids. Three lines of evidence support this contention. First, histone H2A-mediated transfection of IL-2 induces a natural killer cell-induced rejection of primary tumors in contrast to Superfect, which produces only a partial reduction in primary tumor growth. Second, the induction of a T cell-mediated protective tumor immunity following gene transfer of scIL-12 is more efficient with the histone H2A-mediated gene transfer because rejection of a lethal wild-type tumor cell challenge is accompanied by the greatest degree of MHC class I-restricted tumor cell killing in vitro. Third, histone H2A-mediated scIL-12 gene therapy induces the greatest release of mIFN-gamma from splenocytes of vaccinated animals in contrast to Superfect and other controls.

Melanoma immunotherapy by targeted IL-2 depends on CD4(+) T-cell help mediated by CD40/CD40L interaction.

The induction of tumor-protective immunity against malignancies remains a major challenge in cancer immunotherapy. A novel, humanized anti-ganglioside-GD(2)-IL-2 immunocytokine (hu14.18-IL-2) induced CD8(+) T cells to eradicate established pulmonary metastases of B78-D14 murine melanoma, in a process that required help by CD4(+) T cells and was mediated by the CD40/CD40 ligand (CD40L) interaction. The anti-tumor effect was diminished in mice deficient in CD4(+) T-cells. Three lines of evidence show that CD4(+) T-cell help was mediated by CD40/CD40L interaction but not by endogenous IL-2 production. First, the hu14.18-IL-2-induced anti-tumor response is partially abrogated in C57BL/6J CD40L knockout (KO) mice in contrast to C57BL/6J IL-2 KO animals, in which the immunocytokine was completely effective. Second, partial abrogation of the anti-tumor effect is induced with anti-CD40L antibodies to the same extent as with CD4(+) T-cell depletion. Third, a complete anti-tumor response induced by hu14.18-IL-2 can be reconstituted in C57BL/6J CD40L KO mice by simultaneous stimulation with an anti-CD40 mAb. These results suggest that help provided by CD4(+) T cells via CD40/CD40L interactions in our tumor model is crucial for effective immunotherapy with an IL-2 immunocytokine.

What to do with targeted IL-2.

A common strategy in immunotherapy of cancer is the induction of an increased immunogenicity of syngeneic malignancies. A novel approach to achieve this goal is the targeting of cytokines into the tumor microenvironment with antibody-cytokine fusion proteins, called immunocytokines. This report summarizes therapeutic efficacy and immune mechanisms involved in targeting IL-2 to syngeneic tumors and describes their extended use as a synergistic treatment modality for cancer vaccines and antiangiogenesis. Treatment of established melanoma and colon carcinoma metastases with IL-2 immunocytokines resulted in eradication of disease, followed by a vaccination effect protecting mice from lethal challenges with wild-type tumor cells. In a syngeneic neuroblastoma model, targeted IL-2 elicited effective antitumor responses mediated by NK cells in the absence of a T-cell memory. Interestingly, targeted IL-2 was effective in amplification of memory immune responses previously induced by cancer vaccines. Furthermore, a synergistic effect achieved by combining targeted IL-2-immunotherapy with an antiangiogenic inhibitor of integrin alpha(v)beta(3) extends the potential of this immunotherapeutic strategy in combination with antiangiogenesis as demonstrated in three syngeneic tumor models. Based on these findings, targeted IL-2 may provide an effective tool for the adjuvant treatment of cancer either applied as a single strategy or in combination with cancer vaccines and antiangiogenic strategies.

Treatment of children with relapsed soft tissue sarcoma: report of the German CESS/CWS REZ 91 trial.

BACKGROUND: The present study was performed to evaluate the possibilities of relapse treatment in patients heavily pretreated for a soft tissue sarcoma. PATIENTS AND METHODS: Prospective, multicenter study in 44 soft tissue sarcoma (STS) patients with first relapse. Primary diagnosis was embryonal rhabdomyosarcoma (RME) in 17 patients, alveolar rhabdomyosarcoma (RMA) in 13, primitive neuroectodermal tumor (PNET) in 6, and miscellaneous soft tissue sarcomas in 8 patients. Initial chemotherapy consisted of carboplatin/etoposide combination (150 mg/m2 each, days 1 to 4) followed by local therapy including surgical treatment and, whenever possible, radiotherapy. RESULTS: In 11/17 patients without primary tumor resection, CR or PR was achieved following the initial two cycles of chemotherapy (61%). The probability of event-free survival (pEFS) for RME patients was 0.41 +/- 0.12 at 5 years, and 0.25 +/- 0.12 for RMA patients. But, in contrast no PNET patient or patient with another soft-tissue sarcoma achieved long-term remission. Additional local radiotherapy significantly (P = 0.002) improved pEFS (3-year estimates of 0.23 +/- 0.2 vs. 0.1 +/- 0.1 in patients without radiotherapy). CONCLUSIONS: In patients with RME, relapse treatment employing a carboplatin/etoposide combination may induce a second remission in approximately 40% of patients. Surgical excision and additional local radiotherapy seem to be essential to maintain a stable remission. In patients with RMA or PNET, however, this treatment strategy is of no long-term benefit.