Conjugation to octa-arginine via disulfide bonds confers solubility to denatured proteins in physiological solution and enables efficient cell internalization.

Some protein transduction methods have already been developed for regenerative medicine application. These methods can be applied to soluble proteins but not to insoluble proteins, such as those that originate from inclusion bodies, for example, Escherichia coli. We have developed a method that allows the in vitro solubilization of denatured proteins without refolding and their efficient cellular internalization through conjugation to the peptide, octa-arginine (R8), via disulfide bonds with cysteine residues. Ovalbumin (OVA), denatured in urea solution containing dithiothreitol, was used as a model protein. The R8 peptide was conjugated with OVA in urea solution. Denatured OVA was recovered in the insoluble fraction after dialysis against phosphate-buffered saline. However, almost all the R8-conjugated OVA was recovered in the soluble fraction and used for translocation experiments in HeLa, Chinese hamster ovary-K1, Cos-7, and matured dendritic cells, where efficient internalization of the protein conjugate was observed. Furthermore, we formulated R8-conjugated ß-galactosidase and R8-conjugated luciferase using a similar procedure, and investigated how the conjugated proteins are processed after cell internalization. We also observed that only a small fraction of these proteins refolded and almost all underwent intracellular degradation. These results suggest that this method is suitable for the transduction of antigen-presenting cells and will benefit research and innovation in vaccine design and discovery.

A novel strategy of cancer gene therapy by transcriptional targeting of an allogeneic histocompatibility transgene.

BACKGROUND: A drawback of cancer gene therapy is the failure of toxic gene introduction into a proportion of the tumor cells, resulting in re-progression of the disease. Cancer cell-specificity of the gene introduction has also been problematic. MATERIALS AND METHODS: Previously defined promoter/enhancer DNA of Q5 tumor antigen gene was used for the purpose of transcriptional targeting. A replication defective adenovirus carrying H-2D(d) cDNA placed downstream of the Q5 promoter/enhancer (Ad/Q5-H-2D(d)) was constructed and administered systemically to C57Bl/6 (H-2(b)) mice bearing pre-established hepatic metastasis of the syngeneic M5076 tumor. RESULTS: 5 of the 10 mice showed complete regression of the tumor. Combination of the therapy with low dose cyclophosphamide (CY) administration augmented the therapeutic effect. The effect was brought about by tumor cell-specific allograft rejection reactions and by tumor antigen-specific reactions induced as "bystander effect" by the former reactions. CONCLUSION: The results provide an experimental basis of a highly efficient novel strategy of cancer gene therapy.

Tumor cell-specific gene expression by 5'-flanking DNA of murine Q5 gene in an adenoviral vector system.

BACKGROUND: Regulatory DNA that would induce tumor cell-specific gene expression of arbitrary genes in human cells has been sought. We previously reported that the transcription of mouse Q5 gene was tumor-selective and hypothesized that Q5 5'-flanking DNA had a key role in tumor-selective transcription. MATERIALS AND METHODS: Isolation of 3.9 kb Q5 5'-flanking DNA was carried out and the sequence characteristics determined. Reporter plasmids, recombinant reporter adenoviruses and recombinant reporter lentiviruses were prepared that contained the Q5 5'-flanking DNA fragments of various lengths. Expression of these reporter genes was examined in various murine cells in vitro and in vivo and in various human cells in vitro. RESULTS: Adenovirus vectors that had Q5 5'-flanking DNA contained a regulatory region and induced tumor cell-specific gene expression not only in mouse cells but also in human cells. CONCLUSION: Our newly constructed vector system could be utilized in gene therapy of human cancer.

Tumor cell-specific transcription of a murine histocompatibility class Ib Q5 gene.

BACKGROUND: We previously reported that the Q5 gene product (Q5 antigen) was expressed on the surface of various tumor cells derived from H-2k (Qa-2-) mice. The Q5 antigen has tumor-protective antigenicity in the syngeneic mice. MATERIALS AND METHODS: Transcripts of the Qa region genes were analyzed by the RT-PCR method. Cell fractionation was performed with the MACS method and the phenotypes were estimated by flow cytometry analysis. RESULTS: Transcripts of Q5 were produced by all tumor cell lines. The Q5 transcription was detected only in thymocytes and PBMCs of H-2k (AKR and C3H/He) mice. The phenotype of the PBMCs in which Q5 transcription takes place seems to be, at least partly, CD3-48 cells that might be related to CD3-4-8- spontaneous thymoma cells derived from an H-2k mouse. CONCLUSION: The expression of Q5 in tumor cells in general is a result of a change of transcriptional regulation associated with malignant transformation.

Detection of anti-HLA-F antibodies in sera from cancer patients.

BACKGROUND: We previously reported that a Q5 gene product (Q5 antigen) on the surface of tumor cells caused humoral immune reaction in syngeneic tumor-bearing mice. Transcripts of the Q5 gene were observed in various tumor cells. The human counterpart of the Q5 gene has not been clarified yet. MATERIALS AND METHODS: Transcripts of human class Ib genes in various human tumor cell lines were analyzed by the RT-PCR method. Whether anti-HLA-F antibodies were present in sera from cancer patients was investigated through Western blotting with recombinant HLA-F as an antigen. RESULTS: Transcripts of the HLA-F gene were produced by various tumor cell lines. In addition, anti-HLA-F IgG was present in the sera derived from various types of cancer patients (26 positives / 42 tested), but not in the sera derived from healthy donors (0 / 20). CONCLUSION: Anti-HLA-F antibodies should be further investigated as possible diagnostic tumor markers.

Clinical benefit of non-toxic therapy in patients with advanced cancer (opinion).

It has been widely recognized that chemotherapy of cancer has profound toxic side-effects and suffers limitations in efficacy but, as yet, no solution has been found to this conflict. Consequently, many patients with advanced cancer desire treatment by less toxic therapies even if the technique is not established as a conventional cancer therapy. Thus the establishment of less toxic therapies should be considered as a tentative requirement in clinical practice. Adoptive immunotherapy is one such therapy which has enjoyed some popularity in Japan. Clinical experience accumulated over the last 15 years indicates benefits to be gained from the therapy. In 1998, the Ministry of Health, Labor and Welfare of Japan (MHLW) approved portions of adoptive immunotherapy practices in their definition of highly-advanced medical technology. In April 1999, we established a private out-patient clinic specializing in adoptive immunotherapy; this clinic has administered 200 infusions of activated lymphocytes a month. Out of the 188 patients treated, we evaluated the tumors of 57 cases (2 CR, 4 PR, 20 SD and 31 PD). The response rate was 11%. It is noteworthy that in 11 of the 20 SD patients, the disease remained stable for more than six months. The follow-up periods for these 11 patients ranged from 11 to 24 months. During this period tumor progressed in 2, but the remaining 9 were still alive and without noticeable disease progression. Our present experience of adoptive immunotherapy administered to a large number of patients indicates that this non-toxic therapy has some beneficial role for the treatment of refractory cancer.

Insoluble fraction of tumor cell homogenate is a useful material for eliciting cytotoxic T lymphocytes: a unique method for protein solubilization.

BACKGROUND/AIM: Dendritic cell (DC)-based cancer immunotherapy using tumor homogenate has been evaluated. In all previously reported cases, DCs have been pulsed with a soluble fraction (lysate) of the tumor homogenate. The aim of this study was the evaluation of DCs pulsed with solubilized insoluble fraction of tumor cells. MATERIALS AND METHODS: Solubilized recombinant murine TRP-2 and solubilized-insoluble fraction of B16 melanoma was prepared by a novel method using nucleotides. Bone marrow-derived DCs were electroloaded with the solubilized proteins. RESULTS: Cytotoxic T lymphocytes were elicited in the splenocytes of C57BL/6 mice immunized with electroloaded DCs with solubilized rTRP-2. CD8(+) T-cells derived from immunized mice with electroloaded DCs using the solubilized insoluble fraction of B16 melanoma had specific killing activity. The effects were augmented when DCs were electroloaded with both the soluble and insoluble fractions of B16 homogenate. CONCLUSION: Insoluble fraction of tumor cells is a useful material for cancer immunotherapy.