ABSTRACT
Photodynamic therapy (PDT) using the photosensitizer talaporfin sodium (talaporfin) is a new mode of treatment for cancer. However, the metabolic mechanism of talaporfin has not been clarified. Thus, we investigated the uptake, transportation, and elimination mechanisms of talaporfin in carcinoma and sarcoma. The results showed that talaporfin co-localized in early endosomes and lysosomes. Talaporfin uptake was via clathrin- and caveolae-dependent endocytosis and a high amount of intracellular ATP was essential. Inhibition of lysosomal enzymes maintained intracellular talaporfin levels. Inhibition of K-Ras signaling reduced talaporfin uptake in carcinoma and sarcoma cell lines. Talaporfin was taken up by clathrin- and caveolae-dependent endocytosis, translocated from early endosomes to lysosomes, and finally degraded by lysosomes. We also demonstrated that ATP is essential for the uptake of talaporfin and that activation of K-Ras is involved as a regulatory mechanism. These results provide new insights into the metabolism of talaporfin in cancer cells for the enhancement of PDT for carcinoma and sarcoma.
Subject(s)
Carcinoma , Photosensitizing Agents/metabolism , Porphyrins/metabolism , Sarcoma , Cell Line, Tumor , HumansABSTRACT
BACKGROUND: Peptide-vaccination therapy targeting tumour-associated antigens can elicit immune responses, but cannot be used to eliminate large tumour burden. In this study, we developed a therapeutic single-chain variable-fragment (scFv) antibody that recognises the cancer stem-like cell/cancer-initiating cell (CSC/CIC) antigen, DNAJB8. METHODS: We screened scFv clones reacting with HLA-A24:20/DNAJB8-derived peptide (DNAJB8_143) complex using naive scFv phage-display libraries. Reactivity and affinity of scFv clones against the cognate antigen were quantified using FACS and surface plasmon resonance. Candidate scFv clones were engineered to human IgG1 (hIgG1) and T-cell-engaging bispecific antibody (CD3xJB8). Complement-dependent cytotoxicity (CDC) and bispecific antibody-dependent cellular cytotoxicity (BADCC) were assessed. RESULTS: scFv clones A10 and B10 were isolated after bio-panning. Both A10-hIgG1 and B10-hIgG1 reacted with DNAJB8-143 peptide-pulsed antigen-presenting cells and HLA-A24(+)/DNAJB8(+) renal cell carcinoma and osteosarcoma cell lines. A10-hIgG1 and B10-hIgG1 showed strong affinity with the cognate HLA/peptide complex (KD = 2.96 × 10-9 M and 5.04 × 10-9 M, respectively). A10-hIgG1 and B10-hIgG1 showed CDC against HLA-A24(+)/DNAJB8(+) cell lines. B10-(CD3xJB8) showed superior BADCC to A10-(CD3xJB8). CONCLUSION: We isolated artificial scFv antibodies reactive to CSC/CIC antigen DNAJB8-derived peptide naturally present on renal cell carcinoma and sarcoma. Immunotherapy using these engineered antibodies could be promising.
Subject(s)
HLA-A24 Antigen/immunology , HSP40 Heat-Shock Proteins/immunology , Immunotherapy/methods , Molecular Chaperones/immunology , Neoplastic Stem Cells/immunology , Nerve Tissue Proteins/immunology , Protein Engineering/methods , Single-Chain Antibodies/biosynthesis , Antibody Specificity , Antibody-Dependent Cell Cytotoxicity , Antigens, Neoplasm/genetics , Antigens, Neoplasm/metabolism , Bone Neoplasms/immunology , Bone Neoplasms/pathology , Bone Neoplasms/therapy , Cancer Vaccines/biosynthesis , Cancer Vaccines/therapeutic use , Carcinoma, Renal Cell/immunology , Carcinoma, Renal Cell/pathology , Carcinoma, Renal Cell/therapy , Cell Line, Tumor , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , HEK293 Cells , HLA-A24 Antigen/genetics , HLA-A24 Antigen/metabolism , HSP40 Heat-Shock Proteins/genetics , HSP40 Heat-Shock Proteins/metabolism , HT29 Cells , Humans , Kidney Neoplasms/immunology , Kidney Neoplasms/pathology , Kidney Neoplasms/therapy , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Osteosarcoma/immunology , Osteosarcoma/pathology , Osteosarcoma/therapy , Peptide Fragments/immunology , Peptide Fragments/metabolism , Peptide Library , Single-Chain Antibodies/therapeutic useABSTRACT
Development and application of anti-CTLA-4 antibody and anti-PD-1 antibody to cancer immunotherapy brought great survival benefits to advanced cancer patients. They have been applied to various cancers such as melanoma, non-small cell lung cancer, renal cell cancer, Hodgkin's disease, and head and neck cancers, and there is no doubt that immunotherapy is becoming a standard therapy as well as surgery, chemotherapy, and radiotherapy. On the other hand, immune-related adverse events (irAEs) have been increasingly reported. Nevertheless, mechanisms of the immune-mediated toxicities are still unclear. There has been a growing interest in the elucidation of the mechanisms. This review describes the general characteristics of irAEs induced by immune checkpoint inhibitors, especially 1. Heterogeneity, 2. Multiplicity, 3. Durability, and 4. Correlativity.
