Predominant expression of CCL2 at the tumor site of prostate cancer patients directs a selective loss of immunological tolerance to CCL2 that could be amplified in a beneficial manner.

We have previously shown that, during inflammatory autoimmune diseases in humans, the immune system develops a neutralizing auto-Ab-based response to a very limited number of inflammatory mediators, and that amplification of each response could be beneficial for the host. Our working hypothesis has been that this selective breakdown of immunological tolerance is due to a predominant expression of an inflammatory mediator at an immune-restricted site undergoing a destructive process. All three conditions also take place in cancer diseases. In this study, we delineate this hypothesis for the first time in a human cancer disease and then explore its clinical implications. We show that in primary tumor sections of prostate cancer subjects, CCL2 is predominantly expressed at the tumor site over other chemokines that have been associated with tumor development, including: CXCL12, CXCL10, CXCL8, CCL3, and CCL5. Subsequently, the immune response selectivity mounts an Ab-based response to CCL2. These Abs are neutralizing Abs. These findings hold diagnostic and therapeutic implications. The current diagnosis of prostate cancer is based on prostate-specific Ag measurements that do not distinguish benign hypertrophy from malignancy. We show in this study that development of anti-CCL2 Abs is selective to the malignant stage. From a clinically oriented perspective, we show, in an experimental model of the disease, that DNA-based amplification of this response suppresses disease, which has implications for a novel way of therapy in humans.

Complete in vivo reversal of the multidrug resistance phenotype by jet-injection of anti-MDR1 short hairpin RNA-encoding plasmid DNA.

Triggering the RNA interference (RNAi) pathway by inducing the expression of short hairpin RNA (shRNA) molecules has become a promising tool for efficient silencing of a given gene in gene therapy applications. In this study, shRNA encoding DNA was utilized to reverse the classical MDR1/P-glycoprotein (MDR1/P-gp)-mediated multidrug resistance (MDR) phenotype in vivo. For the first time, the nonviral jet-injection technology was applied for delivering naked shRNA-vector constructs for direct intratumoral in vivo transfer. The highly efficient anti-MDR1 shRNA expression vectors were applied twice in the human MDR1/P-gp overexpressing MaTu/ADR cancer xenograft-bearing mice, and twice in the corresponding drug-sensitive parental MaTu tumor xenograft bearing mice as well. Two days after anti-MDR1 shRNA vector injection, the expression level of the MDR1 messenger RNA (mRNA) was decreased by more than 90% and the corresponding MDR1/P-gp protein was no longer detectable in the tumors. Two jet-injections of anti-MDR1 shRNA vectors into the tumors, combined with two intravenous (IV) administrations of doxorubicin, were sufficient to achieve complete reversal of the drug-resistant phenotype. The data show that jet-injection delivery of shRNA-expressing vectors is effective in reversing MDR1/P-gp-mediated MDR in vivo, and is therefore a promising strategy for making tumors with an MDR1/Pgp-dependent MDR phenotype revert to a drug-sensitive state.

Intratumoral injection of IL-12 plasmid DNA--results of a phase I/IB clinical trial.

Effective eradication of established tumor and generation of a lasting systemic immune response are the goals of cancer immunotherapy. The objective of this phase IB study was to assess the safety and toxicity of treatment to metastatic tumor underlying the skin with the DNA encoding interleukin-12 (IL-12). This treatment strategy allowed the patient's own tumor to serve as a source of autologous antigen in the tumor microenvironment. We proposed that IL-12 protein produced by the transfected cells would result in the generation of both a local and systemic antitumor response. The tumor was treated with either three or six intratumoral injections of plasmid containing IL-12 DNA. This treatment strategy resulted in no significant local or systemic toxicity. The treatment did not result in an increase in serum IL-12 protein. The size of the treated lesion decreased significantly (greater than 30%) in five of the 12 patients. However, nontreated subcutaneous lesions or other disease did not decrease in size.

Dendritic cell-based immunotherapy of malignant gliomas.

The failure of conventional treatment modalities for gliomas, in spite of tremendous progress in research in the past two decades, has led to increasing interest in alternative treatment strategies, including immunotherapy. It has become evident that vaccination with dendritic cells (DC), designed to express tumor antigens, is a potent strategy to elicit anti-tumor immune response in both pre-clinical and clinical settings. Various methods have been applied in order to induce DC to express tumor antigens including: pulsing with isolated tumor peptides or whole tumor lysate; fusion with tumor cells; and pulsing with apoptotic tumor cells. Herein, we review the recent progress in DC biology with regard to tumor immunity and discuss current DC-based strategies and future prospects in immunotherapy for malignant gliomas.

Progress in the development of immunotherapy of cancer using ex vivo-generated dendritic cells expressing multiple tumor antigen epitopes.

Immunotherapy with tumor-associated antigen-pulsed, ex vivo-generated dendritic cells (DCs) is a promising approach for the treatment of cancer that has shown efficacy in animal models and is now being tested in the clinic. The majority of studies performed to date make use of a single tumor-associated epitope. However, because of the high rate of mutation in tumor cells allowing for loss of expression of a single antigen, it is likely that use of multiple antigenic epitopes will induce a broader, longer-lasting, and effective tumor-specific immune response. Multiple vehicles for loading DCs with multiple antigenic epitopes are under investigation to determine the most effective method for vaccination, with many of these methods showing promise. These loading methods, as well as other critical considerations for making DC vaccination as efficacious as possible, are discussed in this article.

Peritumoral CpG DNA elicits a coordinated response of CD8 T cells and innate effectors to cure established tumors in a murine colon carcinoma model.

The immune system of vertebrates is able to detect bacterial DNA based on the presence of unmethylated CpG motifs. We examined the therapeutic potential of oligodeoxynucleotides with CpG motifs (CpG ODN) in a colon carcinoma model in BALB/c mice. Tumors were induced by s.c. injection of syngeneic C26 cells or Renca kidney cancer cells as a control. Injection of CpG ODN alone or in combination with irradiated tumor cells did not protect mice against subsequent tumor challenge. In contrast, weekly injections of CpG ODN into the margin of already established tumors resulted in regression of tumors and complete cure of mice. The injection site was critical, since injection of CpG ODN at distant sites was not effective. Mice with two bilateral C26 tumors rejected both tumors upon peritumoral injection of one tumor, indicating the development of a systemic immune response. The tumor specificity of the immune response was demonstrated in mice bearing a C26 tumor and a Renca tumor at the same time. Mice that rejected a tumor upon peritumoral CpG treatment remained tumor free and were protected against rechallenge with the same tumor cells, but not with the other tumor, demonstrating long term memory. Tumor-specific CD8 T cells as well as innate effector cells contributed to the antitumor activity of treatment. In conclusion, peritumoral CpG ODN monotherapy elicits a strong CD8 T cell response and innate effector mechanisms that seem to act in concert to overcome unresponsiveness of the immune system toward a growing tumor.

An optimum anti-melanoma response in mice immunized with fibroblasts transfected with DNA from mouse melanoma cells requires the expression of both syngeneic and allogeneic MHC-determinants.

Most neoplasms do not induce antitumor immune responses that can control tumor growth. Tumor associated antigens (TAAs) are insufficiently immunogenic. A vaccine that augments the immunogenic properties of TAAs could be of importance in the treatment of cancer patients. In an animal model, we prepared a vaccine by transfection of highly antigenic allogeneic mouse fibroblasts (LM; H-2(k)) with DNA from B16 mouse melanoma cells. We then tested the transfected cells' immunogenic properties in C57BL/6 mice, syngeneic with the melanoma (H-2(b)). We hypothesized that the immunogenic properties of 'weak' TAAs formed by the neoplasm would be enhanced if they were expressed by highly antigenic cells. The results indicated that mice with melanoma treated by immunization with the DNA-transfected fibroblasts survived significantly longer than mice in various control groups. To investigate the contribution of MHC determinants expressed by the transfected cells to their immunogenic properties, we compared the antimelanoma responses in mice immunized with transfected cells that expressed allogeneic or syngeneic class I determinants. The results indicated that the immunogenic properties of the DNA-transfected cells were enhanced if the cells expressed allogeneic MHC determinants. The antimelanoma responses of greatest magnitude, however, mediated predominantly by CD8(+) T cells, were in mice immunized with transfected fibroblasts that expressed both syngeneic and allogeneic class I determinants.

Plasmid DNA electrotransfer: a new non viral method for gene therapy in oncology.

With some defined conditions, electric pulses delivery to tissue in vivo can greatly enhance DNA transfection. We here describe the application in oncology of intratumoral or intramuscular DNA electrotransfer by using muscle as a secretory tissue of transgenic proteins displaying anticancer properties.

Generation of polyclonal rabbit antisera to mouse melanoma associated antigens using gene gun immunization.

Lymphocytes from patients with melanoma have been used to clone melanoma associated antigens which are, for the most part, nonmutated melanocyte tissue differentiation antigens. To establish a mouse model for the use of these 'self' antigens as targets for anti-tumor immune responses, we have employed the mouse homologues of the human melanoma antigens Tyrosinase, Tyrosinase Related Protein-1 (TRP-1), gp100, and MART-1. We sought to generate antisera against these proteins for use in the construction of experimental recombinant and synthetic anti-cancer vaccines, and for use in biologic studies. Using genes cloned from the B16 mouse melanoma or from murine melanocytes, we immunized rabbits with plasmid DNAs coated onto microscopic gold beads that were then delivered using a hand-held, helium-driven 'gene gun'. This strategy enabled us to generate polyclonal rabbit sera containing antibodies that specifically recognized each antigen, as measured by immunostaining of vaccinia virus infected cells. The sera that we generated specifically for TRP-1, gp100, and MART-1 recognized extracts of the spontaneous murine melanoma, B16. The identities of the recognized proteins was confirmed by Western blot analysis. The titers and specificities of these antisera were determined using ELISA. Interestingly, serum samples generated against murine MART-1 and gp100 developed antibodies that were cross-reactive with the corresponding human homologues. Recognition of human gp100 and murine Tyrosinase appeared to be dependent upon conformational epitopes since specificity was lost upon denaturation of the antigens. These antisera may be useful in the detection, purification and characterization of the mouse homologues of recently cloned human tumor associated antigens and may enable the establishment of an animal model of the immune consequences of vaccination against 'self antigens.

Immunization with interleukin-2-secreting allogeneic cells transfected with DNA from mouse melanoma cells induces immune responses that prolong the lives of mice with melanoma.

Altered genes in tumor cells specify tumor-associated antigens. Because genetic instability is a characteristic of the malignant cell phenotype, a large number of different, altered genes may be present in a population of neoplastic cells, specifying an array of undefined tumor-associated determinants. We hypothesized that immunogenic cells transfected with DNA from malignant cells will include cells that specify tumor-associated antigens. To test this question, we deliberately mutagenized a population of B16 melanoma cells (H-2b) by ultraviolet-B irradiation. DNA from the surviving cells was used to transfect LM cells (H-2k), a mouse fibroblast cell line modified previously to secrete interleukin-2. The transfected allogeneic cells were then tested for their immunogenic properties in C57BL/6J mice (H-2b) syngeneic with the melanoma. Mice injected with a mixture of the mutagenized B16 cells and the transfected cells survived significantly longer than untreated mice injected with the mutagenized B16 cells alone. Mice injected with a mixture of mutagenized B16 cells and cells transfected with DNA from unirradiated B16 cells died in shorter intervals. Based on the results of cytotoxicity assays performed in vitro, the cellular immune responses of greatest magnitude were directed toward the type of cell from which the DNA was obtained.

Genetic immunization against neu/erbB2 transgenic breast cancer.

erbB2/neu, an overexpressed oncogene product, has been proposed as a human cancer vaccine target. In the present study, transgenic (rat neuNT oncogene) FVB/neu mice, developing metastasizable mammary carcinoma, were immunized with a plasmid DNA encoding are not tolerant to the self antigen and sequences. We report that transgenic tumour-bearing mice, like some breast cancer patients erbB2 + X, develop anti-neu autoimmune responses, which can be boosted and skewed to a Th1 phenotype by DNA immunization. Intramuscular injections of neuNT plasmid drastically reduced (or even prevented in a small number of treated mice) the outgrowth of mammary neoplasms as well as their metastatic penetrance. Furthermore, DNA immunization caused haemorrhagic necrosis of established cancer nests, leaving a greatly reduced portion of the tumour burden for the host to cope with. The antitumour activities we obtained, in this very challenging model for cancer immunotherapy, lay the foundation for DNA-based immunization to control erbB2/neu-overexpressing neoplasms.

Cytokine enhancement of DNA immunization leads to effective treatment of established pulmonary metastases.

DNA immunization can result in the induction of Ag-specific cellular and humoral immune responses and in protective immunity in several Ag systems. To evaluate the utility of DNA-based immunization as a potential cancer treatment strategy, we employed an experimental murine tumor, CT26, expressing the model tumor-associated Ag, beta-galactosidase (beta-gal), designated CT26.CL25. A plasmid expressing beta-gal (pCMV/beta-gal) administered by particle-mediated gene delivery to the epidermis using a hand-held, helium-driven "gene gun" induced beta-gal-specific Ab and lytic responses. Immunization with this construct prevented the growth of pulmonary metastatic tumor, and the adoptive transfer of splenocytes generated by pCMV/beta-gal in vivo immunization and cultured in vitro with the beta-gal876-884 immunodominant peptide reduced the number of established pulmonary nodules. DNA immunization alone had little or no impact on the growth of established lung metastases. To enhance the function of DNA immunization for active immunotherapy, a panel of cytokines was added as adjuvants following DNA administration. Significant reduction in the number of established metastases was observed when human rIL-2, mouse rIL-6, human rIL-7, or mouse rIL-12 were given after DNA inoculation; mouse rIL-12 as an adjuvant had the most profound effect. These findings suggest that the cytokines involved in the activation and expansion of lymphocyte populations may improve the therapeutic effects of DNA immunization. Given the ease with which plasmid DNA can be prepared to high purity for safe use in humans with infectious diseases and cancers, DNA immunization administered together with cytokine adjuvant may be an attractive alternative to recombinant viral vaccines.

Administration of tumor cell chromatin to immunosuppressed and non-immunosuppressed non-human primates.

For decades, developers and regulators of vaccines and other biological products have been concerned about the theoretical risk to patients posed by contaminants derived from the cell substrates used to produce those products. The present study addresses the issue of how risky DNA may be as a residual impurity by injecting both normal and immunosuppressed monkeys with 10(8) genome equivalents of DNA from a human tumor cell line. After more than eight years of observation, none of the animals shows evidence of neoplastic disease. The results of this study along with clinical experiences with already approved products derived from continuous cell lines suggest that he benefits of using such cells for the production of biologicals far outweigh any theoretical risks associated with DNA.

Optical anisotropy of toluidine blue-stained transformed cell nuclei.

Changes in chromatin order with cell transformation were studied in terms of optical anisotropy (birefringence) in toluidine blue-stained NIH 3T3 and Balb/3T3 cells. The transformed NIH 3T3 cell lines used were obtained by transfection with the T24 cell H-ras oncogene and the whole genomic DNA of MCF-7 human breast carcinoma cells. C2PO and PAP2 cell lines were used as representatives of Ha-ras-transformed NIH 3T3 cells with poor and high metastatic ability, respectively. The chromatin of all cells examined exhibited metachromasy and a birefringence of greenish-yellow interference color. The highest birefringence intensity was found in the chromatin granules of the most frequent nuclear phenotype of ras- and MCF-7 DNA-transformed cells, with the exception of the cells with high metastatic ability, in which a faint birefringence was observed. The differences in chromatin birefringence intensity are assumed to indicate differences in chromatin stereoarrangement with cell transformation. In the case of the highly tumorigenic and/or highly metastatic transformants the faint birefringence is assumed to be associated with the heterogeneous and complex physiological processes that may require a relatively less ordered arrangement of the chromatin.

[Effect of exogenous neoplasm DNA on the course of Swetz leukemia in non-inbred rats of different age groups]. / Vliianie ékzogennoi opukholevoi DNK na techenie leikoza Shwetsa u besporodnykh krys razlichnykh vozrastnykh grupp.

Experiments on 150 non-inbred white rats have shown that preliminary administration of high polymer native DNA isolated from Swetz leukemia tumor suppresses the tumor growth. Tumor DNA combined with BCG vaccine in young animals suppresses and in old ones stimulates the tumor growth. These results correlate with DNA and CIC content in the blood serum. Due to the tumor growth suppression the animals' life increases by 40%. Administration of BCG vaccine only does not prevent the development of tumor in usual terms.