Development of 36 clones producing human monoclonal antibodies specific to cancer cells and their ability to inhibit cancer cell growth.

The development of a specific antibody for cancer therapy could enable a potent strategy for overcoming cancer. As ideal immunotherapy, a human monoclonal antibody (HuMoAb) might have a useful antitumor effect without any lethal toxicities. Thirty-six unique clones producing HuMoAbs were successfully developed using tumor infiltrating lymphocytes collected from 28 patients with several malignant solid tumors. The 36 tumor-specific immunoglobins were found among 9,450 clones after 43 fusions by the conventional hybridoma method. Among these 36 HuMoAbs, 9 had a remarkable tumor-specific reaction and no reaction with normal tissues, as determined with quantum dots-streptavidin and a fluorescence microscope. The inhibition of cell proliferation by the HuMoAbs was evaluated with the MTT assay. Over 40% cell growth inhibition was confirmed with 4 of the 36 HuMoAbs. Two of the antibodies had highly-specific reactivity to carcinomatous lesions with strong growth inhibition and up to 94.3% inhibition of the control growth. In conclusion, 36 clones with HuMoAbs that have specific reactions with cancer cells were successfully established. These HuMoAbs might be utilized as either anticancer or drug delivery agents.

A novel human monoclonal antibody derived from tumor-infiltrating lymphocytes in lung cancer inhibits cancer cell growth with morphological changes.

Tumor-infiltrating lymphocytes (TIL) were isolated from surgically resected human samples using hybridoma techniques, and human monoclonal antibodies (HuMoAbs) were produced. Stable antibody-producing hybridoma cell lines were established and, on the basis of reactivity to human cancer cell lines, a clone of HuMoAb, named HoAKs-1, was selected. By confocal microscopy, we confirmed that HoAKs-1 showed specific and intense reactivity to the cell membrane of HLC-1 and PANC-1, wheareas the antibody did not show reactivity to human umbilical vein endothelial cells (HUVECs). Using xenografts formed from epithelial cancer cell lines in nude mice, we also demonstrated a broad spectrum of reactivity of the antibody in 6 out of 14 xenografts. In addition, using surgically resected clinical specimens from two patients with lung cancer, we showed that HoAKs-1 had specific reactivity to cancerous lesions but not to normal sites. In an in vitro experiment, HoAKs-1 induced morphological changes with neurite-like cytoplasmic processes in MKN-45, HLC-1 and PANC-1, whereas no morphological alterations were observed in HUVECs. Growth inhibition rates by HoAKs-1 were 63% in HLC-1 and 47% in MKN-45, while no growth inhibition occurred in normal HUVECs. We confirmed that HoAKs-1 recognized a 55 kDa protein by determining the molecular weight of the HoAKs-1 reacting protein. In conclusion, we successfully produced a novel HuMoAb, HoAKs-1, which reacted specifically to cancer cells and inhibited cell growth with morphological changes. HoAKs-1 may have the potential to be utilized as an anticancer agent, without causing any immunological reaction in humans.

A new immunofluorostaining method using red fluorescence of PerCP on formalin-fixed paraffin-embedded tissues.

Immunofluorostaining, a versatile tissue staining method, is used in biomedical research because of its clear contrast and precise quantification of positive signals. However, its application in clinical diagnosis has been limited. A major obstacle is high fluorescent background of formalin-fixed, paraffin-embedded tissue sections (paraffin sections). On paraffin sections, strong and broad fluorescence of the section overlapping that of conventional fluorescent dyes such as fluorescein isothiocyanate (FITC) prevents detection of target immunofluorescence. To circumvent the background, we selected an albuminous dye, peridinin chlorophyll a protein (PerCP), for immunostaining of human tumor sections with tumor-reactive monoclonal antibodies. Red fluorescence of PerCP clearly distinguished the tumor region within the yellow-green autofluorescence of the section. Furthermore, it was possible to observe tissue morphology simultaneously without any counterstaining; autofluorescence served as counterstaining in this method. Digital quantification of PerCP-stained image intensity correlated (r2>0.99) well with extracted PerCP amount, indicating the usefulness of image quantification. We conclude that this new and simple immunofluorostaining method can be applied to pathological diagnosis of a wide range of conditions, including cancer.

Antitumor effect of MCC-465, pegylated liposomal doxorubicin tagged with newly developed monoclonal antibody GAH, in colorectal cancer xenografts.

MCC-465 is an immunoliposome-encapsulated doxorubicin. The liposome is tagged with polyethylene glycol and the F(ab')2 of a monoclonal antibody named GAH, a human antibody obtained by the hybridoma technique. The epitope recognized by GAH is not well characterized, but human gastric, colorectal, and mammary cancer cells were GAH-positive, while the normal counterparts were GAH-negative. Pegylated liposome doxorubicin (PLD) and MCC-465 did not show significant antitumor activity against GAH-negative Caco-2 xenografts. On the other hand, MCC-465 exhibited significantly superior antitumor effects against GAH-positive WiDr-Tc and SW837 xenografts, compared with PLD. Immunohistochemistry with GAH revealed that 94% (100 of 106) of surgical specimens of colorectal cancer were GAH-positive. These results warrant a phase I clinical trial of MCC-465 for patients with metastatic colorectal cancer.

Establishment and evaluation of cancer-specific human monoclonal antibody GAH for targeting chemotherapy using immunoliposomes.

To establish human monoclonal antibodies suitable for targeting chemotherapy, we prepared a panel of human-mouse hybridomas, using mouse myelomas and lymphocytes of regional lymph nodes excised from cancer patients, and selected antibodies on the basis of their specificity of binding to the surface of viable cancer cells derived from fresh cancer tissues. A selected antibody, named GAH, was found to react with viable cancer cells from 21/22 stomach and 13/20 colon cancer tissues. As for further analysis, complementary DNAs encoding GAH were cloned and recombinant GAH (rGAH) was obtained from established CHO cells transfected with GAH expression vectors. rGAH selectively stained cancer cells in human tissue sections from 13/14 stomach, 4/11 colon, 5/11 mammary, and 0/7 lung cancers, while no positive staining was observed in those of non-tumor and various normal specimens. Notably, using confocal fluorescence microscopy, rGAH was not only bound to the surface of cancer cells, but was also internalized by the cells. The potential of rGAH for intracellular drug delivery was subsequently evaluated using rGAH-conjugated, doxorubicin (DXR)-encapsulated immunoliposomes. The immunoliposomes were also internalized into the cancer cells and finally DXR was delivered to the cell nucleus. Furthermore, the immunoliposomes could inhibit the growth of DXR-insensitive stomach cancer cells (B37) in an in vivo model. These results suggest that a GAH-utilized liposome-targeting technique will provide a potent and useful cancer chemotherapy with broad applications for cancer patients.