PIM-1-specific mAb suppresses human and mouse tumor growth by decreasing PIM-1 levels, reducing Akt phosphorylation, and activating apoptosis.

Provirus integration site for Moloney murine leukemia virus (PIM1) is a proto-oncogene that encodes a serine/threonine kinase with multiple cellular functions. Overexpression of PIM-1 plays a critical role in progression of prostatic and hematopoietic malignancies. Here we describe the generation of a mAb specific for GST-PIM-1, which reacted strongly with most human and mouse cancer tissues and cell lines of prostate, breast, and colon origin but only weakly (if at all) with normal tissues. The mAb binds to PIM-1 in the cytosol and nucleus as well as to PIM-1 on the surface of human and murine cancer cells. Treatment of human and mouse prostate cancer cell lines with the PIM-1-specific mAb resulted in disruption of PIM-1/Hsp90 complexes, decreased PIM-1 and Hsp90 levels, reduced Akt phosphorylation at Ser473, reduced phosphorylation of Bad at Ser112 and Ser136, and increased cleavage of caspase-9, an indicator of activation of the mitochondrial cell death pathway. The mAb induced cancer cell apoptosis and synergistically enhanced antitumor activity when used in combination with cisplatin and epirubicin. In tumor models, the PIM-1-specific mAb substantially inhibited growth of the human prostate cancer cell line DU145 in SCID mice and the mouse prostate cancer cell TRAMP-C1 in C57BL/6 mice. These findings are important because they provide what we believe to be the first in vivo evidence that treatment of prostate cancer may be possible by targeting PIM-1 using an Ab-based therapy.

Anti-Pim-1 mAb inhibits activation and proliferation of T lymphocytes and prolongs mouse skin allograft survival.

Pim-1 is an important signaling molecule mediating cell proliferation and survival. Our previous study identified a Pim-1 specific monoclonal antibody, P9, with significant inhibitory effect on cell proliferation. Herein, we report that P9 inhibited the activation and proliferation of PHA-stimulated human PBMC and induced them to undergo apoptosis. In contrast, P9 showed little effect on freshly isolated human blood T lymphocytes which poorly expressed Pim-1. P9 also detected an up-regulation of Pim-1 in mouse lymphocytes after mitogen stimulation, and showed similar selective inhibition on stimulated cells as observed with hPBMC. Furthermore, P9 inhibited the in vitro mixed lymphocyte reaction and P9 treatment significantly prolonged the survival of mouse skin allografts (P<0.001). It is concluded that Pim-1 expression correlates with lymphocyte proliferation and activation. P9 functions as a Pim-1 antagonist and is potential for immunosuppressive therapy.

Mannan-MUC1-pulsed dendritic cell immunotherapy: a phase I trial in patients with adenocarcinoma.

PURPOSE: Tumor antigen-loaded dendritic cells show promise for cancer immunotherapy. This phase I study evaluated immunization with autologous dendritic cells pulsed with mannan-MUC1 fusion protein (MFP) to treat patients with advanced malignancy. EXPERIMENTAL DESIGN: Eligible patients had adenocarcinoma expressing MUC1, were of performance status 0 to 1, with no autoimmune disease. Patients underwent leukapheresis to generate dendritic cells by culture ex vivo with granulocyte macrophage colony-stimulating factor and interleukin 4 for 5 days. Dendritic cells were then pulsed overnight with MFP and harvested for reinjection. Patients underwent three cycles of leukapheresis and reinjection at monthly intervals. Patients with clinical benefit were able to continue with dendritic cell-MFP immunotherapy. RESULTS: Ten patients with a range of tumor types were enrolled, with median age of 60 years (range, 33-70 years); eight patients were of performance status 0 and two of performance status 1. Dendritic cell-MFP therapy led to strong T-cell IFNgamma Elispot responses to the vaccine and delayed-type hypersensitivity responses at injection sites in nine patients who completed treatments. Immune responses were sustained at 1 year in monitored patients. Antibody responses were seen in three patients only and were of low titer. Side effects were grade 1 only. Two patients with clearly progressive disease (ovarian and renal carcinoma) at entry were stable after initial therapy and went on to further leukapheresis and dendritic cell-MFP immunotherapy. These two patients have now each completed over 3 years of treatment. CONCLUSIONS: Immunization produced T-cell responses in all patients with evidence of tumor stabilization in 2 of the 10 advanced cancer patients treated. These data support further clinical evaluation of this dendritic cell-MFP immunotherapy.

[Relationship between cytokine gene polymorphism and development of gastric adenocarcinoma].

OBJECTIVE: To investigate the relationship between the single nucleotide polymorphisms (SNP) of tumor necrosis factor-alpha (TNF-alpha) promoter genes and susceptibility to gastric adenocarcinoma or gastric adenocarcinoma with helicobacter pylori (Hp) infection. METHODS: The SNPs of the TNF-alpha (-238G/A and -308G/A) were determined by gene chip in 130 patients with gastric adenocarcinoma and 142 healthy controls. The sera concentrations of IgG, IgM and IgA of Hp antibodies were measured by ELISA in all cases and controls. RESULTS: H. pylori infection was detected in 69.2% of the 130 patients and 46.5% of the 142 controls (P < 0.01). The frequencies of TNF-alpha-238GA genotype and A allele in the patients with gastric adenocarcinoma were significantly higher than that in the healthy controls (P < 0.01, P < 0.01). The frequencies of TNF-alpha-238GA genotype and A allele in the patients with gastric adenocarcinoma with Hp infection were significantly higher than that in the Hp-negative patients with gastric adenocarcinoma (P < 0.05, P < 0.01). No association was found between any of the other polymorphisms and the patients with gastric adenocarcinoma or patients with gastric adenocarcinoma with Hp infection. CONCLUSION: TNF-alpha-238GA genotype and A allele are significantly related to susceptibility to gastric adenocarcinoma or susceptibility to gastric adenocarcinoma with Hp infection.

The oncogenic serine/threonine kinase Pim-1 directly phosphorylates and activates the G2/M specific phosphatase Cdc25C.

The proto-oncogene Pim-1 encodes a serine-threonine kinase which is a downstream effector of cytokine signaling and can enhance cell cycle progression by altering the activity of several cell cycle regulators among them the G1 specific inhibitor p21(Waf), the phosphatase Cdc25A and the kinase C-TAK1. Here, we demonstrate by using biochemical assays that Pim-1 can interact with the phosphatase Cdc25C and is able to directly phosphorylate the N-terminal region of the protein. Cdc25C is functionally related to Cdc25A but acts specifically at the G2/M cell cycle transition point and can be inactivated by C-TAK1-mediated phosphorylation. Immuno-fluorescence experiments showed that Pim-1 and Cdc25C co-localize in the cytoplasm of both epithelial and myeloid cells. We find that phosphorylation by Pim-1 enhances the phosphatase activity of Cdc25C and in transfected cells that are arrested in G2/M by bleomycin, Pim-1 can enhance progression into G1. Therefore, we propose that Pim-1 activates Cdc25C by a direct phosphorylation and can thereby assume the function of a positive cell cycle regulator at the G2/M transition.

Pim-1 kinase stability is regulated by heat shock proteins and the ubiquitin-proteasome pathway.

Elevated expression of the serine/threonine kinase Pim-1 increases the incidence of lymphomas in Pim-1 transgenic mice and has also been found to occur in some human cancers. Pim-1 acts as a cell survival factor and may prevent apoptosis in malignant cells. It was therefore of interest to understand to what extent maintenance and degradation of Pim-1 protein is affected by heat shock proteins (Hsp) and the ubiquitin-proteasome pathway in K562 and BV173 human leukemic cells. The half-life of Pim-1 protein in these cells was found to increase from 1.7 to 3.1 hours when induced by heat shock or by treating the cells with the proteasome inhibitor PS-341 (bortezomib). The Hsp90 inhibitor geldanamycin prevented the stabilization of Pim-1 by heat shock. Using immunoprecipitation, it was determined that Pim-1 is targeted for degradation by ubiquitin and that Hsp70 is associated with Pim-1 under these circumstances. Conversely, Hsp90 was found to protect Pim-1 from proteasomal degradation. A luminescence-based kinase assay showed that Pim-1 kinase bound to Hsp70 or Hsp90 remains active, emphasizing the importance of its overall cellular levels. This study shows how Pim-1 levels can be modulated in cells through degradation and stabilization.

Cripto: a novel target for antibody-based cancer immunotherapy.

Cripto, a member of the epidermal growth factor-Cripto-FRL-Criptic (EGF-CFC) family, has been described recently as a potential target for immunotherapy (Adkins et al., J Clin Invest 2003;112:575-87). We have produced rat monoclonal antibodies (mAbs) to a Cripto 17-mer peptide, corresponding to the "EGF-like" motif of Cripto. The mAbs react with most cancers of the breast, colon, lung, stomach, and pancreas but do not react or react weakly with normal tissues. The mAbs inhibit cancer cell growth in vitro, and this effect was greater with cytotoxic drugs such as 5-fluorouracil, epirubicin, and cisplatin. The anti-Cripto mAbs prevent tumor development in vivo and inhibit the growth of established tumors of LS174T colon xenografts in Scid mice. The growth inhibitory effects with these mAbs may be greater than those described elsewhere, possibly because of IgM giving more effective cross-linking or binding to a different epitope (EGF-like region versus CFC region). The mechanism of inhibitory effects of the Cripto mAbs includes both cancer cell apoptosis, activation of c-Jun-NH(2)-terminal kinase and p38 kinase signaling pathways and blocking of Akt phosphorylation. Thus, Cripto is a unique target, and mAbs to Cripto could be of therapeutic value for human cancers.

MDX-060. Medarex.

Medarex is developing the antibody MDX-060 for the potential treatment of CD30+ lymphomas, such as Hodgkin's disease and anaplastic large cell lymphomas. Phase I/II clinical trials were underway by December 2002.

Cripto monoclonal antibodies.

The success of molecular target-based cancer therapy exampled by Herceptin targeting Her2 indicates that cancer immunotherapy involves identifying and targeting key molecular drivers of cancer. Recently, the human Cripto, a founding member of the epidermal growth factor-Cripto-FRL1-Cryptic (EGF-CFC) protein family has been demonstrated to be a unique molecule and could be targeted by anti-Cripto monoclonal antibodies for the treatment of cancer. Cripto plays an important role in embryonic development, tumorigenesis, cancer cell proliferation and survival. Cripto is upregulated in most epithelial cancers but is absent or weakly expressed in normal cells. Cripto expression is associated with tumorigenesis and invasion. Cripto is also involved in tumor metastasis, which is strongly supported by the recent discovery that the phenotypic changes of increased motility and invasiveness of cancer cells are reminiscent of the epithelial mesenchymal transition (EMT) that occurs during embryonic development. In this review, we emphasize that the EGF-like region of Cripto plays a critical role in Cripto signaling-mediated tumor growth and EMT. Therefore the EGF-like region should be regarded as a therapeutic point for monoclonal antibody (MAb) intervention. The mechanisms of action of these MAbs to the EGF-like region of Cripto are most likely through intervention of c-Src signaling. The CFC region of Cripto is another region to be targeted for treatment of cancer, as the Cripto can bind to activin B through the CFC region to stimulate cell proliferation. The MAbs to the CFC region block the binding of Cripto and activin B and result in an inhibition of cell growth. Therefore the MAbs to Cripto may be of value in the treatment of various cancers.

Technology evaluation: R-1549, Antisoma/Roche.

R-1549 (formerly known as pemtumomab), a murine monoclonal antibody (HMFG1) specific for the cancer antigen PEM and radiolabeled with yttrium-90, is under development by Antisoma and Roche as a potential treatment for ovarian and gastric cancer. In July 2003, enrollment for a phase III trial in ovarian cancer was completed.

Discovery and validation of new molecular targets for ovarian cancer.

The occult progression of tumors within the peritoneal cavity results in the late diagnosis and high mortality rate of ovarian cancer. An improved understanding of the molecular biology of ovarian cancer has led to the discovery of novel molecules as targets for the treatment of ovarian cancer. This review highlights the latest advance of mucins, which are upregulated in cancer cells and interact with the epidermal growth factor receptor family to regulate cell behavior via signaling pathways associated with malignant transformation, invasion and metastasis. Disruption of these molecules might represent a novel therapeutic approach in treating ovarian cancer. We also describe the recent development and validation of the most studied mucins (MUC1 and MUC16), ErbB-2 (Her2/neu) and folate binding protein as target-based immunotherapy of ovarian cancer.

Pim-1 expression and monoclonal antibody targeting in human leukemia cell lines.

OBJECTIVE: Based on our previous findings that Pim-1 was expressed on the cell surface and could be targeted with a highly specific anti-Pim-1 monoclonal antibody (P9), this study aims to evaluate the possibility that Pim-1 could be targeted for the treatment of human leukemia. MATERIALS AND METHODS: Pim-1 expression was investigated in a series of human leukemia cell lines with immunohistochemistry and flow cytometry. The inhibitory effect of P9 on cell proliferation was evaluated with (3)H-thymidine incorporation assay. Cell apoptosis was assayed with Annexin-V/propidium iodide dual staining. The in vivo effect of P9 was evaluated with xenograft tumor models in severe combined immunodeficient mice. RESULTS: Pim-1 expression varied depending on the cell lines and correlated with the inhibitory effects mediated by P9. An association between Pim-1 expression and drug resistance was observed. Although the drug-resistant CEM/A7R cells were highly resistant to cytotoxic P-glycoprotein substrates, their growth was inhibited by P9 as demonstrated by in vitro proliferation assay and in vivo inhibition of xenograft tumors. P9 had little effect on P-glycoprotein expression and intracellular Rhodamine 123 accumulation, but it inhibited the phosphorylation of Bad and induced apoptosis. CONCLUSIONS: Pim-1 is variably expressed in leukemia cell lines and associated with drug resistance. Targeting Pim-1 with monoclonal antibody could be explored for the treatment of leukemia and may represent a novel strategy to overcome drug resistance.

Carbohydrate residues downstream of the terminal Galalpha(1,3)Gal epitope modulate the specificity of xenoreactive antibodies.

Carbohydrates are involved in many immunological responses including the rejection of incompatible blood, tissues and organs. Carbohydrate antigens with Galalpha(1,3)Gal epitopes are recognized by natural antibodies in humans and pose a major barrier for pig-to-human xenotransplantation. Genetically modified pigs have been established that have no functional alpha1,3-galactosyltransferase (alpha1,3GT), which transfers alphaGal to N-acetyllactosamine (LacNAc) type oligosaccharides. However, a low level of Galalpha(1,3)Gal is still expressed in alpha1,3GT knockout animals in the form of a lipid, isoglobotrihexosylceramide (iGb3), which is produced by iGb3 synthase on lactose (Lac) type core structures. Here, we define the reactivity of a series of monoclonal antibodies (mAb) generated in alpha1,3GT-/- mice immunized with rabbit red blood cells (RbRBC), as a rich source of lipid-linked antigens. Interestingly, one mAb (15.101) binds weakly to synthetic and cell surface-expressed Galalpha(1,3)Gal on LacNAc, but strongly to versions of the antigen on Lac cores, including iGb3. Three-dimensional models suggest that the terminal alpha-linked Gal binds tightly into the antibody-binding cavity. Furthermore, antibody interactions were predicted with the second and third monosaccharide units. Collectively, our findings suggest that although the terminal carbohydrate residues confer most of the binding affinity, the fine specificity is determined by subsequent residues in the oligosaccharide.

MUC1 cytoplasmic tail: a potential therapeutic target for ovarian carcinoma.

Ovarian cancer is often a lethal disease, since the occult progression of the tumor within the peritoneal cavity results in late diagnosis and treatment failure. The identification of molecular events specific to metastasis is critical for the development of effective therapies. MUC1 is aberrantly overexpressed by most ovarian cancer and regarded as a molecular target for ovarian cancer. This review focuses on the latest advances regarding a signaling region in the MUC1 C-terminal subunit-mediated c-Src signaling pathways in malignant transformation, invasion and metastasis. Disruption of MUC1-C-terminal subunit-associated c-Src signaling by targeting the specific sites might represent a novel immunotherapeutic approach for the treatment of ovarian cancer.

The molecular basis for galalpha(1,3)gal expression in animals with a deletion of the alpha1,3galactosyltransferase gene.

The production of homozygous pigs with a disruption in the GGTA1 gene, which encodes alpha1,3galactosyltransferase (alpha1,3GT), represented a critical step toward the clinical reality of xenotransplantation. Unexpectedly, the predicted complete elimination of the immunogenic Galalpha(1,3)Gal carbohydrate epitope was not observed as Galalpha(1,3)Gal staining was still present in tissues from GGTA1(-/-) animals. This shows that, contrary to previous dogma, alpha1,3GT is not the only enzyme able to synthesize Galalpha(1,3)Gal. As iGb3 synthase (iGb3S) is a candidate glycosyltransferase, we cloned iGb3S cDNA from GGTA1(-/-) mouse thymus and confirmed mRNA expression in both mouse and pig tissues. The mouse iGb3S gene exhibits alternative splicing of exons that results in a markedly different cytoplasmic tail compared with the rat gene. Transfection of iGb3S cDNA resulted in high levels of cell surface Galalpha(1,3)Gal synthesized via the isoglobo series pathway, thus demonstrating that mouse iGb3S is an additional enzyme capable of synthesizing the xenoreactive Galalpha(1,3)Gal epitope. Galalpha(1,3)Gal synthesized by iGb3S, in contrast to alpha1,3GT, was resistant to down-regulation by competition with alpha1,2fucosyltransferase. Moreover, Galalpha(1,3)Gal synthesized by iGb3S was immunogenic and elicited Abs in GGTA1 (-/-) mice. Galalpha(1,3)Gal synthesized by iGb3S may affect survival of pig transplants in humans, and deletion of this gene, or modification of its product, warrants consideration.

Delivery of tumor associated antigens to antigen presenting cells using penetratin induces potent immune responses.

Cytoplasmic delivery of proteins or CTL epitopes is crucial for the presentation of antigen for the generation of CTL. We previously described the use of the 16-amino acid peptide penetratin from the Drosophila Antennapedia domain (Int) to transport CTL epitopes into cells. Here we show that, Int, incorporating MUC1 CTL epitopes in tandem is able to facilitate their rapid uptake by macrophages and dendritic cells (DC) in an energy-dependent endocytic pathway. We also demonstrate for the first time that Int conjugated proteins are also able to be efficiently taken up by DC. Furthermore, C57BL/6 and HLA-A2 transgenic mice immunized with the Int-peptides or Int-proteins induce strong IFN-gamma secreting T cells and weak IgG1 antibodies. Immunized C57BL/6 mice were protected against the growth of a MUC1(+) tumor cell line.

Enhanced immunogenicity of heat shock protein 70 peptide complexes from dendritic cell-tumor fusion cells.

We have developed a molecular chaperone-based tumor vaccine that reverses the immune tolerance of cancer cells. Heat shock protein (HSP) 70 extracted from fusions of dendritic (DC) and tumor cells (HSP70.PC-F) possess superior properties such as stimulation of DC maturation and T cell proliferation over its counterpart from tumor cells. More importantly, immunization of mice with HSP70.PC-F resulted in a T cell-mediated immune response including significant increase of CD8 T cells and induction of the effector and memory T cells that was able to break T cell unresponsiveness to a nonmutated tumor Ag and provide protection of mice against challenge with tumor cells. By contrast, the immune response to vaccination with HSP70-PC derived from tumor cells is muted against such nonmutated tumor Ag. HSP70.PC-F complexes differed from those derived from tumor cells in a number of key manners, most notably, enhanced association with immunologic peptides. In addition, the molecular chaperone HSP90 was found to be associated with HSP70.PC-F as indicated by coimmunoprecipitation, suggesting ability to carry an increased repertoire of antigenic peptides by the two chaperones. Significantly, activation of DC by HSP70.PC-F was dependent on the presence of an intact MyD88 gene, suggesting a role for TLR signaling in DC activation and T cell stimulation. These experiments indicate that HSP70-peptide complexes (PC) derived from DC-tumor fusion cells have increased their immunogenicity and therefore constitute an improved formulation of chaperone protein-based tumor vaccine.

Cripto as a target for cancer immunotherapy.

One of the recent, significant advances in cancer immunotherapy is the identification of molecules as targets which regulate cell growth by induction of proliferation and survival signalling pathways. Among them, epidermal growth factor receptor and Her2 have been effectively targeted by monoclonal antibodies. Currently, the treatment of cancer has limitations and most cancer deaths result from the local invasion and distant metastasis of tumour cells. An important insight for the understanding of tumour invasion and metastasis came from the recent discovery that the phenotypic changes of increased motility and invasiveness of cancer cells are reminiscent of the epithelial-mesenchymal transition (EMT) that occurs during embryonic development. The human Cripto, a member of the epidermal growth factor-Cripto, Frl1, and Cryptic (EGF-CFC) protein family and a signalling protein during early embryonic development, plays an important role in cancers. Cripto is attached to the cell membrane through a glycosyl-phosphatidylinositol motif, and is upregulated in a wide range of epithelial cancers. In this paper the authors review the role of Cripto expression in tumourigenesis and in EMT to promote tumour invasion, with emphasis that the unique EGF-like region of Cripto plays a critical role in Cripto signalling-mediated tumour growth and EMT. Therefore, the region should be regarded as a therapeutic point for interruption of the oncogenic and metastatic potential of Cripto for cancer immunotherapy.

The oncogenic serine/threonine kinase Pim-1 phosphorylates and inhibits the activity of Cdc25C-associated kinase 1 (C-TAK1): a novel role for Pim-1 at the G2/M cell cycle checkpoint.

The Pim-1 oncogene encodes a serine-threonine kinase that relays signals from cytokine receptors and contributes to the formation of lymphoid tumors when expressed at high levels. Here we show that the protein kinase Cdc25 C-associated kinase 1 (C-TAK1) is a binding partner and a substrate of Pim-1. A physical interaction of Pim-1 and C-TAK1 could be shown biochemically and in yeast two-hybrid assays. Immunofluorescence experiments suggested that Pim-1.C-TAK1 complexes are predominantly cytoplasmic. When transiently transfected, Pim-1 was also found in the nucleus and could recruit C-TAK1 to this compartment. Both Pim-1 and C-TAK1 underwent autophosphorylation, but only Pim-1 was able to phosphorylate C-TAK1 but not vice versa. Mass spectrometry analysis of C-TAK1 suggested that the sites of autophosphorylation and Pim-1-mediated phosphorylation are distinct and not overlapping. Phosphorylation by Pim-1 decreased C-TAK1 kinase activity significantly, in particular its ability to phosphorylate and inactivate Cdc25C, a protein that actively promotes cell cycle progression at the G(2)/M phase. Hence our findings directly suggest a novel role for Pim-1 as a positive regulator at the G(2)/M transition of the cell cycle.