The generation of monoclonal antibodies against human pancreatic exocrine cancer: a study of six different immunisation regimes

Six different immunisation regimes have been used to generate spleen cells with reactivity against human pancreatic exocrine cancer. Immunised spleen cells were fused with an NSO/1 myeloma line and supernatants from these hybridomas selectively screened for monoclonal antibodies which bound predominantly to a pancreatic cancer cell line (GER). The spleen cells from hairy litter mates immunised with pancreatic cancer xenograft homogenates and viable GER cells generated 13% of hybridoma supernatants which showed some selectivity for GER pancreatic cancer cells in a fixed cell ELISA assay. The other methods produced only 4% of hybrids with selectivity for GER cells. The antigen distribution on gluteraldehyde fixed cells was similar to that found for viable cell monolayers but many antigens were unstable on formalin fixation. Immunohistochemical staining of GER cells grown on glass slides showed a heterogeneity of antigen distribution with up to 70% of the cells exhibiting a vesicular pattern of staining. Fifty percent of the antibodies which bound to GER cells were also reactive against antigens in formalin-fixed paraffin-embedded tissue sections of the original GER tumour. Monoclonal antibody DD9E7 identified an antigen expressed on 12/14 pancreatic adenocarcinomas. The antibody showed strong staining of malignant luminal membranes and cytoplasm. The antigen was also present in normal salivary and sweat glands, and colon and breast carcinomas, but its tissue distribution was unlike that of CEA or EMA. The expression of this antigen in 12/14 of pancreatic carcinomas suggests that DD9E7 may be a useful reagent for pancreatic tumour detection.     

Growth of human digestive-tumour xenografts in athymic nude rats.

The athymic nude rat rnu/rnu has been established as an in vivo model for the acceptance of human digestive-tumour xenografts. We report the successful xenografting of 7/12 (58%) primary explants from patients with digestive cancer. Successful xenografting also occurred in 21/25 (84%) pancreatic tumours derived from a pancreatic exocrine adenocarcinoma (GER) maintained in cell culture; 2 of those have been successfully passaged in nude rats. The simultaneous implantation of these tumours into nude mice led to an almost identical take rate. Passage of one colonic and one pancreatic xenograft from nude rats into nude mice, and transplantation back into nude rats, increased the take rates. The critical period for the establishment of primary tumour growth was usually 28-42 days. The xenografts maintained histological and cytological characteristics of the primary explants or of the original tumour from which the cell line derived. The karyotype of the cell line was also maintained in the solid tumour. Three murine tumours were successfully grown as xenografts. Despite their immunoincompetence, the rats in this study showed no increased morbidity or mortality when kept in conventional conditions, compared with animals housed in isolators. The athymic nude rat will become a valuable complementary tool to the nude mouse for the establishment and maintenance of human digestive tumours and for surgical and serial serological studies.     

Production of antibodies against antigens released from human pancreatic tumour xenografts

Antibodies directed against the antigen released from viable tumour cells during growth have been raised by cross-immunization of immunocompetent hairy litter-mates with serum from nude mice bearing pancreatic tumour xenografts. Antisera raised against the components released from a primary pancreatic tumour xenograft (WB) or from a tumour cell-line xenograft (GER) showed a titre greater than 1:625 against cultured pancreatic tumour cells by an I125-binding assay. Five out of 14 of the hairy litter-mates immunized with serum from the same tumour (GER) produced antisera that bound more strongly to pancreatic cancer cells than to 13 other non-pancreatic cell lines (binding ratio greater than 2). Absorption of the antisera with pure CEA reduced the level of binding by 11-25% without affecting the specificity for pancreatic tumour cells. The antibody response could be completely removed by absorption with pancreatic tumour cells, whereas 50% and 18% of the activity remained after absorption with normal pancreas homogenate and a mixed non-pancreatic tumour-cell pool, respectively. Immunofluorescent staining of pancreatic tumour sections indicated that the antibody was localized on the membrane of ductular epithelial cells. Challenge of immunocompetent mice using this procedure has produced a polyspecific antiserum with signs of selectivity for the circulating antigens released from pancreatic cancer cells, and may provide a route to the production of antibody against specific tumour components.     

Athymic rats in preclinical immunodiagnostic studies

The usefulness of nude rat xenograft systems in immunolocalization studies was investigated using the monoclonal antibody 9.2.27 which binds to melanomas and osteosarcomas. Three human tumors, two melanomas (LOX and FEMX-I) and one osteosarcoma (OHSX), were used. They were established as s.c. xenografts in congenitally athymic (rnu/rnu) nude rats. These serially transplantable tumors showed the same morphology, take rate and growth properties in nude rats as in nude mice. Radiolabeled 9.2.27 F(ab')2 fragments injected i.v. into nude rats were concentrated in s.c. LOX and OHSX xenografts, reaching tumor to blood ratios of up to 30 after 3-4 days. However, the injected antibody failed to concentrate in FEMX-I xenografts, in contrast to previous findings in mice. This discrepancy could be attributed neither to significant differences in in vivo distribution of the labeled antibody nor to the presence of blocking factors in the serum of nude rats. In immunoscintigraphic studies clear images of s.c. LOX tumors were obtained, whereas lung colonies were less well visualized. Biodistribution studies showed a low tumor to blood ratio of about 4 in the latter animals, suggesting a tumor site-dependent variation in homing of labeled antibodies. Radiography was found to be superior to immunoscintigraphy in detecting the lung tumors. The present findings demonstrate that results of immunolocalization studies in nude mice cannot readily be extrapolated to other species. For the purpose of preclinical evaluation of new methods in cancer diagnosis and treatment, tumor xenografts in nude rats may represent a valuable complement to nude mouse models.     

Reliable establishment of human sarcoma xenografts in the nude rat

Purpose. The ability to establish consistent human tumor xenografts in experimental animals is a crucial part of preclinical investigations.The goal of this study was to develop a method of establishing a human tumor xenograft in the leg of a nude rat for evaluation of new surgical and molecular methods of treatments of human extremity sarcoma.Methods and results. Initial attempts to produce sarcoma nodules by subcutaneous injection of a human leiomyosarcoma tumor cell suspension (SKLMS-1) resulted in tumor nodule formation in only four of 10 sites (40%).The xenograft method was modified to include younger nude rats of a different source and substrain (HSD:rnu/rnu, 5-9 weeks old), treated with 500 cGy whole-body irradiation, and the transplantation of tumor cells or small tumor fragments which had been embedded in Matrigel.These changes improved the tumor take rate per site to 52/52 (100%).Tumor nodules demonstrated rapid and progressive growth and histological features consistent with the original human sarcoma.Discussion. Successful human leiomyosarcoma establishment in these nude rats permits the investigation of sarcoma biology and treatment with surgical procedures for which a mouse model would be inadequate. In this study we identified modifications in technique which enhanced the xenografting of a leiomyosarcoma cell line in nude rats; these techniques may increase tumor take rates for other tumor types as well.     

Decrease of human pancreatic cancer cell tumorigenicity by alpha1,3galactosyltransferase gene transfer

The enzyme alpha1,3galactosyltransferase synthesizes the alphaGal epitope, a carbohydrate structure (Galalpha1,3Galbeta1,4GlcNAc-R), on glycoconjugates in lower mammals. The enzyme is absent in humans but large amounts of natural antibodies that recognize alphaGal epitopes are present in human serum. It is likely that these antibodies contribute to the host defense and participate in the hyperacute rejection of xenograft. Previous studies indicated that the glycosyltransferase gene transfer into tumoral cells can modify the structure of glycoconjugates at the cell surface and, as a consequence, modulates the metastatic and tumorigenic behaviors of these cells. The aim of our study was to determine whether the expression of alphaGal epitope can modify the tumorigenicity of human pancreatic cancer cells. The expression of alphaGal epitopes in the human pancreatic cancer cell lines BxPC-3 and Panc-1 was obtained by selecting stable cell clones transfected with murine alpha1,3galactosyltransferase gene. The expression of the enzyme activity in BxPC-3 and Panc-1 cells resulted in the formation at the cell surface of alphaGal epitopes that are recognized by human anti-alphaGal antibodies. alphaGal epitope expression at the surface of pancreatic cancer cells was associated with the fixation of complement 1q to human anti-alphaGal antibodies. The alphaGal epitope expression also resulted in a delay in the tumoral development of BxPC-3 and Panc-1 cells in vivo after xenograft transplantation of nude mice. In addition to the impairment of the metastatic potential of murine tumor cell lines and the activation of immune response, our study provides evidence that the cell surface expression of alphaGal epitopes also modulates the tumorigenic behavior of human pancreatic cancer cells.     

Restoration of alpha(1,2) fucosyltransferase activity decreases adhesive and metastatic properties of human pancreatic cancer cells

The expression of alpha(1,2) fucosyltransferases that catalyze the fucose transfer to galactose of the N-acetyl(iso)lactosamine chain is decreased in human metastatic pancreatic cancer cells. alpha(2,3) Sialyltransferases catalyze the transfer of sialic acid to the same substrate to form, with alpha(1,3/1,4) fucosyltransferases, sialyl-Lewis a and sialyl-Lewis x determinants on cell surface that are involved in pancreatic metastatic invasion. The aim of this study was to determine whether this decrease of alpha(1,2) fucosyltransferase expression can favor the alpha(2,3) sialyltransferase activity to form metastatic sialyl-Lewis antigens. Restoration of alpha(1,2) fucosyltransferase activity in the human pancreatic cancer cell line BxPC-3 was obtained by selecting stable transfectants expressing FUT1. Overexpression of FUTI in BxPC-3 cells resulted in a substantial reduction of sialyl-Lewis antigen expression that correlated with an increase of expression of Lewis y and H-type antigens on cell surface. The modified oligosaccharide structures were preferentially restricted to three major glycoproteins, which could in part be related to mucin-type glycoproteins. The reduction of sialyl-Lewis antigen expression was associated with an inhibition of adhesive properties to E-selectin and a decrease of gastrointestinal metastatic power of BxPC-3 cells after xenograft transplantation into nude mice. This study provides evidence that the expression level of alpha(1,2) fucosyltransferase may regulate the expression of sialyl-Lewis a and sialyl-Lewis x antigens and consequently could play an important role in metastatic properties of human pancreatic cancer cells.     

A small interfering RNA targeting proteinase-activated receptor-2 is effective in suppression of tumor growth in a Panc1 xenograft model

Proteinase-activated receptor-2 (PAR-2), which is a G protein-coupled receptor, is activated in inflammatory processes and cell proliferation. We previously demonstrated that an anti-PAR-2 antibody suppresses proliferation of human pancreatic cells in vitro. However, there have been no studies of PAR-2 signaling pathways in vivo. The aim of this study was to determine whether blockade of PAR-2 by RNA interference influences pancreatic tumor growth. We originally constructed small interfering RNAs (siRNAs) targeting human PAR-2, and performed cell proliferation assays of Panc1 human pancreatic cancer cell line with these siRNAs. Intratumoral treatment with these PAR-2 siRNAs and atelocollagen was also performed in a xenograft model with nude mice and Panc1 cells. siRNAs against human PAR-2 inhibited proliferation of Panc1 cells, whereas control scramble siRNAs had no effect on proliferation. The PAR-2 siRNAs dramatically suppressed tumor growth in the xenograft model. PAR-2-specific siRNA inhibited growth of human pancreatic cancer cells both in vitro and in vivo. Blockade of PAR-2 signaling by siRNA may be a novel strategy to treat pancreatic cancer.     

Relationship between alphaGal epitope expression and decrease of tumorigenicity in pancreatic adenocarcinoma model

The alphaGal epitope is a carbohydrate structure, Galalpha1,3Galbeta1,4GlcNAc-R, synthesized on glycoconjugates in many mammals by alpha1,3galactosyltransferase. Humans do not express this epitope and present in serum large amounts of naturally occuring antibodies, which recognize the alphaGal epitopes and participate in the hyperacute rejection of xenograft. Studies indicated that the fundamental mechanism of hyperacute rejection involving the alphaGal epitope expression can be used in cancer therapy. We have previously suggested that the alphaGal epitope expression by human pancreatic tumoral cells could decrease the tumorigenic behavior of these cells. To determine whether the expression of the alphaGal epitope can modify the tumorigenicity of pancreatic cancer cells, we used a Syrian golden hamster pancreatic adenocarcinoma experimental model. The expression of alphaGal epitopes in the Syrian golden hamster pancreatic cancer cell line HaP-T1 was obtained by selecting stable cell clones transfected with murine alpha1,3galactosyltransferase gene. The alphaGal epitope expression resulted in a delay in the tumoral development of HaP-T1 cells in vivo after allograft transplantation of Syrian golden hamsters (2.5-fold, P < 0.05) and of nude mice. This result is associated with an 100% increase in survival time of nude mice bearing tumors expressing the alphaGal epitope. Our results confirm that the cell surface expression of alphaGal epitope decreases the tumorigenic behavior of pancreatic cancer cells. This novel property may be useful for the development of cancer gene immunotherapy strategy.     

ESTABLISHMENT OF A HUMAN PANCREATIC ADENOCARCINOMA CELL LINE （JF305） WITH p53 EXPRESSION

李昕，王宏，姜奕，王晓华，贾兰玲，张宝庚ＥＳＴＡＢＬＩＳＨＭＥＮＴＯＦＡＨＵＭＡＮＰＡＮＣＲＥＡＴＩＣＡＤＥＮＯＣＡＲＣＩＮＯＭＡＣＥＬＬＬＩＮＥ（ＪＦ３０５）ＷＩＴＨｐ５３ＥＸＰＲＥＳＳＩＯＮ￥ＬｉＸｉｎ；ＷａｎｇＨｏｎｇ；ＪｉａｎｇＹｉ；Ｗａｎｇ...     

Human alpha-lactalbumin made lethal to tumor cells (HAMLET) kills human glioblastoma cells in brain xenografts by an apoptosis-like mechanism and prolongs survival

Malignant brain tumors present a major therapeutic challenge because no selective or efficient treatment is available. Here, we demonstrate that intratumoral administration of human alpha-lactalbumin made lethal to tumor cells (HAMLET) prolongs survival in a human glioblastoma (GBM) xenograft model, by selective induction of tumor cell apoptosis. HAMLET is a protein-lipid complex that is formed from alpha-lactalbumin when the protein changes its tertiary conformation and binds oleic acid as a cofactor. HAMLET induces apoptosis in a wide range of tumor cells in vitro, but the therapeutic effect in vivo has not been examined. In this study, invasively growing human GBM tumors were established in nude rats (Han:rnu/rnu Rowett, n = 20) by transplantation of human GBM biopsy spheroids. After 7 days, HAMLET was administered by intracerebral convection-enhanced delivery for 24 h into the tumor area; and alpha-lactalbumin, the native, folded variant of the same protein, was used as a control. HAMLET reduced the intracranial tumor volume and delayed the onset of pressure symptoms in the tumor-bearing rats. After 8 weeks, all alpha-lactalbumin-treated rats had developed pressure symptoms, but the HAMLET-treated rats remained asymptomatic. Magnetic resonance imaging scans revealed large differences in tumor volume (456 versus 63 mm(3)). HAMLET caused apoptosis in vivo in the tumor but not in adjacent intact brain tissue or in nontransformed human astrocytes, and no toxic side effects were observed. The results identify HAMLET as a new candidate in cancer therapy and suggest that HAMLET should be additionally explored as a novel approach to controlling GBM progression.     

Tumor localization by combinations of monoclonal antibodies in a new human colon carcinoma cell line (LIM1899)

One of the problems of in vivo diagnosis and therapy of tumors with monoclonal antibodies is their heterogeneity with respect to antigen expression, with some cells expressing no antigen and others being weakly or strongly positive. Selected mixtures of antibodies to different antigens are therefore likely to react with more cells than single antibodies and be more effective for imaging and therapy. With this in mind, we have examined a new human colon cancer cell line (LIM1899) which has a heterogeneous expression of several cell surface molecules: by flow cytometry 38% were carcinoembryonic antigen positive; 64%, human milk fat globule positive, and 73%, CD46 positive; 87% of tumor cells bound a mixture of all three antibodies in vitro. Some blocking of the binding of anti-human milk fat globule antibody by the anti-CD46 antibody was noted. LIM1899 was established as a xenograft in nude mice and in vivo biodistribution studies performed using antibodies alone or in combination. Mixtures of antibodies clearly showed a higher percentage of injected dose of antibody in the tumor than did single antibodies: one antibody gave 10%; two together, 17 to 21%; and all three together gave 29% of the injected dose in the tumor. Tumor:blood ratios were also superior for combinations of antibodies, provided that low doses of the antibodies were used; at higher doses the effect was lost. The study demonstrates that combinations of antibodies are better than single antibodies for localization, provided that the dose used is carefully selected.     

An investigation of cellular components released from human renal cancer and foetal kidney xenografts in nude mice (nu/nu) by cross-immunization of hairy littermate relatives

The release of components from human kidney tumour xenografts (GYL) and human foetal kidney explants maintained in nude mice has been studied. The GYL tumour released antigens into the serum which could be detected by the generation of antibodies following cross-immunisation of closely related hairy litter mate (HLM) mice. The production of anti-GYL antibody was monitored by an I125 binding assay using viable GYL tumour cells. In 2/16 hairy litter mate mice, cell surface antibody binding by GYL cells was twice that found with 8 other human tumour cell lines (including 2 other kidney cancer cell lines). Absorption of these antisera with 10(7) GYL tumour cells completely abolished this response, where 50%, 38% and 25% of activity remained following absorption with; a normal kidney cell line, a homogenate of normal kidney and a mixed pool of human tumour cells. Six out of 8 GYL tumour bearing nude mice tested had elevated plasma levels of HCG. Absorption of the HLM antisera with an excess of commercial HCG abrogated I125 binding by only 15%, suggesting that antibody production was not directed primarily against ectopic HCG.     

Depression of T cell-mediated immunity reduces sulfadimethoxine-induced capsular inflammation and inhibits associated development of invasive thyroid follicular cell carcinomas in rats

We previously demonstrated that thyroid capsular inflammation induced by continuous treatment with the antithyroidal agent sulfadimethoxine is associated with development of invasive follicular cell carcinomas in rats initiated with N-bis(2-hydroxypropyl)nitrosamine (DHPN). The inflammatory changes are characterized by large numbers of macrophages and lymphocytes as well as fibroblasts and we hypothesized that it might be enhanced by interplay between macrophages and T cells. To clarify this hypothesis, a comparative study was conducted between athymic nude (rnu/rnu) rats and euthymic (rnu/+) littermates initiated with DHPN (2800 mg/kg, s.c.) followed by sulfadimethoxine treatment in drinking water (0.1%) for 10 weeks. In rnu/+rats, marked capsular thickening with inflammation was induced along with invasive follicular cell carcinomas (2.8 +/- 1.3/rat). In rnu/rnu rats, limited fibrous capsular thickening was noted with or without minimal inflammatory change, and the multiplicity of invasive carcinomas was significantly lower (1.1 +/- 1.0/rat, P < 0.01). Inducible nitric oxide synthase expression in the inflamed lesions was detected in three of 10 rnu/+rats but in none of the rnu/rnu animals. The results thus suggest that development of invasive carcinomas is enhanced by capsular inflammation mediated by T cells, and inducible nitric oxide synthase induction may play a role in tumor progression.     

T-cell lineage of a Friend virus-induced lymphatic leukemia in athymic rats

Athymic (rnu/rnu) and euthymic rats inoculated with the Friend virus-associated lymphatic leukemia virus developed lymphocytic leukemia. Neoplastic cells from these animals were evaluated by means of indirect immunofluorescence and flow cytofluorometry with monoclonal antibodies Ox-1, Ox-7, and W3/25, which react with surface antigens present on normal rat lymphoid cell populations. Lymphoid cells from leukemic animals revealed characteristic alterations in cell surface fluorescence profiles when compared to normal, healthy controls. Athymic and euthymic leukemic rats were similar in that many cells from both the spleen and bone marrow had markers on the cell surface normally found on thymocytes but not on mature peripheral lymphocytes. These studies provided evidence supporting the presence of T-lineage lymphocytes in the athymic rat. Further, this population of early or "pre"-T-lymphocytes included the predominant leukemia cell type induced by the Friend virus-associated lymphatic leukemia virus.     

Blood flow, oxygen consumption, and tissue oxygenation of human breast cancer xenografts in nude rats

Human breast cancer xenografts in T-cell-deficient rnu/rnu rats permit the detailed and systematic study of blood flow, oxygen supply, and characterization of the cellular microenvironment of human tumors in vivo. Using an epigastric pouching technique, it is possible to obtain a tissue-isolated preparation which makes direct studies of blood flow and oxygen supply in human tumors feasible. So far, medullary and squamous cell carcinomas of the breast from patients have been investigated under well-defined systemic conditions. At comparable tumor sizes, the average blood flow rate through human breast cancer xenografts is higher in medullary than in squamous cell carcinomas (0.17 versus 0.10 ml X g-1 X min-1). Blood flow per unit tumor mass significantly decreases with increasing wet weight. No significant differences are obvious when comparing the flow values of pre- and postmenopausal tumors or of cancer tissues with different hormone receptor capacities. On the average, the oxygen consumption rates of human breast cancer xenografts are 10.4 in medullary and 7.7 microliter O2 X g-1 X min-1 in squamous cell carcinomas. With increasing tumor mass, the O2 consumption rate per unit weight significantly decreases. This decrease parallels the respective decline of tumor blood flow, implying that the O2 consumption rate of the cancer cells in vivo is mostly limited by the nutritive blood flow, i.e., by the O2 availability to the tumors. Due to a restricted blood supply, the O2 utilization of human breast cancer xenografts is high. Tissue oxygenation in microareas of human breast cancers xenotransplanted s.c. into nude rats is mostly inadequate. As a consequence, tissue hypoxia and anoxia are common findings even in very early growth stages. Due to marked intra- and intertumor variabilities in blood flow, heterogeneities in the tissue oxygenation are characteristic features of human breast cancer xenografts. From the results obtained it is concluded that human breast cancers growing as xenografts in rnu/rnu rats may be useful tools for cancer research, especially for investigations of blood flow, tissue oxygenation, and substrate turnover.     

Generation and evaluation of a chimeric antibody against coxsackievirus and adenovirus receptor for cancer therapy

Coxsackievirus and adenovirus receptor (CAR) is a single‐pass transmembrane protein that is associated with adenoviral infection. CAR is involved in the formation of epithelial tight junctions and promotes tumor growth in some cancers. Previously, we developed mouse monoclonal antibodies against human CAR and found that one, mu6G10A, significantly inhibited tumor growth in xenografts of human cancer cells. Herein, we generated and characterized a mouse‐human chimeric anti‐CAR antibody (ch6G10A) from mu6G10A. ch6G10A had binding activity, inducing antibody‐dependent cellular cytotoxicity and complement‐dependent cytotoxicity, and in vivo anti‐tumor activity against CAR‐expressing prostate cancer DU‐145 cells. In addition, cancer tissue array analysis confirmed that CAR is highly expressed in neuroendocrine lung cancers including small cell lung cancer, and treatment with ch6G10A effectively inhibited in vivo subcutaneous tumor growth of NCI‐H69 small cell lung cancer cells in nude mice. Moreover, treatment with mu6G10A effectively inhibited both in vivo orthotopic tumor growth and distant metastatic formation in mouse xenograft models of a highly metastatic subline of human small cell lung cancer DMS273 cells. These results suggest that targeting therapy to CAR with a therapeutic antibody might be effective against several cancer types including small cell lung cancer.     

Adhesion of human gastric and pancreatic cancer cells to peritoneal mesothelial cells is mediated by CD44 and beta(1) integrin

Peritoneal dissemination is a common cause of the recurrence of gastric or pancreatic cancer after patients have undergone surgery. The presence of peritoneal metastasis after surgery affects the prognosis of patients with gastric or pancreatic cancer. Very little is known about the biochemical processes involved in the initial attachment of cancer cells to peritoneal mesothelial cells. We conducted in vitro and in vivo studies to assess the role of adhesion molecules in this process, using 5 cell lines derived from human gastric and pancreatic cancers. NUGC-4 and SW1990 cells, which disseminate earlier than the other 3 types of cancer cells after inoculation into the abdominal cavity of nude mice, express large amounts of CD44H. We found that NUGC-4 and SW1990 cells adhere to monolayers of mesothelial cells more firmly than the other cell lines, as shown by adhesion assays performed at 4 degrees C. The adhesion of NUGC-4 and SW1990 cells to mesothelial cells was partially inhibited by antibodies against CD44H or the beta(1) subunit of integrin, and they almost completely blocked adhesion when these 2 antibodies were used in combination in vitro. These 2 antibodies also inhibited the peritoneal metastasis of NUGC-4 and SW1990 cells and prolonged their mean survival time in vivo. These findings suggest that CD44H and beta(1) integrin play important roles in the initial attachment of gastric and pancreatic cancer cells to mesothelial cells. Our results suggest that changes in the expression of CD44H and beta(1) integrin in cancer cells is associated with their ability to adhere to peritoneal mesothelial cells, and thus with the peritoneal metastatic ability of gastric and pancreatic cancer cells. Therefore, the expression of CD44H and beta(1) integrin in gastric and pancreatic cancers could be used as prognostic indicators of peritoneal metastasis. It is possible that a treatment strategy that interferes with the functions of CD44H or beta(1) integrin may result in decreased intra-abdominal spread of cancer.     

Mapping tumor epitope space by direct selection of single-chain Fv antibody libraries on prostate cancer cells

The identification of tumor-specific cell surface antigens is a critical step toward the development of targeted therapeutics for cancer. The epitope space at the tumor cell surface is highly complex, composed of proteins, carbohydrates, and other membrane-associated determinants including post-translational modification products, which are difficult to probe by approaches based on gene expression. This epitope space can be efficiently mapped by complementary monoclonal antibodies. By selecting human antibody gene diversity libraries directly on the surface of prostate cancer cells, we have taken a functional approach to identifying fully human, tumor-specific monoclonal antibodies without prior knowledge of their target antigens. Selection conditions have been optimized to favor tumor-specific antibody binding and internalization. To date, we have discovered >90 monoclonal antibodies that specifically bind and enter prostate cancer cells, with little or no binding to control cells. These antibodies are able to efficiently deliver intracellular payloads when attached to nanoparticles such as liposomes. In addition, a subset of the antibodies displayed intrinsic antiproliferative activity. These tumor-specific internalizing antibodies are likely to be useful for targeted therapeutics either alone or in combination with effector molecules. The antigens they bind constitute a tumor-specific internalizing epitope space that is likely to play a significant role in cancer cell homeostasis. Targeting components of this epitope space may facilitate development of immunotherapeutic and small molecule-based strategies as well as the use of other therapeutic agents that rely upon delivery to the interior of the tumor cell.