Hironori Abe

Generation, immunologic characterization and antitumor effects of human monoclonal antibodies for carcinoembryonic antigen

We generated fully human mAbs (HmAbs) to carcinoembryonic antigen (CEA) using the KM mouse, which carries a human chromosome 14 fragment containing the entire Ig H chain loci and human κ L chain segments in the mouse genome. Forty‐six hybridoma clones producing HmAbs to CEA were thus obtained by fusing the P3‐U1 mouse myeloma cells with splenocytes of the KM mice immunized with CEA. Among them, 22 clones produced HmAbs that reacted with CEA but not with 3 other CEA‐related cell adhesion molecule (CEACAM) family members, CEACAM1, CEACAM6 and CEACAM8. In 12 HmAbs examined, 8 were IgG4, 2 were IgG3, 1 was IgG2, and the other was IgG1. The affinity constants for CEA of these HmAbs were comparable to those of the previously prepared mouse anti‐CEA mAbs (MmAbs). BIAcore analyses revealed that 1 and 2 of the 22 HmAbs react with 2 epitopes defined by MmAbs on the domain N and the domain A1 or B1 of CEA, respectively. In the presence of human complement in vitro, 2 HmAbs tested showed substantial cytotoxicity, namely, 50–65%, against CEA‐expressing tumor cells. With human lymphokine‐activated killer cells in vitro, 3 HmAbs tested exhibited 40–65% Ab‐dependent cell‐mediated cytotoxicity against the tumor cells. Moreover, one of the HmAbs induced a significant inhibition of tumor growth when administered to mice xenografted with the CEA‐expressing cells. Considering their lack of immunogenicity to humans, these CEA‐specific HmAbs may be useful for immunotherapeutic approaches as well as for immunodiagnosis.

Preparation of recombinant MK-1/Ep-CAM and establishment of an ELISA system for determining soluble MK-1/Ep-CAM levels in sera of cancer patients.

The MK-1 antigen, also termed as Ep-CAM, is a membrane glycoprotein that is overexpressed on the majority of tumor cells of epithelial origin and thereby can be used as a target of immunodetection and immunotherapy of cancer. It has previously been shown that several type-I transmembrane proteins, including E-cadherin, ErbB-2 and intercellular adhesion molecule-1 (ICAM-1), may be useful as tumor markers because they are released into the circulation of many cancer patients. To address the question of whether MK-1, the same type-I membrane protein, is also released into the sera, we developed a sandwich-type enzyme-linked immunosorbent assay (ELISA) system by preparing a recombinant MK-1 protein and two anti-MK-1 monoclonal antibodies with different epitope specificities. Using this ELISA, we found that the MK-1 levels in serum samples from healthy volunteers were all less than 2 ng/ml, whereas the Mk-1 levels in sera of about 10% of patients with malignant tumors of various tissue origins were increased to 2-78 ng/ml, indicating that MK-1 is released from tumor cells into the circulation under certain conditions. These findings should be borne in mind when trying to perform passive antibody therapy for cancer using anti-MK-1 antibody.

Identification and comparison of residues critical for cell-adhesion activities of two neutrophil CD66 antigens, CEACAM6 and CEACAM8.

CEACAM6 (CD66c) and CEACAM8 (CD66b) are cell‐adhesion proteins onneutrophils that belong to the human carcinoembryonic antigen (CEA)family. CEACAM6 reveals homophilic adhesion and heterophilic adhesionto other CEACAM family antigens including CEACAM8, CEACAM1, and CEA, whereas CEACAM8 exhibits only heterophilic adhesion to CEACAM6. Here, we investigated and compared structural requirements for the homophilicadhesion of CEACAM6 and heterophilic adhesion between CEACAM6 andCEACAM8 at the amino acid level by using CHO transfectants expressingtheir mutant and chimeric proteins. The NH2‐terminal domain(N‐domain) of CEACAM6 expressed on a CHO cell was suggested to bind the N‐domain of CEACAM6 or CEACAM8 on the opposing cell. Byhomologue‐scanning mutagenesis, we found that the locations of thesequences critical for the adhesion of CEACAM6 to itself and to CEACAM8are overlapped and that they are highly similar but not identical tothe locations of the residues previously shown to be essential for thebinding of CEACAM antigens to Opa proteins of pathogenicNeisseriae. Our findings imply that subtle differences inthe N‐domain sequences determine the specificity of the CEACAM antigenson neutrophils for interaction with the same or different CEACAMantigens and the bacterial proteins.

Opposite effects of thrombospondin-1 via CD36 and CD47 on homotypic aggregation of monocytic cells.

Thrombospondin-1 (TSP-1), an extracellular matrix protein, has a multimodular structure and each domain specifies a distinct biological function through interaction with a specific ligand. In this study we found that exogenously added TSP-1 inhibits phorbol myristate acetate (PMA)/LPS-induced homotypic aggregation of human monocytic U937 cells, whereas the 70-kDa fragment of TSP-1 generated by the proteolytic cleavage of the intact molecule promotes the homotypic aggregation. The aggregation was also inhibited by anti-CD47 mAb or the 4N1K peptide, of which sequence is derived from the CD47-binding site of TSP-1 and absent in the 70-kDa fragment. In contrast, the augmented cell aggregation by the 70-kDa fragment was hampered by anti-CD36 mAb or antibody against the CD36-binding site of TSP-1. The cell aggregation of U937 cells was completely blocked, even in the presence of the 70-kDa fragment, by mAb against leukocyte function associated antigen-1 (LFA-1) or intercellular adhesion molecule-1 (ICAM-1). We therefore propose that TSP-1 may regulate LFA-1/ICAM-1-mediated cell adhesion of monocytes/macrophages by either the inhibitory effect through CD47 or the promoting effect through CD36 depending on which domain/fragment is functional in a given biological setting.

Subcutaneous penectomy and phalloplasty using a testicle for invasive urethral carcinoma.

Invasive bulbomembranous urethral carcinoma appears highly aggressive and requires radical local excision to cure. The standard surgical therapy for this tumor includes en bloc resection of the penis, urethra, scrotum and pubic bone with radical cystoprostatectomy.1 However, recently it has become increasingly important to also consider quality of life.2,3 We report on a patient with invasive bulbous urethral carcinoma, who has been satisfied 5 years after surgery with the surgical and cosmetic results of the methods used for penis and bladder preservation.