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

Division of Antibody Project, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192, Japan.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 06/2008; 105(20):7287-92. DOI: 10.1073/pnas.0712202105
Source: PubMed

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 IgG(1) 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 IgG(1) 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.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The search for effective antibodies (Ab) for curable cancer immunotherapy has been a quest of many research groups in order to find an effective target that exists on the cancer cell surface. So far there have been no conclusive answers to shed light on the search. This study therefore aimed to bridge the gap of cancer therapy. Screening against 49 kinds of cell lines belonging to 11 kinds of solids cancers was performed. Isolation and characterization for approximately 4200 monoclonal antibodies (mAb) was also performed thereafter. Of those mAb 488 clones that turned out to bind to 29 tumor-associated antigens (TAA) were subjected to immunohistochemical (IHC) analyses. Selection of target antigens (Ag) and a potential antibody for cancer therapy was conducted prior to clinical examinations. In order to find predictably effective targets for therapeutic Ab against solid cancers, expression of the Ag on the surface of cancer and normal cells was extensively examined by IHC analyses using fresh cancer specimens resected from patients. In this study, the tendencies of all staining patterns and distribution of the Ab are reported. While all of the TAA appeared to be involved in tumorigenesis, their expression was not restricted to some specific tumor types but rather randomly distributed among various cancers. Some kinds of Ab including anti-epidermal growth factor receptor (EGFR) and anti-human epidermal growth factor receptor 2 (HER2) indicated the frequency of expression in normal cells was generally low. We concluded that identification of 488 mAb and the accumulated results of IHC analyses in this study could be the key for further therapeutic Ab against cancers. The targets that showed cancer-specific expression are expected to be better for therapeutic Ab than the other Ab. Moreover, further investigation into the growth of cancer cell lines using full human IgG form of Ab shows available efficacy in specific cases.
    Cancer Science 09/2010; 102(1):175-81. · 3.48 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Human antibodies specific for HCMV are currently considered as potential anti-HCMV therapeutic agents. In this study, we used a combinatorial human antibody library to isolate and characterize complete human monoclonal antibodies that effectively neutralize HCMV in a complement-dependent manner. One hundred and six clones were isolated in two independent screens using HCMV virions and recombinant glycoprotein B, gB654, as antigens. All of the clones recognized the same molecule gB and were classified into 14 groups based on the amino acid sequence of the V(H) region. Seven representative clones from these 14 groups had a strong gB654 binding affinity by surface plasmon resonance (SPR). A pairwise binding competition analysis suggested that there were three groups based on differences in the gB recognition sites. Although Fab fragments of the seven groups showed strong affinity for gB, none of the Fab fragments neutralized HCMV infectivity in vitro. In contrast, complete human IgG(1) antibodies of at least three groups neutralized HCMV in a complement-dependent manner. These data suggest that potent therapeutic antibodies can be obtained from a human antibody library, including most of the functional antibodies that mediate humoral immunity to the selected pathogen.
    Microbes and Infection 08/2009; 11(13):1029-36. · 2.92 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: High-throughput generation of antibodies against cellular components is currently a challenge in proteomics, therapeutic development and other biological applications. It is particularly challenging to raise antibodies that target membrane proteins due to their insolubility in aqueous solutions. To address these issues, a yeast display library of human single-chain antibody fragments (scFvs) was efficiently screened directly against detergent-solubilized and biotinylated lysates of a target cell line, thereby avoiding issues with membrane protein insolubility and eliminating the need for heterologous expression or purification of antigens. Antibody clones that specifically bind plasma membrane proteins or intracellular proteins were identified, depending on the biotinylation method applied. Antibodies against a predetermined target could also be identified using cell lysate as an antigen source as demonstrated by selecting an scFv against the transferrin receptor (TfR). When secreted from yeast and purified, the selected scFvs are active under physiological conditions in the absence of detergents. In addition, this method allows facile characterization of target antigens because it is compatible with yeast display immunoprecipitation. We expect that this method will prove useful for multiplex affinity reagent generation and in targeted antibody screens.
    Protein Engineering Design and Selection 05/2010; 23(7):567-77. · 2.59 Impact Factor

Full-text (2 Sources)

Available from
May 27, 2014