Tumor-targeting CTL expressing a single-chain Fv specific for VEGFR2.

Cytotoxic T lymphocytes (CTL) are critical effector cells in tumor immunity. Adoptive transfer therapy with in vitro-expanded tumor-specific CTL is a promising approach for preventing cancer metastasis and recurrence. Transferred CTL are not effective in clinical trials, however, due to inadequate tumor-infiltration. Therefore, the development of functionally modified CTL, such as tumor-targeting CTL, is widely desired. Here, we designed the tumor-targeting CTL expressing a single-chain antibody fragment (scFv-CTL) specific for vascular endothelial growth factor receptor 2 (VEGFR2/flk1) by transducing the CTL with a retroviral vector. The scFv-CTL bound to VEGFR2/flk1-expressing cells and retained their cytotoxic activity against tumor cells. In addition, adoptive transfer of scFv-CTL into tumor-bearing mice effectively suppressed tumor growth due to the augmented accumulation of the transferred CTL in the tumor tissue. These findings indicate that the creation of CTL capable of targeting tumor vascular endothelial cells by scFv-expression technique is considerably promising for improvement of efficacy in adoptive immunotherapy.

NK cells are migrated and indispensable in the anti-tumor activity induced by CCL27 gene therapy.

Natural killer (NK) cells have been demonstrated could play an important role in the treatment of a number of tumors in mice. In the present study, chemokine CCL27, which be considered only selectively chemoattracts cutaneous lymphocyte antigen positive memory T cells and Langerhans cells, firstly demonstrated that it could induce the accumulation of NK cells into tumor by the intratumoral injection of CCL27-encoding fiber-mutant vector, AdRGD-CCL27. Experiments using spleen cell fractionation and RT-PCR showed CCL27 receptor, mCCR10, was strongly expressed in NK cells, suggesting the accumulation of NK cells in tumor was attributed to chemoattractant activity of CCL27 itself. Moreover, the combination of AdRGD-CCL27 and AdRGD-IL-12 induced the synergistic anti-tumor activity via NK-dependent manner and induced more NK cells infiltration into tumor nodule than that induced by AdRGD-CCL27 alone or AdRGD-IL-12 alone.

Antitumor mechanism of intratumoral injection with IL-12-expressing adenoviral vector against IL-12-unresponsive tumor.

Interleukin-12 (IL-12) has potent antitumor activities through the activation of natural killer cells and cytotoxic T lymphocytes. Some tumors, however, are resistant to IL-12; for example, in murine Meth-A fibrosarcoma, an IL-12-unresponsive tumor model, tumors do not regress following IL-12 administration due to the fact that few T cells migrate to tumor sites. Transduction of IL-12 genes into preexisting Meth-A tumors using RGD fiber-mutant adenoviral vectors (AdRGD), however, enhances tumor infiltration by T cells, thereby inducing tumor regression due to enhanced tumor infiltration by T cells. Here, we demonstrated the mechanism underlying the anti-tumor effect induced by intratumoral injection of AdRGD encoding IL-12 (AdRGD-IL12). The anti-tumor activity of the IL-12 treatment was T cell-dependent, requiring mainly CD8(+) cytotoxic T lymphocytes in the effector phase, as confirmed by analysis using BALB/c nude mice and an in vivo depletion assay. Additionally, intratumoral injection of AdRGD-IL12 promoted cellular immunity, as determined by the frequency of interferon-gamma-positive cells in regional lymph nodes, and changed the tumor microenvironment to an immunologic activation state with enhanced expression of lymphocyte activation markers and cell adhesion molecules. The present data provide evidence that the antitumor effect of IL-12 gene transduction in a local tumor site is dependent on induced alterations of the tumor microenvironment and systemic immunity.

Factors involved in the maturation of murine dendritic cells transduced with adenoviral vector variants.

Adenoviral vector (Ad)-mediated gene transfer is an attractive method for manipulating the immunostimulatory properties of dendritic cells (DCs) for cancer immunotherapy. DCs treated with Ad have phenotype alterations (maturation) that facilitate T cell sensitization. We investigated the mechanisms of DC maturation with Ad transduction. Expression levels of a maturation marker (CD40) on DCs treated with conventional Ad, fiber-modified Ads (AdRGD, AdF35, AdF35DeltaRGD), or a different serotype Ad (Ad35) were correlated with their transduction efficacy. The alphav-integrin directional Ad, AdRGD, exhibited the most potent ability to enhance both foreign gene expression and CD40 expression, and induced secretion of interleukin-12, tumor necrosis factor-alpha, and interferon-alpha in DCs. The presence of a foreign gene expression cassette in AdRGD was not necessary for DC maturation. Maturation of DCs treated with AdRGD was suppressed by destruction of the Ad genome, inhibition of endocytosis, or endosome acidification, whereas proteasome inhibition increased CD40 expression levels on DCs. Moreover, inhibition of alphav-integrin signal transduction and blockade of cytokine secretion affected the maturation of DCs treated with AdRGD only slightly or not at all, respectively. Thus, our data provide evidence that Ad-induced DC maturation is due to Ad invasion of the DCs, followed by nuclear transport of the Ad genome, and not to the expression of foreign genes.

Combination of two fiber-mutant adenovirus vectors, one encoding the chemokine FKN and another encoding cytokine interleukin 12, elicits notably enhanced anti-tumor responses.

For achieving optimal cancer immunotherapy, it is anticipated that both the activation and infiltration of immune cells into tumor are indispensable. In the present study, fiber-mutant adenovirus vectors (Ad) encoding chemokine FKN, (AdRGD-FKN), and cytokine interleukin 12, (AdRGD-IL-12), were constructed. The in vivo gene expression of AdRGD was confirmed and the combination of both FKN and IL-12 encoding Ad elicited synergistic anti-tumor activity in ovarian carcinoma, which induced tumor regression in all tumor-bearing mice, while using FKN alone did not show notable tumor-suppressive effect. The treatment with both IL-12 and FKN induced long-term specific immunity against OV-HM tumors in tumor-rejected mice. The results of immunohistochemical staining for CD3+ and perforin-positive cells suggested that the failure of using FKN alone was because of the inactivation of infiltrated immune cells. In contrast, cotransduction with IL-12 and FKN could induce more activated tumor-infiltrating immune cells than that transducted with FKN or IL-12 alone. The results indicated that using both chemokine and cytokine might be a powerful tool and a promising way for effective cancer immunotherapy.

CC-chemokine ligand 17 gene therapy induces tumor regression through augmentation of tumor-infiltrating immune cells in a murine model of preexisting CT26 colon carcinoma.

Chemokines, which regulate leukocyte trafficking and infiltration of local sites, are attractive candidates for improving the efficacy of cancer immunotherapy by enhancing the accumulation of immune cells in tumor tissue. Herein, we evaluated the antitumor effects of intratumoral injection of RGD fiber-mutant adenoviral vectors (AdRGDs) encoding the chemokines CCL17, CCL19, CCL20, CCL21, CCL22, CCL27, XCL1 or CX3CL1 in a murine model of preexisting CT26 colon carcinoma. Among these 8 chemokine-expressing AdRGDs, injection of AdRGD-CCL17 most effectively induced tumor regression and generated specific immunity in rechallenge experiments. Tumor elimination activity by intratumoral injection of AdRGD-CCL17 depended on both the vector dose and the number of injections, and mainly required CD8+ CTLs in an effector phase as confirmed by analysis using BALB/c nude mice and an in vivo depletion assay. In addition, CCL17 gene transduction induced significant increases in the number of infiltrating macrophages and CD8+ T cells in CT26 tumors, and changed the tumor microenvironment to an immunologic activation state in which there was enhanced expression of lymphocyte activation markers and cell adhesion molecules. Thus, our data provide evidence that CCL17 gene transduction of local tumor sites is a promising approach for the development of a cancer immunogene therapy that can recruit activated tumor-infiltrating immune effector cells.

Design of a practical fluorescent probe for superoxide based on protection-deprotection chemistry of fluoresceins with benzenesulfonyl protecting groups.

A strategy for designing probes based on protection-deprotection chemistry involving fluoresceins and their benzenesulfonyl (BES) derivatives has led to the development of a much more practical superoxide (O(2) (-.)) probe than the previously reported bis(2,4-dinitro-BES) tetrafluorofluorescein (6 a). Examination of various BES derivatives, developed from the starting point of the prototype probe 6 a, yielded 4,5-dimethoxy-2-nitro-BES tetrafluorofluorescein (BESSo; 7 j) as the optimal reagent. A microtiter plate assay with BESSo showed a tenfold improved detection limit for O(2) (-.) compared with such an assay based on 6 a. BESSo showed markedly better specificity for O(2) (-.) than for GSH or other reactive oxygen species, and this specificity was significantly higher than that of Fe(2+) and some reducing enzymes. These features have resulted in the development of an assay based on BESSo that is capable of providing more unambiguous results for O(2) (-.) release from neutrophils, with or without stimulation by phorbol myristate acetate, as compared with an assay based on 6 a. Intracellular generation of O(2) (-.) in human Jurkat T cells stimulated by butyric acid has been measured by using flow cytometry and fluorescence microscopy utilizing the acetoxymethyl derivative of BESSo.

Anti-tumor responses induced by chemokine CCL19 transfected into an ovarian carcinoma model via fiber-mutant adenovirus vector.

Considerable attention has recently been paid to the application of chemokines to cancer immunotherapy because of their chemotactic affinity for a variety of immune cells and because several chemokines are strongly angiostatic. In the present study, the recombinant adenovirus vectors encoding chemokine CCL19 or XCL1 in an E1 cassette (AdRGD-mCCL19 and AdRGD-mXCL1) were developed. The constructed fiber-mutant adenovirus vector, which contained the integrin-targeting Arg-Gly-Asp (RGD) sequence in the fiber knob, notably enhanced the transfection efficiency to OV-HM ovarian carcinoma cells compared to that induced by conventional adenovirus vector. The results of an in vitro chemotaxis assay for chemokine-encoding vector demonstrated that both AdRGD-mCCL19 and AdRGD-mXCL1 could induce the migration of cells expressing specific chemokine receptors. Of the two chemokine-encoding vectors evaluated in vivo, AdRGD-mCCL19 showed significant tumor-suppressive activity in B6C3F1 mice via transduction into OV-HM cells, whereas XCL1 did not exhibit any notable anti-tumor effects, suggesting that CCL19 may be a candidate for cancer immunotherapy.

A single intratumoral injection of a fiber-mutant adenoviral vector encoding interleukin 12 induces remarkable anti-tumor and anti-metastatic activity in mice with Meth-A fibrosarcoma.

Cytokine-encoding viral vectors are considered to be promising in cancer gene immunotherapy. Interleukin 12 (IL-12) has been used widely for anti-tumor treatment, but the administration route and tumor characteristics strongly influence therapeutic efficiency. Meth-A fibrosarcoma has been demonstrated to be insensitive to IL-12 treatment via systemic administration. In the present study, we developed an IL-12-encoding fiber-mutant adenoviral vector (AdRGD-IL-12) that showed enhanced gene transfection efficiency in Meth-A tumor cells, and the production of IL-12 p70 in the culture supernatant from transfected cells was confirmed by ELISA. In therapeutic experiments, a single low-dose (2 x 10(7) plaque-forming units) intratumoral injection of AdRGD-IL-12 elicited pronounced anti-tumor activity and notably prolonged the survival of Meth-A fibrosarcoma-bearing mice. Immunohistochemical staining revealed that the IL-12 vector induced the accumulation of T cells in tumor tissue. Furthermore, intratumoral administration of the vector induced an anti-metastasis effect as well as long-term specific immunity against syngeneic tumor challenge.