In the present study, we describe the production of transgenic silkworms expressing a recombinant mouse mAb in their cocoons. Two transgenic lines, L‐ and H‐, were generated that carried cDNAs encoding the L‐ and H‐chains of a mouse IgG mAb, respectively, under the control of the enhancer‐linked sericin‐1 promoter. Cocoon protein analysis indicated that the IgG L‐ or H‐chain was secreted into the cocoons of each line. We also produced a transgenic line designated L/H, which carried both cDNAs, by crossing the L‐ and H‐lines. This line efficiently produced the recombinant mAb as a fully assembled H2L2 tetramer in its cocoons, with negligible L‐ or H‐chain monomer and H‐chain dimer production. Thus, the H2L2 tetramer was synthesized in, and secreted from, the middle silk gland cells. Crossing of the L/H‐line with a transgenic line expressing a baculovirus‐derived trans‐activator produced a 2.4‐fold increase in mAb expression. The recombinant mAb was extracted from the cocoons with a buffer containing 3 m urea and purified by protein G affinity column chromatography. The antigen‐binding affinity of the purified recombinant mAb was identical to that of the native mAb produced by a hybridoma. Analysis of the structure of the N‐glycans attached to the recombinant mAb revealed that the mAb contained high mannose‐, hybrid‐ and complex‐type N‐glycans. By contrast, insect‐specific paucimannose‐type glycans were not detected. Fucose residues α‐1,3‐ and α‐1,6‐linked to the core N‐acetylglucosamine residue, both of which are found in insect N‐glycans, were not observed in the N‐glycans of the mAb.

Abstract. In order to identify new serum markers of esophageal squamous cell carcinoma (SCC), we performed serological identification of antigens by recombinant cDNA expression cloning (SEREX). E. coli was transformed with a ÏZAPII phage cDNA library prepared from mRNA of an esophageal cancer cell line (T.Tn), and IPTG-induced cDNA products were screened for interaction with antibodies in allogeneic sera of patients with esophageal SCC. We identified myomegalin (MMGL, phosphodiesterase 4D interacting protein/PDE4DIP) as a new SEREX antigen for esophageal SCC. Western blot analysis revealed that serum anti-myomegalin antibodies (s-MMGL-Abs) were present in 43 (47%) of 91 patients, but in only one (2.2%) of 45 healthy controls. Of the 21 patients with stage I disease, 8 (38%) were sero-positive. The positive rate of s-MMGL-Abs was greater than those of other conventional tumor markers. Reverse transcription-PCR analysis suggested that alternative splicing from myomegalin variant 1 to variant 5 may explain, in part, the development of s-MMGL-Abs. Although the presence of s-MMGL-Abs was not related to any clinicopathological features of the patients, multivariate analysis indicated that the presence of s-MMGL-Abs was significantly associated with a favorable prognosis. Consequently, s-MMGL-Abs may be a useful tumor marker to diagnose and establish a prognosis in patients with esophageal SCC. IntroductionEsophageal squamous cell carcinoma (SCC) is a deadly disease. Late presentation in a group of elderly patients with co-morbid illnesses resulted in a poor outcome (1,2). The aggressive behavior of esophageal SCC has been associated with systemic involvement at diagnosis, and the poor prognosis has been attributed largely to a delay in diagnosis (2). Although several serum markers have been reported to be clinically useful for diagnosing esophageal SCC (3,4), these serum markers are limited in terms of detecting early esophageal cancers, except the serum p53 IgG antibody (5,6). In order to identify new tumor-associated antigens that may generate new serum markers for esophageal SCC, we used the method of serological identification of antigens by recombinant cDNA expression cloning (SEREX) which involves the immunoscreening of cDNA libraries prepared from tumor specimens using autologous or allogeneic sera (7). Chen et al (8) and our group (9-13) have succeeded in the identification of several tumor antigens for esophageal SCC. The identification of human tumor antigens recognized by the autologous host is yielding an array of target molecules for the diagnosis, monitoring, and immunotherapy of human cancer (14-16).In the present study, we identified myomegalin as a new SEREX antigen of esophageal SCC, observed that serum anti-myomegalin antibodies (s-MMGL-Abs) were present frequently in esophageal SCC patients, and associated s-MMGL-Abs with a favorable prognosis in patients with esophageal SCC. Materials and methods Human esophageal squamous cell carcinoma cells and tissues.Seven human esophageal squamous cell carcin...

Since benzolamide had effects similar to acetazolamide, inhibition of membrane-bound CA appears to be sufficient to enhance subretinal fluid absorption and retinal adhesiveness. Membrane-specific CA inhibitors may therefore be of clinical value if they minimize side-effects from intracellular CA inhibition.

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