Sonodynamic therapy of cancer using novel sonosensitizers.

Sonodynamic therapy (SDT) of cancer is based on preferential uptake and/or retention of a sonosensitizing drug (sonosensitizer) in tumor tissues and subsequent activation of the drug by ultrasound irradiation. Ultrasound can penetrate deeply into tissues and can be focused into a small region of a tumor to activate a sonosensitizer. This is a unique advantage in the non-invasive treatment of nonsuperficial tumors when compared to laser light used for photodynamic therapy. Recently, it has been found that photochemically active porphyrins also show significant antitumor effects when activated with ultrasound. The mechanism of sonodynamic action has been suggested to involve photoexcitation of the sensitizer by sonoluminescent light, with subsequent formation of singlet oxygen. This mini-review provides a brief overview of the following four sonosensitizers useful in SDT: i) a homogeneous complex of oligomers of hematoporphyrin, Photofrin II; ii) a gallium porphyrin complex, ATX-70; iii) a hydrophilic chlorin derivative, A7X-S10, and iv) a novel porphyrin derivative devoid of photosensitivity, DCPH-P-Na (I).

Selective up-regulation of claudin-1 and claudin-2 in colorectal cancer.

BACKGROUND: To understand the molecular and morphological alterations in the tight junction in colorectal cancer (CRC) tissues, the expression of eight tight junction proteins in normal and cancer colorectal tissues were compared. PATIENTS AND METHODS: Adenocarcinoma tissues and paired normal mucosa were resected from surgical specimens of CRC patients. The expression of occludin, ZO-1, ZO-2, and claudin-1 -5 was analyzed at the mRNA level by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and at the protein level by immunohistochemistry. RESULTS: The expression of claudin-1 and claudin-2 in cancer tissues was upregulated 40- and 49.2-fold, respectively, at the mRNA level, as compared with that in normal tissues. The up-regulation of these two claudins was also observed at the protein level and it appeared to depend on the depth of tumor invasion. CONCLUSION: Claudin-1 and claudin-2 were found to be overexpressed in CRC tissues. They may be useful as tumor markers and targets for the treatment of colorectal cancer.

Sonodynamic therapy of cancer using a novel porphyrin derivative, DCPH-P-Na(I), which is devoid of photosensitivity.

To improve the efficacy of sonodynamic therapy of cancer using photosensitizers, we developed a novel porphyrin derivative designated DCPH-P-Na(I) and investigated its photochemical characteristics and sonotoxicity on tumor cells. DCPH-P-Na(I) exhibited a minimum fluorescent emission by excitation with light, compared with a strong emission from ATX-70, which is known to reveal both photo- and sonotoxicity. According to this observation, when human tumor cells were exposed to light in the presence of DCPH-P-Na(I) in vitro, the least phototoxicity was observed, in contrast to the strong phototoxicity of ATX-70. However, DCPH-P-Na(I) exhibited a potent sonotoxicity on tumor cells by irradiation with ultrasound in vitro. This sonotoxicity was reduced by the addition of L-histidine, but not D-mannitol, thus suggesting that singlet oxygen may be responsible for the sonotoxicity of DCPH-P-Na(I). DCPH-P-Na(I) demonstrated significant sonotoxicity against a variety of cancer cell lines derived from different tissues. In addition, in a mouse xenograft model, a potent growth inhibition of the tumor was observed using sonication after the administration of DCPH-P-Na(I) to the mouse. These results suggest that sonodynamic therapy with DCPH-P-Na(I) may therefore be a useful clinical treatment for cancers located deep in the human body without inducing skin sensitivity, which tends to be a major side-effect of photosensitizers.

Possible applications of antibodies or their genes in cancer therapy.

In this review article the possible applications of anti-tumor-associated antigen (TAA) antibodies in the therapy of cancer have been summarized. First, recombinant monoclonal antibodies (MAbs) are increasingly being used as therapeutic agents, especially in combination with anti-cancer drugs. Second, conjugation of antibody therapy with toxins or radioisotopes offers more therapeutic approaches. Third, development of cytotoxic T-lymphocyte (CTL) or natural killer (NK)-cell populations with anti-TAA antibody activity may be important for the success of cancer immunotherapy because the downregulated HLA class I molecules and the non-ubiquitous expression of NK receptor ligands in tumor tissues constitute the major tumor escape mechanism facing tumor-specific CTL- and/or NK-cell-mediated responses. Finally, in cancer gene therapy, the strategies to target viral vectors carrying therapeutic genes to tumor tissues by modifying the tropisms with MAbs or their genes against TAAs are also very promising.

A fully human chimeric immune receptor for retargeting T-cells to CEA-expressing tumor cells.

BACKGROUND: Recombinant chimeric immune receptors (CIRs) with anti-CEA specificity can retarget grafted T-cells to CEA-expressing tumors in an HLA-independent manner. To reduce the immunogenicity of conventional CIR in humans, an attempt was made to generate a CIR encoded by all human genes. MATERIALS AND METHODS: A single-chain variable fragmented (scFv) antibody gene was prepared from variable region genes of the C2-45 human mAb clone specific for CEA. The scFv gene was connected to a gene construct comprised of the cDNAs for the human CD8a hinge region, the human CD28 transmembrane and cytoplasmic domains, and the human CD3zeta intracellular domain. The resulting human CIR gene, designated L45scFv-CIR, was inserted into the pcDNA3.1 expression vector and transfected into human primary T-cells. RESULTS: Flow cytometric analysis using allophycocyanin-labeled CEA demonstrated the expression of the L45scFv-CIR protein on the T-cells and its specific antigen binding activity. CONCLUSION: This L45scFv-CIR gene, consisting of four human genes, may be a useful tool for eradication of CEA-expressing but HLA-downregulated tumor cells.

Re-targeting of cytotoxic T lymphocytes and/or natural killer cells to CEA-expressing tumor cells with anti-CEA antibody activity.

Cellular immunity, in which cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are main effector cells, plays an important role in the antitumor defense mechanism. T cell immunotherapy is based on the assumption that tumor antigen (TA) peptides are correctly presented by HLA class I molecules on target tumor cells, while NK cell immunotherapy is based on the hypothesis that cell surface TAs or ligands for NK receptors are widely expressed in tumor cells. However, human tumor cells are well known to often lose HLA class 1 molecules, and target cell ligands for NK receptors are not always expressed in human tumor cells. This altered HLA class 1 expression and non-ubiquitous distribution of NK receptor ligands constitute the major tumor escape mechanism facing tumor-specific CTL and/or NK cell-mediated responses. These facts also indicate that it is not easy to eliminate the target tumors only by activating tumor-specific CTLs or NK cells. On the other hand, although the protective role of humoral immunity in cancer seems not to be imperative, it is easily confirmed by immunostaining whether or not antibody-recognized TAs such as carcinoembryonic antigen (CEA) exist on the cell surface of target tumor cells. Therefore, endowing CTLs or NK cells with antigen-binding specificity of anti-TA antibody is promising for re-targeting the activities of these effector cells to tumor cells in an HLA-independent manner. This mini-review provides a brief overview of the following four technologies for re-targeting CTLs or NK cells to CEA-expressing tumor cells with anti-CEA antibody activity: i) bispecific antibody technology, ii) antibody-cytokine fusion protein technology, iii) chimeric immune receptor technology, and iv) antibody-HLA/peptide complex technology.

Preparation of human IgG and IgM monoclonal antibodies for MK-1/Ep-CAM by using human immunoglobulin gene-transferred mouse and gene cloning of their variable regions.

For antibody-based therapy of cancer, monoclonal antibodies (mAbs) of human origin are superior to mouse, mouse/human chimeric or humanized mAbs, because of their minimum immunogenicity to humans and their efficient collaboration with human effector cells. In the present study, human mAbs were prepared against a pancarcinoma antigen, MK-1 (Ep-CAM), using a genetically-engineered mouse (KM mouse) that contains the human immunoglobulin genes. Spleen cells from KM mice, immunized with recombinant MK-1, were fused with P3-U1 mouse myeloma cells. Of 44 anti-MK-1 clones analyzed, two were of IgG4 and the others of IgM clones. Although the two IgG4 clones were suggested to recognize the same antigenic determinant or two closely located determinants, their VK regions were encoded by different light-chain genes while their VH sequences were identical. The two IgG4 and one of the IgM clones tested revealed antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity, respectively, against MK-1-expressing cells in vitro, suggesting that these fully human mAbs produced against MK-1 and their V-region genes, which are applicable for the preparation of engineered antibody fragments that may be useful for antibody-based therapy of cancer.

Strategies to endow cytotoxic T lymphocytes or natural killer cells with antibody activity against carcinoembryonic antigen.

Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are main effecter cells in cellular immunity against tumor cells. T-cell immunotherapy is based on the assumption that tumor(-associated) antigen (TA) peptides are correctly presented by HLA class I molecules on target tumor cells, and NK cell immunotherapy is based on the hypothesis that cell surface TAs or ligands for NK receptors are widely expressed in tumor cells. However, human tumor cells often lose HLA class I molecules, and target cell ligands for NK receptors are not always expressed in human tumor cells. These altered HLA class I phenotypes and non-ubiquitous expression of NK receptor ligands constitute the major tumor escape mechanism facing tumor-specific CTL and/or NK cell mediated responses. These facts also indicate that it is not easy to eliminate the target tumors only by activating tumor-specific CTLs or NK cells with cancer vaccine treatments. On the other hand, it is easily confirmed by immunohistochemistry whether or not antibody-recognized TAs exist on the cell surface of target tumor cells. Therefore, endowing CTLs or NK cells with antigen-binding specificity of anti-TA antibody is a promising approach for re-targeting the activities of these effector cells to tumor cells in an HLA-independent manner. This review summarizes the following four new strategies for re-targeting CTLs or NK cells to carcinoembryonic-antigen-expressing tumor cells: (1) bispecific antibody technology; (2) antibody-cytokine fusion protein technology; (3) chimeric immune receptor technology, and (4) antibody-HLA/peptide complex technology.

Cloning and sequencing of variable region cDNAs of a novel human monoclonal antibody to carcinoembryonic antigen, and generation of a single chain variable fragmented antibody.

BACKGROUND: Taking into consideration immunogenicity in humans, human antibodies and their derivatives have potential advantages for cancer immunotherapy and/or gene therapy over those from different species such as mouse. Recently, we generated 22 human monoclonal antibodies (HmAbs) specific for human carcinoembryonic antigen (CEA) using KM mouse, which carries human antibody genes. In the present study, we tried to clone the variable (V) region genes of the C2-45 HmAb, with the highest affinity for CEA and to generate a human single-chain variable fragmented (scFv) antibody. MATERIALS AND METHODS: Using RT-PCR methods, we cloned and sequenced cDNAs encoding V regions of C2-45, and constructed an scFv gene including a signal sequence for secreting into the periplasmic space of E. coli. RESULTS: Soluble scFv antibody, designated 45KHscFv, was secreted in the periplasmic space and purified by Ni2+ affinity column from the supernatant of E. coli after osmotic shock. When analyzed by SDS-PAGE, the 45KHscFv antibody showed a band corresponding to a calculated molecular weight of 30 kDa. The 45KHscFv antibody also retained a high reactivity with CEA in ELISA using immobilized CEA. CONCLUSION: These results indicate that the nonimmunogenic 45KHscFv antibody could be advantageously used for fusing with other human functional proteins such as cytokines.

Tumor-targeting of viral vectors for cancer gene therapy by using antibodies or their genes against tumor-associated antigens.

Gene therapy is a promising modality for the treatment of malignant tumors for which conventional therapies are often inadequate. Various therapeutic genes have shown promise for tumor cell killing. However, successful gene therapy depends on the development of efficient and targeted gene transfer vectors. The aim of this review is to summarize the tumor-targeting viral vectors for cancer gene therapy by utilizing antibodies or genes against tumor-associated antigens.