RESUMEN
BACKGROUND: CD44 is a major cellular receptor for hyaluronic acids. The stem structure of CD44 encoded by ten normal exons can be enlarged by ten variant exons (v1-v10) by alternative splicing. We have succeeded in preparing MV5 fully human IgM and its class-switched GV5 IgG monoclonal antibody (mAb) recognizing the extracellular domain of a CD44R1 isoform that contains the inserted region coded by variant (v8, v9 and v10) exons and is expressed on the surface of various human epithelial cancer cells. METHODS AND PRINCIPAL FINDINGS: We demonstrated the growth inhibition of human cancer xenografts by a GV5 IgG mAb reshaped from an MV5 IgM. The epitope recognized by MV5 and GV5 was identified to a v8-coding region by the analysis of mAb binding to various recombinant CD44 proteins by enzyme-linked immunosorbent assay. GV5 showed preferential reactivity against various malignant human cells versus normal human cells assessed by flow cytometry and immunohistological analysis. When ME180 human uterine cervix carcinoma cells were subcutaneously inoculated to athymic mice with GV5, significant inhibition of tumor formation was observed. Furthermore, intraperitoneal injections of GV5markedly inhibited the growth of visible established tumors from HSC-3 human larynx carcinoma cells that had been subcutaneously transplanted one week before the first treatment with GV5. From in vitro experiments, antibody-dependent cellular cytotoxicity and internalization of CD44R1 seemed to be possible mechanisms for in vivo anti-tumor activity by GV5. CONCLUSIONS: CD44R1 is an excellent molecular target for mAb therapy of cancer, possibly superior to molecules targeted by existing therapeutic mAb, such as Trastuzumab and Cetuximab recognizing human epidermal growth factor receptor family.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Receptores de Hialuranos/metabolismo , Neoplasias/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Antineoplásicos/inmunología , Línea Celular Tumoral , Epítopos/genética , Epítopos/inmunología , Epítopos/metabolismo , Femenino , Citometría de Flujo , Variación Genética , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/inmunología , Inmunohistoquímica , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Ratones , Ratones Desnudos , Neoplasias/inmunología , Neoplasias/metabolismo , Células Madre Neoplásicas/inmunología , Células Madre Neoplásicas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismo , Ratas , Ratas Endogámicas F344 , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Projectin is a giant protein related to twitchin and titin/connectin, that is found in arthropod striated muscle. The complete sequence of a 1 MDa projectin from Drosophila muscle was recently deduced from a thorough analysis of the genomic DNA (Southgate and Ayme-Southgate, 2001). Here we report the complete sequence for projectin from crayfish claw closer muscle (8625 residues; 962,634 Da). The N-terminal sequence contains 12 unique 19-residue repeats rich in glutamic acid (E) and lysine (K). This region, termed the EK region, is clearly distinguishable from the PEVK-like domain of Drosophila projectin. The sequence of crayfish flexor projectin differs from that of closer muscle projectin in that there is a 114-residue deletion and a 35-residue insertion in the N-terminal region. Immunofluorescence microscopy demonstrated that projectin is mainly localized within the sarcomeric A band in both closer and flexor muscles, although the N-terminal region was shown to extrude into the I band region. In the closer muscles, invertebrate connectin (D-titin) connects the Z line to the edge of the A band (Fukuzawa et al., 2001). We have shown that invertebrate connectin is also present in flexor muscle sarcomeres, although in very low abundance.