1. Establishment of P38Bf, a Core-Fucose-Deficient Mouse-Canine Chimeric Antibody Against Dog Podoplanin
Yukinari Kato, Takuya Mizuno, Shinji Yamada, Takuro Nakamura, Shunsuke Itai, Miyuki Yanaka, Masato Sano, Mika K Kaneko Monoclon Antib Immunodiagn Immunother. 2018 Nov;37(5):218-223. doi: 10.1089/mab.2018.0035.
Podoplanin (PDPN), a type I transmembrane sialoglycoprotein, is expressed in normal tissues, including lymphatic endothelial cells, pulmonary type I alveolar cells, and renal podocytes. The overexpression of PDPN in cancers is associated with hematogenous metastasis by interactions with the C-type lectin-like receptor 2 (CLEC-2). We have previously reported the development of a mouse monoclonal antibody (mAb) clone, PMab-38 (IgG1, kappa), against dog PDPN (dPDPN). PMab-38 reacted strongly with canine squamous cell carcinomas and melanomas, but not with lymphatic endothelial cells, indicating its cancer specificity. In this study, we developed and produced several mouse-canine chimeric antibodies originating from PMab-38. A mouse-canine chimeric antibody of subclass A (P38A) and a mouse-canine chimeric antibody of subclass B (P38B) were transiently produced using ExpiCHO-S cells. Core-fucose-deficient P38B (P38Bf) was developed using FUT8 knockout ExpiCHO-S cells. We compared the binding affinities, antibody-dependent cellular cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC) of P38A, P38B, and P38Bf against Chinese hamster ovary (CHO)/dPDPN cells. Flow cytometry analysis showed that the KD of P38A, P38B, and P38Bf were 1.9 × 10-7, 5.2 × 10-9, and 6.5 × 10-9, respectively. Both P38B and P38Bf revealed high ADCC activities against CHO/dPDPN cells; P38Bf demonstrated significantly higher ADCC compared with P38B, especially at low concentrations. P38B and P38Bf exhibited higher CDC activities against CHO/dPDPN cells. Conversely, P38A did not exhibit any ADCC or CDC activity. In summary, P38Bf is a good candidate for antibody therapy against dPDPN-expressing canine cancers.
2. Effects of systemic interferon-alpha (IFN-alpha) on the antigenic phenotype of melanoma metastases. EORTC melanoma group cooperative study No. 18852
U von Stamm, E B Bröcker, M von Depka Prondzinski, D J Ruiter, P Rümke, C Broding, S Carrel, F J Lejeune Melanoma Res. 1993 Jun;3(3):173-80. doi: 10.1097/00008390-199306000-00005.
In order to investigate the effects of in vivo treatment with interferon-alpha (IFN-alpha) on melanoma antigens, a clinical EORTC trial (No. 18852) was accompanied by an immunohistological study. Twenty patients with melanoma metastases of skin and soft tissues, eventually also of the lung, who were treated with systemic IFN-alpha, were evaluated for a comparison of metastases before (40) and during (42) treatment. Representative cryostat sections were studied immunohistologically with a panel of monoclonal antibodies against differentiation antigens (HMW-MAA, K-1-2, NKI-beteb, M-2-10-15), progression markers (transferrin receptor, ICAM-1, VLA-2), histocompatibility antigens (HLA-A, B, C, HLA-DR) and the proliferation-associated nuclear antigen Ki67. We found an overall reduction of the proliferation-associated antigen Ki-67 (p < 0.01), and an increase in expression of HLA-DR (p < 0.05) and ICAM-1 (trend) during treatment. The intensity of expression of HLA-A, B and C antigens as well as pigmentation (p < 0.01) was found to be increased. Early progression (< or = 8 weeks after onset of treatment) was associated with a lack of phenotypic changes. The data suggest an independent modulation of proliferation, pigmentation, and antigen expression by systemic treatment of metastatic melanoma with IFN-alpha.
3. A high affinity nanobody against endothelin receptor type B: a new approach to the treatment of melanoma
Lili Ji, Changsheng Dong, Reiwen Fan, Shuhui Qi Mol Biol Rep. 2020 Mar;47(3):2137-2147. doi: 10.1007/s11033-020-05313-w. Epub 2020 Feb 20.
The aim of the study was to produce a single-domain antibody (nanobody) specific for endothelin receptor type B (EDNRB) which has high expression in melanoma. Cultured human melanoma cells were used as antigens to immunize alpacas. After antibody generation was verified in alpaca serum, total RNA was extracted from alpaca lymphocytes and the target VHH fragment was amplified by two-step PCR, cloned in the pCANTAB5E phagemid vector, and used to transform Escherichia coli TG1 cells to obtain a phage-display nanobody library, which was enriched by panning. The results indicated successful construction of a phage-display anti-human melanoma A375 nanobodies library with a size of 1.2 × 108/ml and insertion rate of 80%. After screening, eight positive clones of anti-EDNRB nanobodies were used to infect E. coli HB2151 for production of soluble nanobodies, which were identified by ELISA. Finally, we obtained a high-affinity anti-EDNRB nanobody, which consisted of 119 amino acids (molecular weight: 12.97 kDa) with 22 amino acids in CDR3 and had good affinity in vitro. The results suggest that the nanobody may be potentially used for the treatment of human melanoma.
