Hyaluronan-estradiol nanogels as potential drug carriers to target ER+ breast cancer cell line.

An innovative hyaluronan-based nano-delivery system is proposed for the active targeting towards ER+ breast cancer. Hyaluronic acid (HA), an endogenous and bioactive anionic polysaccharide, is functionalized with estradiol (ES), a sexual hormone involved in the development of some hormone-dependent tumors, to give an amphiphilic derivative (HA-ES) able to spontaneously self-assemble in water to form soft nanoparticles or nanogels (NHs). The synthetic strategy used to obtain the polymer derivatives and the physico-chemical properties of the obtained nanogels (ES-NHs) are reported. ES-NHs ability to entrap hydrophobic molecules has also been investigated, by loading curcumin (CUR) and docetaxel (DTX), both able to inhibit the growth of ER+ breast cancer. The formulations are studied for their capability to inhibit the growth of the MCF-7 cell line, thus evaluating their efficacy and potential as a selective drug delivery systems. Our results demonstrate that ES-NHs have not toxic effects on the cell line, and that both ES-NHs/CUR and ES-NHs/DTX treatments inhibit MCF-7 cell growth, with ES-NHs/DTX effect higher than that of free DTX. Our findings support the use of ES-NHs to deliver drugs to ER+ breast cancer cells, assuming a receptor-dependent targeting.

Chloroquine supplementation increases the cytotoxic effect of curcumin against Her2/neu overexpressing breast cancer cells in vitro and in vivo in nude mice while counteracts it in immune competent mice.

Autophagy is usually a pro-survival mechanism in cancer cells, especially in the course of chemotherapy, thus autophagy inhibition may enhance the chemotherapy-mediated anti-cancer effect. However, since autophagy is strongly involved in the immunogenicity of cell death by promoting ATP release, its inhibition may reduce the immune response against tumors, negatively influencing the overall outcome of chemotherapy. In this study, we evaluated the in vitro and in vivo anti-cancer effect of curcumin (CUR) against Her2/neu overexpressing breast cancer cells (TUBO) in the presence or in the absence of the autophagy inhibitor chloroquine (CQ). We found that TUBO cell death induced by CUR was increased in vitro by CQ and slightly in vivo in nude mice. Conversely, CQ counteracted the Cur cytotoxic effect in immune competent mice, as demonstrated by the lack of in vivo tumor regression and the reduction of overall mice survival as compared with CUR-treated mice. Immunohistochemistry analysis revealed the presence of a remarkable FoxP3 T cell infiltrate within the tumors in CUR/CQ treated mice and a reduction of T cytotoxic cells, as compared with single CUR treatment. These findings suggest that autophagy is important to elicit anti-tumor immune response and that autophagy inhibition by CQ reduces such response also by recruiting T regulatory (Treg) cells in the tumor microenvironment that may be pro-tumorigenic and might counteract CUR-mediated anti-cancer effects.

Curcumin induces apoptosis in breast cancer cell lines and delays the growth of mammary tumors in neu transgenic mice.

Breast cancer is a leading cancer in women and despite the benefits of the current therapies a significant number of patients with this tumor is at risk of relapse. Some of the alterations taking place in breast cancer cells are currently exploited by molecularly targeted drugs. Different drugs have been developed which target a single molecule but, given that the tumor originates from the dysregulation of many genes, there is the need to find new drugs that have more than one molecular target. Curcumin [1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione] (CUR), a polyphenolic compound found in the spice turmeric, is a pleiotropic molecule able to interact with a variety of molecular targets and has antitumor, anti-inflammatory, antioxidant, immunomodulatory and antimicrobial activities. Here we demonstrate that CUR inhibits the growth of breast cancer cell lines in a dose dependent manner, with IC50 values in the micromolar range, and induces an increase in the percentage of cells in sub-G0 phase, representing the apoptotic cell population. The activation of apoptosis was confirmed by PARP-1 cleavage and by the increased ratio between the pro-apoptotic Bax and the anti-apoptotic Bcl-2 protein. In addition, in CUR-treated cells the activity of ERK1/ERK2 MAP kinases was down-regulated. The cytotoxic effects of CUR were observed in breast cancer cells expressing either high or low levels of ErbB2/neu. The in vivo antitumor activity of CUR was tested in BALB-neuT mice transgenic for the neu oncogene, which develop atypical hyperplasia of the mammary gland at 6 weeks of age and invasive carcinoma at 16 weeks of age. CUR, administered to mice both early and in an advanced stage of mammary carcinogenesis, induced a significant prolongation of tumor-free survival and a reduction of tumor multiplicity. In addition, CUR administration was safe, since no modification of hematological and clinical chemistry parameters could be observed in BALB-neuT and BALB/c mice treated with this compound for several weeks. These findings support further studies on the therapeutic potential of CUR in combination with standard therapies in breast cancer patients.

Changes in serum levels of TNF-alpha, IL-6, OPG, RANKL and their correlation with radiographic and clinical assessment in fragility fractures and high energy fractures.

Stages of bone turnover during fracture repair can be assessed employing serum markers of osteoblastic and osteoclastic activity, inflammatory cytokines, clinical evaluation and imaging instruments. Our study compare the fracture healing process in fragility fractures and high energy fractures by evaluating serum changes of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), osteoprotegerin (OPG) and receptor activator of the nuclear factor-kB ligand (RANKL) in combination with radiographic (Radiographic Union Scale for Tibial fractures, RUST) and clinical (Lower extremity measure, LEM) assessments. We enrolled 56 patients divided into four corresponding groups: group A with high energy trauma fracture (tibial/femoral shaft); group B with low energy trauma fracture (femoral fractures); healthy (control A) and osteoporotic subjects (control B). Blood samples were collected before surgery (T0) and after 10 weeks (T10). Serum concentrations of IL-6, TNF-alpha, RANKL and OPG were quantified using commercial enzyme-linked immunosorbent assay (ELISA) kits. Our results show that RANKL values are significantly higher at T10 than at T0 in low energy trauma fractures (group B). OPG is significantly lower in each control group than that of the respective fractured group and its concentration at T0 and at T10 is significantly lower in high than in low energy fractures. RANKL/OPG ratio is significantly higher in both controls than in fractured groups, and significantly increases after 10 weeks. IL-6 and TNF-alpha concentrations significantly decrease during fracture healing and are higher in high (group A) than in low energy fractures (group B). Significant differences were also found in both RUST score and LEM between groups A and B. Changes in TNF-alpha and IL-6 levels correlate with RUST and LEM in fragility and high energy fractures, while RANKL/OPG ratio is associated with these clinical parameters only in fragility fractures. These findings suggest that serum levels of IL-6, TNF-alpha, RANKL and OPG might be used to monitor the stages of fracture repair. Further studies will be needed to confirm the role of these cytokines in fracture repair.

Alpha fetoprotein is more than a hepatocellular cancer biomarker: from spontaneous immune response in cancer patients to the development of an AFP-based cancer vaccine.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide, with a poor prognosis and limited therapeutic options. Due to its overexpression in the majority of HCCs, alpha-fetoprotein (AFP) represents one of the most useful markers for hepatocarcinomas and for monitoring patients' response to therapy. Although it was earlier reported that AFP has immunosuppressive properties, it has been recently demonstrated that AFP induces spontaneous T and B cells responses in HCC patients. The characterization of AFP-immunogenic epitopes gives the opportunity to design AFP-based cancer vaccines for human HCC. The activity of AFP-based vaccines has been investigated in HCC mouse models in order to develop novel strategies to treat patients with HCC. This review will discuss the rationale for using the AFP-based vaccination strategy and recent results corroborating the usefulness of AFP vaccines as a potential tool for cancer therapy.

Placenta growth factor is a survival factor for human malignant mesothelioma cells.

Placenta growth factor (PlGF) is a key regulator of pathological angiogenesis and its overexpression has been linked to neoplastic progression. To assess whether PlGF could have a role in malignant mesothelioma (MM), we analyzed the expression of PlGF, VEGF, and their cognate receptors (VEGF-R1 and VEGF-R2) and co-receptors (neuropilin-1 and neuropilin-2) in MM cell lines as well as in resected MM tissues, hyperplastic/reactive mesothelium and normal mesothelium. MM cell cultures expressed both ligands and the associated receptors to a variable extent and released different amounts of PlGF. As assessed by immunohistochemistry, PlGF expression was switched on in hyperplastic/reactive compared to normal mesothelium. Moreover, 74 and 94 percent of MM tissues overexpressed PlGF and VEGF-R1, respectively (p<0.05 MM vs normal mesothelium). Administration of recombinant PlGF-2 did not elicit a significant stimulation of MM cell growth, while it was associated with a transient phosphorylation of Akt, suggesting that PlGF-2 could activate downstream effectors of proliferative and cytoprotective signals via VEGF-R1 in MM cells. Indeed, the administration of an anti-PlGF antibody was found to cause a significant reduction of MM cell survival. In conclusion, our data demonstrate that, by acting as a survival factor, PlGF can play a role which goes beyond the stimulation of angiogenesis in MM. This evidence could help the rational design of new therapeutic interventions for this aggressive tumor.

A common repertoire of autoantibodies is shared by cancer and autoimmune disease patients: Inflammation in their induction and impact on tumor growth.

The repertoire of autoantibodies found in cancer patients partly overlaps with that typical of patients with autoimmune diseases. Beside the biochemical and immunological properties of the target antigens and their altered expression in tumor tissues, the intratumoral inflammatory context can play a key role in the induction of autoimmune disease-associated autoantibodies in cancer patients. Furthermore, the impact of such antibodies on cancer growth and progression can be deeply influenced by the interplay with inflammation. The characterization of the spontaneous humoral responses occurring in cancer patients, of the mechanisms that trigger and sustain the autoantibody response and of the biological effects of such autoantibodies may help the rational design of anti-cancer immunotherapeutic protocols.

Identification and characterization of the product encoded by ORF69 of Kaposi's sarcoma-associated herpesvirus.

We report the identification and characterization of p33, the product of Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 69 (ORF69), a positional homolog of the conserved herpesvirus protein UL31. p33 is expressed upon induction of viral lytic cycle with early kinetics. Immunofluorescence analysis revealed that in infected cell lines, the protein is localized in the nucleus, both in dotted spots and along the nuclear membrane. Nuclear fractionation experiments showed that p33 partitions with the nuclear matrix, and both immunoblotting of purified virions and immunoelectron microscopy indicated that the novel protein is not a component of the mature virus. Following ectopic expression in KSHV-negative cells, the protein was never associated with the nuclear membrane, suggesting that p33 needs to interact with additional viral proteins to reach the nuclear rim. In fact, after cotransfection with the ORF67 gene, the KSHV positional homolog of UL34, the p33 intranuclear signal changed and the two proteins colocalized on the nuclear membrane. A similar result was obtained when ORF69 was cotransfected with BFRF1, the Epstein-Barr virus (EBV) positional homolog of UL34 and ORF67. Finally, upon cotransfection, ORF69 significantly increased nuclear membrane reduplications induced by BFRF1. The above results indicate that KSHV p33 shares many similarities with its EBV homolog BFLF2 and suggest that functional cross-complementation is possible between members of the gammaherpesvirus subfamily.

Targeting cytokine/chemokine receptors: a challenge for molecular nuclear medicine.

Radiolabelled cytokines and chemokines are a group of radiopharmaceuticals that, by highlighting in vivo the binding to specific high-affinity receptors expressed on selected cell populations, allow the molecular and functional characterisation of immune-mediated processes Recently, several authors have described the use of radiolabelled cytokines and chemokines not only for imaging of inflammation and infection, but also as an approach to study in vivo the biology of primary and metastatic cancer cells. The latter avenue of research has been pursued particularly to help oncologists in therapeutic decision making and to follow up the efficacy of new immune therapies. In this paper we describe the characteristics of cytokines and chemokines, focussing on their role as radiopharmaceuticals for the imaging of cancer cells in vivo, a new challenge for molecular nuclear medicine.

Co-localization of multiple ErbB receptors in stratified epithelium of oral squamous cell carcinoma.

The expression of all four ErbB receptors was compared by immunohistochemistry, using receptor-specific polyclonal antisera, in 32 invasive, 11 in situ carcinomas, six benign lesions, and 22 samples of histologically normal mucosa adjacent to specimens of carcinoma originating from oral cavity epithelium. Among invasive and in situ carcinoma, EGFR expression was the most prevalent (in 29/32 and 8/11 cases, respectively) followed by ErbB2 (17/32 and 2/11) and ErbB4 (9/32 and 1/10), while ErbB3 was only detected in invasive tumours (12/32). Specific patterns included invasive tumours with expression of EGFR (8/32) or ErbB4 (1/32) alone, as well as different receptor combinations (EGFR+ErbB2, EGFR+ErbB4, EGFR+ErbB2+ErbB3, EGFR+ErbB2+ErbB4, and all four receptors). Simultaneous expression of three or four ErbB receptors correlated with tumour invasion (p=2.2x10(-4)) and localized in the intermediate epithelial cell layer of well and moderately differentiated tumours. No other significant correlation with clinico-pathological features was noticed. Some benign lesions and histologically normal mucosa adjacent to carcinomas showed weak immunostaining of EGFR (10/28), ErbB2 (4/28) or ErbB4 (3/28). By comparison, overexpression, as indicated by increased staining intensity, was observed in invasive tumours for EGFR (18/32), ErbB2 (8/32), ErbB4 (3/32), and ErbB3 (3/32). Statistical evaluation demonstrated a significant association of EGFR or ErbB2 overexpression with invasive carcinoma when compared with benign lesions and apparently normal epithelium (p=5.2x10(-7) and p=5x10(-3), respectively). Tumour-specific overexpression of ErbB receptors and their co-expression, most frequently involving EGFR and ErbB2, in the same cell layer of neoplastic epithelium, implicate receptor heterodimers in the pathogenesis of oral squamous carcinoma.

DNA vaccination against rat her-2/Neu p185 more effectively inhibits carcinogenesis than transplantable carcinomas in transgenic BALB/c mice.

The ability of vaccination with plasmids coding for the extracellular and the transmembrane domain of the product of transforming rat Her-2/neu oncogene (r-p185) to protect against r-p185(+) transplantable carcinoma (TUBO) cells and mammary carcinogenesis was evaluated. In normal BALB/c mice, DNA vaccination elicits anti-r-p185 Ab, but only a marginal CTL reactivity, and protects against a TUBO cell challenge. Massive reactive infiltration is associated with TUBO cell rejection. In BALB/c mice transgenic for the rat Her-2/neu gene (BALB-neuT), DNA vaccination elicits a lower anti-r-p185 Ab response, no CTL activity and only incompletely protects against TUBO cells, but markedly hampers the progression of carcinogenesis. At 33 wk of age, when control BALB-neuT mice display palpable tumors in all mammary glands, about 60% of immunized mice are tumor free, and tumor multiplicity is markedly reduced. Tumor-free mammary glands still display the atypical hyperplasia of the early stages of carcinogenesis, and a marked down-modulation of r-p185, along with a massive reactive infiltrate. However, BALB-neuT mice protected against mammary carcinogenesis fail to efficiently reject a TUBO cell challenge. This suggests that the mechanisms required for the rejection of transplantable tumors may not coincide with those that inhibit the slow progression of carcinogenesis.

ErbB2 immune response in breast cancer patients with soluble receptor ectodomain.

Investigation of ErbB2 immunity in human breast cancer employing recombinant expression sources in immunoblot analysis revealed ErbB2-specific antibodies of the IgG isotype in sera of 14 of 71 cancer patients and 1 of 31 normal donors. Reactivity was confirmed on ErbB2-specific immunoprecipitates. Independent evidence of existing ErbB2 immunity was obtained after in vitro transformation of peripheral blood leukocytes from six positive patients. Furthermore, in vitro immortalization of B-lymphocytes unmasked existent ErbB2 immunity in 1 of 8 patients negative for ErbB2 serum antibodies. Determining shed ErbB2 extracellular domain as an indirect measure of tumor burden in ErbB2-positive malignancy, elevated serum levels were observed in 16 of 71 breast cancer and 1 of 31 normal donor sera. Strikingly, existing ErbB2 immunity correlated significantly with elevated shed ErbB2 ectodomain among the patients analyzed. Incidence of both ErbB2 immunity and elevated ErbB2 extracellular domain increased with a progressed disease stage and was significantly associated with metastatic breast cancer. These observations implicate soluble ErbB2 amounts in vivo in the development of ErbB2 immunity in breast cancer. They further project serum analysis of ErbB2 immunity and soluble ectodomain as potential markers of disease progression in ErbB2-positive malignancy.

The BFRF1 gene of Epstein-Barr virus encodes a novel protein.

Computer analysis of the Epstein-Barr virus (EBV) genome indicates there are approximately 100 open reading frames (ORFs). Thus far about 30 EBV genes divided into the categories latent and lytic have been identified. The BamHI F region of EBV is abundantly transcribed during lytic replication. This region is highly conserved among herpesviruses, thus suggesting that some common function could be retained in the ORFs encompassed within this viral fragment. To identify putative novel proteins and possible new markers for viral replication, we focused our attention on the first rightward ORF in the BamHI F region (BFRF1). Histidine and glutathione S-transferase-tagged BFRF1 fusion proteins were synthesized to produce a mouse monoclonal antibody (MAb). Analysis of human sera revealed a high seroprevalence of antibodies to BFRF1 in patients affected by nasopharyngeal carcinoma or Burkitt's lymphoma, whereas no humoral response to BFRF1 could be detected among healthy donors. An anti-BFRF1 MAb recognizes a doublet migrating at 37 to 38 kDa in cells extracts from EBV-infected cell lines following lytic cycle activation and in an EBV-negative cell line (DG75) transfected with a plasmid expressing the BFRF1 gene. Northern blot analysis allowed the detection of a major transcript of 3.7 kb highly expressed in EBV-positive lytic cycle-induced cell lines. Treatment with inhibitors of viral DNA polymerase, such as phosphonoacetic acid and acyclovir, reduced but did not abolish the transcription of BFRF1, thus indicating that BFRF1 can be classified as an early gene. Cell fractionation experiments, as well as immunolocalization by immunofluorescence microscopy, immunohistochemistry, and immunoelectron microscopy, showed that BFRF1 is localized on the plasma membrane and nuclear compartments of the cells and is a structural component of the viral particle. Identification of BFRF1 provides a new marker with which to monitor EBV infection and might help us better understand the biology of the virus.

Cryptic epitopes on alpha-fetoprotein induce spontaneous immune responses in hepatocellular carcinoma, liver cirrhosis, and chronic hepatitis patients.

To determine alpha-fetoprotein (AFP) immunogenicity in vivo, the presence of antibodies in sera of 60 hepatocellular carcinoma, 15 liver cirrhosis, and 15 chronic hepatitis patients was evaluated by Western blotting and immunoprecipitation analyses using purified human AFP. High titers of anti-AFP immunoglobulins were detected in 14 hepatocellular carcinomas (P = 0.0006), 3 liver cirrhosis (P = 0.0173), and 1 chronic hepatitis patient, but they were not detected in 40 healthy individuals. Therefore, spontaneous immune responses to AFP are significantly associated to liver diseases (P = 0.0015). Patient immunoglobulins recognized proteic linear epitopes that were cryptic in the native protein, as demonstrated by their restricted reactivity with denatured deglycosylated AFP. Thus, in pathological liver conditions, tolerance to this self-molecule is circumvented. The identification of AFP immunogenic epitopes may contribute to defining novel immunotherapeutic strategies targeting this antigen.

Immune responses to all ErbB family receptors detectable in serum of cancer patients.

Employing NIH3T3 transfectants with individual human ErbB receptor coding sequences as recombinant antigen sources, we detected by immunoblot analysis specific immunoreactivity against all four ErbB receptors among 13 of 41 sera obtained from patients with different types of epithelial malignancies. Overall, serum positivity was most frequently directed against ErbB2 followed by EGFR, ErbB3 and ErbB4. Specificity patterns comprised tumor patients with unique serum reactivity against ErbB2 or ErbB4. Moreover, approximately half of the positive sera exhibited concomitant reactivity with multiple ErbB receptors including EGFR and ErbB2, EGFR and ErbB4, ErbB2 and ErbB3 or EGFR, ErbB2 and ErbB3. Serum reactivity was confirmed for the respective ErbB receptors expressed by human tumor cells and corroborated on receptor-specific immunoprecipitates. Positive sera contained ErbB-specific antibodies of the IgG isotype. Representative immunohistochemical analysis of tumor tissues suggested overexpression of ErbB receptors for which serum antibodies were detectable in five of six patients. These findings implicate multiple ErbB receptors including ErbB3 and ErbB4 in addition to EGFR and ErbB2 in primary human cancer. Heterogeneity of natural ErbB-specific responses in cancer patients warrants their evaluation in light of immunotherapeutic approaches targeting these receptors.

The use of a cationic liposome formulation (DOTAP) mixed with a recombinant tumor-associated antigen to induce immune responses and protective immunity in mice.

The cationic liposome DOTAP is a well-known transfection reagent. It has been manufactured and approved for clinical use, is readily available, and can be easily used as an adjuvant. These characteristics prompted us to investigate the effectiveness of DOTAP as an adjuvant to induce immune responses and protective immunity in mice using baculovirus-derived carcinoembryonic antigen (bV-CEA) as a model antigen. Two routes of administration and a dose-response study of bV-CEA were used in BALB/c mice to define the magnitude of the immune response as well as the most effective route of immunization. The results demonstrate differences in antibody titers, immunoglobulin (Ig)G isotype, and T-cell responses between the intravenous (i.v.) or subcutaneous (s.c.) route of immunization. The titer of the anti-CEA antibodies induced by the s.c. immunization was greater than the response by i.v. immunization. The s.c. route enhanced the IgG2a/2b isotype, whereas i.v. immunization elicited primarily IgG1. T-cell proliferation responses and cytokine production paralleled the humoral response (i.e., production was higher in the s.c. immunized animals). No differences in immunological responses were seen using either 25 or 10 microg of bV-CEA three times. An amount of 25 microg of bV-CEA/DOTAP given by s.c. immunization was sufficient in protecting mice from the transplant of syngeneic tumor cells transduced with the human CEA gene. We conclude that the cationic liposome DOTAP may be a useful immunoadjuvant for active anti-tumor immunotherapy in future clinical trials. This study will help to define the most effective way to use such an adjuvant.

Evaluation of serum and tissue levels of alpha-tocopherol.

This study was designed to test the effect of supplementation of several antioxidants, including alpha-tocopherol, on the clinical reduction of premalignant oral lesions. Samples of oral mucosa and serum were taken from baseline to 9 months of supplementation from patients with premalignant oral lesions and analyzed for alpha-tocopherol by HPLC. Statistical increases in both serum and tissue alpha-tocopherol were found after supplementation. There was no statistical relationship between alpha-tocopherol and beta-carotene levels.

Cell mediated cytotoxicity of human colon carcinoma cells by a monoclonal antibody (R4) recognizing the carcinoembryonic antigen (CEA) and CEA-related molecules.

We report a novel anti-CEA monoclonal antibody (MAb) designated R4, which mediates antibody-dependent cell-mediated cytotoxicity (ADCC) of human colon carcinoma cells and displays differential reactivity for human carcinomas versus the normal counterparts. R4 (IgG1) reacted with the cell surface of 6 colon carcinoma cell lines expressing CEA. Western blot analysis and epitope mapping using native and baculovirus recombinant CEA and non specific cross-reacting antigen (NCA) demonstrated that MAb R4 recognizes a proteinic epitope located on the 3' end of the domain I shared by CEA and NCA molecules. Immunohistochemical analysis demonstrated an intense staining of MAb R4 with the majority of the neoplastic tissues tested, including colon (13/13), stomach (2/2), breast (9/10), lung (7/10) and endometrial (2/4) carcinomas, whereas no reactivity with the correspondent normal tissues was observed. Using human PBLs from healthy donors as effector cells, we have shown that MAb R4 mediated antibody dependent-cell mediated cytotoxicity (ADCC) of human carcinoma cells LS-174T, CBS and WiDr. This activity was enhanced after PBLs activation with interleukin-2 (IL-2). The specificity of MAb R4 for an epitope shared by two tumor overexpressed antigens, CEA and NCA, resulting in an intense reactivity with neoplastic cells and the peculiar property to mediate ADCC, indicate that MAb R4 might be a novel powerful reagent for diagnostic and immunotherapy of carcinoma patients.

Immunohistochemical analysis of epidermal growth factor receptor (EGF-R) and c-erbB-2 and correlation with pathological prognostic variables in endometrial carcinomas.

In this study the immunohistochemical expressions of epidermal growth factor receptor (EGF-R) and erbB-2 in endometrial carcinomas at different stages of disease progression were evaluated and correlated with clinicopathological prognostic variables. Our results indicate that EGF-R and erbB-2 molecules are expressed in normal endometrium as well as in the majority of the carcinomas evaluated (83% and 92% respectively). The intensity of the immunostaining varied greatly (1+ to 3+) in the different tumors. Within these tumors we focused on those characterized by high levels of expression (i.e. overexpression). Expression and overexpression of EGF-R has been associated with poorly differentiated tumors and with a 3.3 times higher risk of a more rapid disease relapse. Overexpression of the erbB-2 oncoprotein was significatively correlated with depth of myometrial invasion (M0-M1 vs M2-M3 p=0.05), pathological stage (stage I vs II-IV p=0.02) and grade of tumor differentiation (G1 vs G2-G3 p=0.001). In particular, c-erbB-2 overexpression was able to discriminate between the different subsets of stage I carcinomas. None of the IA tumors overexpressed the oncoprotein, as compared to IB and IC (41% and 100% respectively, p=0.04). This finding highlights a role for erbB-2 protein as a prognostic parameter in stage I endometrial carcinoma patients.

Baculovirus expression of a functional single-chain immunoglobulin and its IL-2 fusion protein.

The baculovirus expression system has been used for the production of a variety of proteins, including antibodies. Two single-gene constructs encoding single-chain immunoglobulins have recently been developed. The antibody employed was monoclonal antibody (MAb) CC49 which reacts with the pancarcinoma antigen, tumor associated glycoprotein, TAG-72. One, single-chain construct designated SCA delta CLCH1 (SCIg), consists of the CC49 sFv covalently joined to the human Fc (gamma 1) through the hinge region. The other, SCA delta CLCH1-IL-2 (SCIg-IL-2), has a human IL-2 molecule attached to the carboxyl end of the SCIg. These constructs have been used to test the feasibility of producing biologically active antibodies using the baculovirus expression system. Both constructs have been successfully expressed in insect cells and purified. The baculovirus recombinant single-chain antibodies have been designated, bV-SCA delta CLCH1 (bV-SCIg) and bV-SCA delta CLCH1-IL-2 (bV-SCIg-IL-2) they have been shown to be secreted in the culture supernatant as dimeric molecules of approximately 115 kDa and 140 kDa, respectively. The specificity and antibody dependent cellular cytolytic activity of the baculovirus recombinant single-chain antibodies were shown to be similar to that of the myeloma derived molecules. Glycosylation analysis showed that baculovirus derived proteins were N-glycosylated, but carried few if any high mannose residues. The biological activity of the IL-2 moiety was retained in bV-SCIg-IL-2, as evidenced by its stimulatory effect on the proliferation of the IL-2 dependent cell line HT-2. The observation that a significantly shorter time is required to develop baculovirus recombinant molecules as compared to myeloma derived molecules and that insect cells express single chain MAbs at acceptable levels may have implications for the production of these molecules for clinical use.