Cell‐surface molecules containing growth factor receptors, adhesion molecules and transporter proteins are often over‐expressed in various cancer cells, and could be regarded as suitable targets for therapeutic monoclonal antibodies (mAb). Anti‐cancer therapeutic mAb are claimed to bind these cell‐surface molecules on viable cancer cells: therefore, it is necessary to produce mAb recognizing epitopes on the extracellular domains of native but not denatured proteins. We have experienced difficulty in obtaining mAb bound to viable cancer cells using synthetic peptides or recombinant proteins produced in bacteria as immunogens, although these immunogens are relatively easy to prepare. In this context, we have concluded that viable cancer cells or cells transfected with cDNA encoding target proteins are suitable immunogens for the production of anti‐cancer therapeutic mAb. Furthermore, we selected rats as the immunized animals, because of their excellent capacity to generate diverse antibodies. Because many target candidates are multi‐pass (type IV) membrane proteins, such as 7‐pass G protein‐coupled receptors and 12‐pass transporter proteins belonging to the solute carrier family, and their possible immunogenic extracellular regions are very small, production of specific mAb was extremely difficult. In this review, we summarize the successful preparation and characterization of rat mAb immunized against the extracellular domain of type I, type II and type IV membrane oncoproteins fused to green fluorescent protein as an approach using reverse genetics, and also introduce the discovery of cell‐death‐inducing antibodies as an approach using forward genetics and a strategy to produce reshaped antibodies using mimotope peptides as the immunogen. (Cancer Sci 2011; 102: 25–35)

C luster of differentiation 98/4F2 is a heterodimeric protein with a relative molecular mass of 125 000 (GP125), comprising a 90-kDa hc and 35-kDa lc.(1-3) CD98 was originally identified as a cell-surface antigen associated with the activation of lymphocytes (2) and is expressed on the basal layer of the squamous epithelium and a wide variety of tumors,suggesting its functional involvement in lymphocyte activation, cell proliferation, and malignant transformation. In fact, mAb against rat and human CD98 hc inhibits the activation of lymphocytes and proliferation of tumor cells. (5,6) In addition, NIH3T3 and Balb3T3 cells transfected with cDNA of human and rat CD98 hc have shown various malignant phenotypes.(7-9) CD98 lc have been revealed to be amino acid transporters, (3,10) and multiple functions of CD98 hc, such as cell fusion, (11) regulation of β 1 integrin activation, (12) and induction of apoptosis, (13) have been demonstrated. Transporters corresponding to the amino acid transport system L, y + L, , and Asc have been shown to be CD98 lc, which require CD98 hc for their membrane-based expression. (3,9,14) Six amino acid transporters (LAT1, LAT2, y + LAT1, y + LAT2, Asc-1, and xCT) that belong to the SLC7 family, have been identified to be CD98 lc, and all CD98 lc are believed to be sorted to the plasma membrane via association with CD98 hc. (15 -21) l-type amino-acid transporter 1 is a 12-membrane pass non-glycosylated protein that was first identified as CD98 lc associated with CD98 hc glycoprotein, and mediates Na + -independent large amino acid transport (system L). (3,22) It is reported that mRNA of LAT1 is expressed widely on tumor cells in addition to in the testis, ovary, and brain. (3,(23)(24)(25) However, because specific mAb recognizing the extracellular domain of native human LAT1 protein have not been obtained until now, the precise expression profile of LAT1 protein in normal and cancer cells remains unsolved. In the present paper, we report the successful production of specific mAb against human LAT1 protein, and discuss the specificity and usefulness of anti-LAT1 mAb in cancer therapy. Materials and MethodsCell culture. Human leukemia cells (Molt-4, Jurkat, Daudi, Raji, CCRF-SB, K562, and U937), mouse myeloma cells (P3 × 63Ag8.653), and peripheral blood leukocytes from healthy volunteers were cultured in RPMI-1640 medium (Sigma-Aldrich, St Louis, MO, USA). Human tumor cell lines from the tongue (HEp2), larynx (HSC-3), lung (A549), esophagus (TE-3), breast , liver (HepG2, Hep3B, and HLF), pancreas (PK-1 and PaCa-1), stomach , colon (SW1116, HT29, DU145, and LS-174T), cervix (HeLa and ME180), prostate (PC-3), kidney (ACHN and TOS-1), and bladder (T24, J82, KU-1, KK47, and MGH-U1), glioblastoma cells (KNS-42), melanoma cells (SK-MEL-37), neuroblastoma cells (Tagawa), HEK293F human embryonic kidney cells (Invitrogen, Carlsbad, CA, USA), Int407 embryonic intestine cells, RH7777 rat hepatoma cells (kindly donated by Mitsubishi Tanabe Pharma, Yokohama, Japan), and RenCa mouse renal carcinoma ce...

Although cancer metastasis is associated with poor prognosis, the mechanisms of this event, especially via lymphatic vessels, remain unclear. Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE‐1) is expressed on lymphatic vessel endothelium and is considered to be a specific marker of lymphatic vessels, but it is unknown how LYVE‐1 is involved in the growth and metastasis of cancer cells. We produced rat monoclonal antibodies (mAb) recognizing the extracellular domain of mouse LYVE‐1, and investigated the roles of LYVE‐1 in tumor formation and metastasis. The mAb 38M and 64R were selected from hybridoma clones created by cell fusion between spleen cells of rats immunized with RH7777 rat hepatoma cells expressing green fluorescent protein (GFP)‐fused mouse LYVE‐1 proteins and mouse myeloma cells. Two mAb reacted with RH7777 and HEK293F human embryonic kidney cells expressing GFP‐fused mouse LYVE‐1 proteins in a GFP expression‐dependent manner, and each recognized a distinct epitope. On immunohistology, the 38M mAb specifically stained lymphatic vessels in several mouse tissues. In the wound healing assay, the 64R mAb inhibited cell migration of HEK293F cells expressing LYVE‐1 and mouse lymphatic endothelial cells (LEC), as well as tube formation by LEC. Furthermore, this mAb inhibited primary tumor formation and metastasis to lymph nodes in metastatic MDA‐MB‐231 xenograft models. This shows that LYVE‐1 is involved in primary tumor formation and metastasis, and it may be a promising molecular target for cancer therapy.

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BackgroundCD44 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 FindingsWe 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.ConclusionsCD44R1 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.