Comprehensive screening for antigens overexpressed on carcinomas via isolation of human mAbs that may be therapeutic

Abstract

Although several murine mAbs that have been humanized became useful therapeutic agents against a few malignancies, therapeutic Abs are not yet available for the majority of the human cancers because of our lack of knowledge of which antigens (Ags) can become useful targets. In the present study we established a procedure for comprehensive identification of such Ags through the extensive isolation of human mAbs that may become therapeutic. Using the phage-display Ab library we isolated a large number of human mAbs that bind to the surface of tumor cells. They were individually screened by immunostaining, and clones that preferentially and strongly stained the malignant cells were chosen. The Ags recognized by those clones were isolated by immunoprecipitation and identified by MS. We isolated 2,114 mAbs with unique sequences and identified 21 distinct Ags highly expressed on several carcinomas. Of those 2,114 mAbs 356 bound specifically to one of the 21 Ags. After preparing complete IgG1 Abs the in vitro assay for Ab-dependent cell-mediated cytotoxicity (ADCC) and the in vivo assay in cancer-bearing athymic mice were performed to examine antitumor activity. The mAbs converted to IgG1 revealed effective ADCC as well as antitumor activity in vivo. Because half of the 21 Ags showed distinct tumor-specific expression pattern and the mAbs isolated showed various characteristics with strong affinity to the Ag, it is likely that some of the Ags detected will become useful targets for the corresponding carcinoma therapy and that several mAbs will become therapeutic agents.phage Ab library ͉ therapeutic Ab ͉ tumor-associated antigen S ince the discovery of a method to produce mAbs numerous scientists have been trying to identify and produce mAbs that could be used for immunotherapy against various malignancies. The success for example of alemtuzumab against CD52, trastuzumab against HER2, and rituximab against CD20 for treatment of chronic lymphocytic leukemia, breast cancer, and nonHodgkins lymphoma, respectively (1-3), suggests that mAbs are likely to become very important therapeutic agents also against a wider range of cancers. However, for the majority of the human cancers useful therapeutic Abs are not yet available because of our lack of knowledge of which antigens (Ags) are likely to become useful targets (4). Therefore, several groups of investigators have been trying to identify other potential Ags as targets for immunotherapy using microarray technology (5, 6). Although many differences in transcripts have been revealed between malignant cells and the normal counterpart cells, it will take more time and laborious work to examine which Ags could be targets and to prepare therapeutic Abs against them. Furthermore, the presence of a large amount of transcripts does not always indicate expression of a large amount of the proteins.Our experimental approach was designed in the opposite way to the strategy with the microarray technology mentioned above and was based on the phage-display technology (7). First we isolate...

Clinical significance of CADM1/TSLC1/IgSF4 expression in adult T-cell leukemia/lymphoma

“…17 To confirm CADM1 expression on the cell surface of ATLL cells, we examined CADM1 membrane expression by flow cytometry with an Alexa 488-labeled human anti-CADM1 antibody generated by phage-display technology. 22 Four ATLL cell lines were used for flow cytometry: CADM1-negative ED and CADM1-positive KOB, KK1 and S1T cell lines. In all three CADM1-positive cell lines, the fluorescence intensity of CADM1 expression was two logs greater than that of the isotype immunoglobulin G control (Figure 1b, upper panels), while only background levels of fluorescence could be seen in the CADM1-negative ED-ATLL cell line, which had high levels of DNA methylation in the CADM1 promoter region.…”

Selecting an Optimal Antibody for Antibody- Drug Conjugate Therapy

“…Antigens are frequently identified by immunoprecipitation with the isolated cell-surface-binding antibodies, followed by proteolytic digestion and mass spectrometry to identify bound peptides (Kurosawa et al 2008); see also (Poul et al 2000;Goenaga et al 2007). Overexpression of the candidate gene on a test cell or reduction of the candidate gene expression via small interfering RNA (siRNA) can be used to confirm the identity of the candidate antigen.…”

A 3D matrix platform for the rapid generation of therapeutic anti-human carcinoma monoclonal antibodies

“…Recent interest has focused on designing unbiased phenotypic screens wherein the identification of function-blocking effects precede efforts to dissect the underlying molecular mechanisms that give rise to the desired outcomes (13,15,31,32). With increasing evidence that cell behavior in 3D culture systems more faithfully recapitulates in vivo function, greater emphasis has been placed on developing improved in vitro models for screening purposes, including the use of basement membranelike gels, pepsin extracts of dermal collagen, and synthetic hydrogels (1, 2, 6, 33-35).…”