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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.

The L-type amino acid transporter-1 (LAT1, SLC7A5) is upregulated in a wide range of human cancers, positively correlated with the biological aggressiveness of tumors, and a promising target for both imaging and therapy. Radiolabeled amino acids such as O-(2-[18F]fluoroethyl)-L-tyrosine (FET) that are transport substrates for system L amino acid transporters including LAT1 have met limited success for oncologic imaging outside of the brain, and thus new strategies are needed for imaging LAT1 in systemic cancers. Here, we describe the development and biological evaluation of a novel zirconium-89 labeled antibody, [89Zr]DFO-Ab2, targeting the extracellular domain of LAT1 in a preclinical model of colorectal cancer. This tracer demonstrated specificity for LAT1 in vitro and in vivo with excellent tumor imaging properties in mice with xenograft tumors. PET imaging studies showed high tumor uptake, with optimal tumor-to-non target contrast achieved at 7 days post administration. Biodistribution studies demonstrated tumor uptake of 10.5 ± 1.8 percent injected dose per gram (%ID/g) at 7 days with a tumor to muscle ratio of 13 to 1. In contrast, the peak tumor uptake of the radiolabeled amino acid [18F]FET was 4.4 ± 0.5 %ID/g at 30 min after injection with a tumor to muscle ratio of 1.4 to 1. Blocking studies with unlabeled anti-LAT1 antibody demonstrated a 55% reduction of [89Zr]DFO-Ab2 accumulation in the tumor at 7 days. These results are the first report of direct PET imaging of LAT1 and demonstrate the potential of immunoPET agents for imaging specific amino acid transporters.