RESUMEN
A dry enzyme mixture was added to the diets of lactating cows and growing lambs to evaluate its ability to improve milk production and nutrient digestibility, respectively. The enzyme mixture contained xylanase and cellulase activity over a broad range of pH (tested from 4 to 7). Twenty-four lactating cows between 50 and 150 d in milk and averaging about 40 kg of milk/ d were fed a total mixed ration (TMR) consisting of 26% [dry matter (DM) basis] corn silage, 17% alfalfa silage, 7% chopped alfalfa hay, and 50% concentrate. One-half of the cows were fed the TMR without supplementation and the remaining half of the cows were fed the same TMR supplemented with 10 g of the enzyme mixture/ cow per day. After 21 d, the treatments were crossed over for a second 21-d period. The dry enzyme mixture had no effect on DM intake, milk production, or milk composition. Addition of various concentrations of the enzyme mixture did not improve the in vitro digestion of neutral detergent fiber from the TMR. In a digestion trial, lambs were fed a commercial diet supplemented with 4 g of the enzyme mixture/lamb per day, and total feces and urine were collected. Although the ratio of enzyme to feed was much higher than it was in the experiment with lactating cows, addition of the enzyme mixture had no effect on the apparent digestion of DM, acid detergent fiber, neutral detergent fiber, or N in the diet.
Asunto(s)
Bovinos/metabolismo , Celulasa/farmacología , Dieta/veterinaria , Digestión/efectos de los fármacos , Endo-1,4-beta Xilanasas/farmacología , Lactancia/efectos de los fármacos , Ovinos/metabolismo , Alimentación Animal/análisis , Animales , Celulasa/administración & dosificación , Celulasa/metabolismo , Fibras de la Dieta/metabolismo , Digestión/fisiología , Endo-1,4-beta Xilanasas/administración & dosificación , Endo-1,4-beta Xilanasas/metabolismo , Femenino , Concentración de Iones de Hidrógeno , Lactancia/fisiología , Masculino , Leche/química , Leche/metabolismoRESUMEN
Expression of the mucin-associated sialyl-Tn (STn) antigen has been associated with a decreased survival in patients with colorectal, gastric, and ovarian cancer. To better understand the role of STn antigen in tumor biology, we developed STn(+) (called LP) and STn(-) (called LN) clonal cell lines from a parental metastatic rat colon carcinoma cell line (LMCR). Both derivative cell lines exhibited identical proliferation rates in vitro. LP cells strongly expressed STn antigen both in vitro and in vivo, and were poorly tumorigenic when given to syngeneic rats. LN cells did not express STn antigen in vitro, but as in vivo tumors these cells rapidly acquired STn expression, readily formed tumors, and were highly lethal. When rats were given an otherwise lethal inoculum of i.p. LN cells, pre-immunization with synthetic STn antigen conjugated to keyhole limpet hemocyanin (STn-KLH) resulted in a 60% survival rate. When LN cells were injected subcutaneously in the presence of STn-KLH-sensitized lymphocytes, tumor growth was decreased. Distribution of STn antigen in normal organs of host rats is quite similar to that of humans. This model mimics human disease and should facilitate studies of mucin-associated antigens in tumor biology and the development of immunotherapeutic agents based on mucin-related antigens.
Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/inmunología , Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Neoplasias del Colon/inmunología , Animales , Formación de Anticuerpos , Antígenos de Carbohidratos Asociados a Tumores/genética , Vacunas contra el Cáncer , División Celular/fisiología , Neoplasias del Colon/patología , Neoplasias del Colon/terapia , Modelos Animales de Enfermedad , Glicoconjugados , Hemocianinas/inmunología , Inmunoterapia/métodos , Inyecciones Intraperitoneales , Trasplante de Neoplasias , Ratas , Ratas Endogámicas , Tasa de Supervivencia , Distribución Tisular , Células Tumorales CultivadasRESUMEN
The purpose of this study was to evaluate the toxicity and potential efficacy of administering the THERATOPE STn-KLH cancer vaccine to ovarian and breast cancer patients after an autologous stem cell transplant. Forty patients (11 high-risk stage II/III breast cancer, 22 stage IV breast cancer, and seven stage III/IV ovarian cancer patients) were treated with high-dose chemotherapy followed by autologous/syngeneic stem cell rescue and vaccination with THERATOPE STn-KLH (Sialyl-Tn-KLH with Detox-B Stable Emulsion). Each patient was scheduled to receive a total of five vaccinations beginning on days 30-151 after stem cell infusion. The vaccine was well tolerated. Induration and erythema at the site of injection were the most common side-effects. When one compares the outcome of patients vaccinated with 66 breast and ovarian cancer patients who were not, following risk-adjustment analysis, vaccinated patients appeared more likely to survive (P = 0.07) and less likely to relapse (P = 0. 10). Vaccinated patients with the greatest specific lytic activity against STn+OVCAR tumor cells relative to nonspecific killing of Daudi cells tended to remain in remission longer than patients who displayed less specific immune activity (P = 0.057). We conclude that the THERATOPE STn-KLH cancer vaccine is well tolerated in breast and ovarian cancer patients after autologous transplant and, while not statistically significant, the trends in data support the concept that THERATOPE vaccine may decrease the risk for relapse and death and thus warrants further study. Bone Marrow Transplantation (2000) 25, 1233-1241.
Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Neoplasias de la Mama/terapia , Vacunas contra el Cáncer/administración & dosificación , Trasplante de Células Madre Hematopoyéticas , Hemocianinas/administración & dosificación , Neoplasias Ováricas/terapia , Adulto , Neoplasias de la Mama/patología , Terapia Combinada , Femenino , Humanos , Persona de Mediana Edad , Neoplasias Ováricas/patología , Trasplante Autólogo , Resultado del TratamientoRESUMEN
Sialyl-Tn (STn) is an O-serine- or O-threonine-linked disaccharide [NeuAcalpha(2-->6)GalNAcalpha-O-Ser/Thr) expressed on mucins of most types of adenocarcinoma as single STn or clustered STn [STn(c)] epitopes. Though STn is expressed on some normal tissues it is relatively tumor-specific, especially in the clustered conformation. Clinical trials with STn-keyhole limpet hemocyanin (KLH) conjugate vaccines, prepared using reductive amination with a two-carbon linker group, have resulted in high titers against STn but lower titers against natural forms of STn (ovine submaxillary mucin, or tumor cells). To obtain antibodies of more appropriate specificity, we attempted to prepare STn(c)-KLH conjugates to establish their immunogenicity in mice in preparation for clinical trials; however, conjugation efficiency was poor when the same two-carbon linker was used, presumably because of steric hindrance. STn-KLH and STn(c)-KLH conjugates were prepared using the regular two-carbon or the recently developed more efficient longer heterobifunctional 4-(4-maleimidomethyl)cyclohexane-1-carboxyl hydrazide (MMCCH) linkers, and the resulting immunogenicities in mice were compared. The highest titers against STn were seen with the STn-KLH conjugate with the two-carbon linker, and the highest titers against STn(c) were seen with STn(c)-KLH with the MMCCH linker. Conjugation with MMCCH resulted in the highest conjugation efficiency (yield) and the highest titers against ovine submaxillary mucin and STn-positive tumor cells, and is the method of choice for the preparation of STn(c) vaccine for clinical trials.
Asunto(s)
Anticuerpos Antineoplásicos/biosíntesis , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Vacunas contra el Cáncer/inmunología , Reactivos de Enlaces Cruzados/química , Inmunoconjugados/inmunología , Maleimidas/química , Mucinas/inmunología , Neoplasias Experimentales/inmunología , Animales , Anticuerpos Antineoplásicos/sangre , Vacunas contra el Cáncer/síntesis química , Vacunas contra el Cáncer/farmacología , Secuencia de Carbohidratos , Ensayo de Inmunoadsorción Enzimática , Femenino , Hemocianinas/inmunología , Hemocianinas/farmacología , Inmunoconjugados/química , Inmunoconjugados/farmacología , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/sangre , Inmunoglobulina M/biosíntesis , Inmunoglobulina M/sangre , Ratones , Datos de Secuencia Molecular , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/terapia , Ovinos , Glándula Submandibular/inmunología , Glándula Submandibular/metabolismoRESUMEN
Seven ovarian and 33 breast high-risk stage II/III and stage IV cancer patients received high-dose chemotherapy followed by stem cell rescue. Thirty to 151 days after stem cell transplantation, the patients received their first immunotherapy treatment with Theratope STn-KLH cancer vaccine. Most patients developed increasing IgG anti-STn titers to a sustained peak after the fourth or fifth immunizations. Only one patient had elevated CA27.29 (MUC1 mucin) serum levels at trial entry. Five of the seven patients with preimmunotherapy elevated serum CA125 levels demonstrated decreasing CA125 levels during immunotherapy, consistent with an antitumor response. Evidence of STn antigen-specific T-cell proliferation was obtained from 17 of the 27 evaluable patients who received at least three immunotherapy treatments. Eleven of the 26 patients tested had evidence of an anti-STn TH1 antigen-specific T-cell response as determined by interferon-gamma, but not interleukin (IL)-4, production. After immunization, lytic activity of peripheral blood lymphocytes (PBLs) tested against a lymphokine activated killer (LAK)-sensitive cell line, a natural killer (NK)-sensitive cell line, and an STn-expressing cancer cell line (OVCAR) increased significantly. In vitro IL-2 treatment of the PBLs after vaccination greatly enhanced killing of the STn+ cancer cell line. Evidence of the development of OVCAR specific killing activity, over and above that seen due to LAK or NK killing, is presented. These studies provide the strongest evidence in humans of the development of an antitumor T-cell response after immunization with a cancer-associated carbohydrate antigen.
Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/inmunología , Neoplasias de la Mama/terapia , Vacunas contra el Cáncer/inmunología , Hemocianinas/inmunología , Neoplasias Ováricas/terapia , Linfocitos T/inmunología , Antígenos de Carbohidratos Asociados a Tumores/administración & dosificación , Antígenos de Carbohidratos Asociados a Tumores/uso terapéutico , Biomarcadores de Tumor/sangre , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/inmunología , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/uso terapéutico , Terapia Combinada , Citotoxicidad Inmunológica/efectos de los fármacos , Femenino , Trasplante de Células Madre Hematopoyéticas , Hemocianinas/administración & dosificación , Hemocianinas/uso terapéutico , Humanos , Inmunoglobulinas/sangre , Inmunoterapia , Interferón gamma/biosíntesis , Interleucina-2/farmacología , Activación de Linfocitos/efectos de los fármacos , Mucinas/inmunología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/inmunología , Células Tumorales CultivadasRESUMEN
MHC class I molecules were isolated from the MUC1-positive human breast adenocarcinoma cell line MCF-7 by immunoaffinity using the panreactive anti-class I monoclonal antibodies (MAb) W6/32. Acid-eluted peptides from the class I molecules were separated twice by high-performance liquid chromatography and tested for reactivity with the MAb BCP8, which reacts with the minimal MUC1 core peptide sequence PDTRPA. A peak with strong and specific BCP8 reactivity was found in fractions eluting at 16.5-17.5 min. The protocol used for the MUC1+ pancreatic adenocarcinoma cell line CAPAN-1 (HLA.A2) was to perform sequential affinity purifications of class I molecules using MAb W6/32, followed by affinity purification of HLA.A2 molecules by the HLA.A2.1-specific MAb, MA2.1, and high-performance liquid chromatography fractionation of the acid-eluted material. A single peak with MAb BCP8 reactivity was noted at 18-19 min. The protocol for the MUC1+ breast adenocarcinoma cell line SKBr-3 (HLA.A11,B40), which used A11- and B40-specific MAbs, also resulted in the detection of BCP8-specific peaks at approximately 18-19 min. A preliminary mass spectral analysis of BCP8 affinity-purified class I associated material surprisingly revealed the presence of two 3-mer MUC1 amino acid sequences and one 6-mer sequence. A synthetic 9-mer MUC1 peptide, TSAPDTRPA, containing the isolated fragments was found to cause strong class I up-regulation in T2 cells as well as to serve as an epitope for CTL generated in a primary in vitro immune response. These studies suggest that MUC1-derived peptides are processed and presented in the context of MHC class I molecules on the surface of tumor cells and support the use of MAb BCP8 to further define MHC class I associated MUC1 motifs.
Asunto(s)
Anticuerpos Monoclonales , Antígenos de Neoplasias/química , Antígenos de Histocompatibilidad Clase I/química , Mucina-1/inmunología , Fragmentos de Péptidos/aislamiento & purificación , Antígenos de Neoplasias/inmunología , Antígeno HLA-A2/química , Antígenos HLA-B/química , Antígeno HLA-B40 , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Fragmentos de Péptidos/inmunología , Células Tumorales CultivadasRESUMEN
Synthetic human MUC1 peptides are important candidates for therapeutic cancer vaccines. To explore whether a human MUC1 peptide BP25 (STAPPAHGVTSAPDTRPAPGSTAPP) can be rendered immunogenic by incorporation in liposomes, the effects of physical association of the peptide with liposomes on immune responses were investigated. Lipid conjugated and nonconjugated MUC1 peptides were incorporated in liposomes with a composition of distearoylphosphatidylcholine/cholesterol/dimyristoylphosphatidylglyc erol (3:1:0.25, molar ratio) containing monophosphoryl lipid A (1% w/w of the total lipids). Liposomes were characterized for peptide retention by HPLC and for surface peptide display of MUC1 epitopes by flow cytometry. C57BL/6 mice were immunized with lipopeptide alone, peptide mixed with peptide-free liposomes, and peptide associated with liposomes in entrapped or surface-exposed forms. T cell proliferative responses, cytokine patterns, and antibody isotypes were studied. Results showed that immune responses were profoundly influenced by the liposome formulations. Physically associated, either encapsulated or surface-exposed, peptide liposomes elicited strong antigen-specific T cell responses, but not lipopeptide alone or peptide mixed with peptide-free liposomes. Analysis of the cytokines secreted by the proliferating T cells showed a high level of IFN-gamma and undetectable levels of IL-4, indicating a T helper type 1 response. Thus, physical association of the peptide with liposomes was required for T cell proliferative responses, but the mode of association was not critical. On the other hand, the nature of the association significantly affected humoral immune responses. Only the surface-exposed peptide liposomes induced MUC1-specific antibodies. A domination of anti-MUC1 IgG2b over IgG1 (94 versus 6%) was observed. Our results support the hypothesis that different immune pathways are stimulated by different liposome formulations. This study demonstrated that a liposome delivery system could be tailored to induce either a preferential cellular or humoral immune response.
Asunto(s)
Formación de Anticuerpos/efectos de los fármacos , Mucina-1/administración & dosificación , Mucina-1/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Química Farmacéutica , Citocinas/metabolismo , Portadores de Fármacos , Epítopos/análisis , Femenino , Humanos , Liposomas , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mucina-1/inmunología , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/farmacología , Propiedades de Superficie , Células TH1/efectos de los fármacos , Células TH1/inmunología , Células TH1/metabolismoRESUMEN
Sixteen metastatic breast cancer patients were immunized with a low dose (5 micrograms) of a 16 amino acid MUC1 peptide (GVTSAPDTRPAPGSTA) conjugated to KLH (BP16-KLH) plus DETOX adjuvant and evaluated for antibody titers against MUC1 peptide and KLH and for cytotoxic lymphocyte (CTL) activity using class 1 HLA-matched MUC1-positive tumor targets. All patients generated strong anti-KLH IgG responses. Only 3 patients developed an anti-MUC1 IgG response, which was weak in magnitude. As it is controversial whether human cancer patients generate class-1-restricted CTL against MUC1, we examined anti-MUC1 CTL activity of PBLs following 4 immunizations with BP16-KLH. The generation of MUC1-specific CTLs required only a 6-day in vitro stimulation of patients' T-cells with synthetic MUC1-peptide-pulsed autologous APCs. The assay for CTL activity was a 4 hour 51Cr release from labeled adenocarcinoma target cells. Eleven of the 16 immunized patients were tested for CTL activity using class-1-matched adenocarcinoma target cell lines. Evidence for class-1-restricted killing of MUC1-expressing tumor cell lines was obtained in 7 of these 11 patients.
Asunto(s)
Neoplasias de la Mama/inmunología , Antígenos de Histocompatibilidad Clase I/fisiología , Mucina-1/inmunología , Linfocitos T Citotóxicos/fisiología , Femenino , Humanos , Inmunización , Inmunoglobulina G/sangre , Metástasis de la NeoplasiaRESUMEN
Monoclonal antibodies TKH2 and B72.3, which react with the mucin-associated sialyl-Tn(STn) antigen, preferentially bind to cancerous but not normal colonic tissues. If O-acetyl groups are removed by saponification of tissues, MAb TKH2 will react with normal colonocytes, whereas MAb B72.3 remains non-reactive. To explain this difference in binding specificity, we tested both MAbs against synthetic constructs of single (monomeric) or clustered (trimeric) STn epitopes by enzyme immunoassay. Both MAb TKH2 and MAb B72.3 reacted with trimeric STn, but MAb TKH2 demonstrated greater binding than MAb B72.3 to monomeric STn. This suggests that normal colonic mucosa expresses monomeric STn epitopes, but that with transformation to malignancy, clustered STn epitopes appear. The appearance of clustered STn epitopes during colonic carcinogenesis represents a novel pattern of carbohydrate antigen expression and implicates alterations at the level of apomucins and/or glycosyltransferases responsible for cluster epitope formation.
Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Colon/metabolismo , Neoplasias del Colon/inmunología , Mucosa Intestinal/metabolismo , Mucinas/metabolismo , Anticuerpos Monoclonales/inmunología , Anticuerpos Antineoplásicos/inmunología , Biomarcadores de Tumor , Transformación Celular Neoplásica , Neoplasias del Colon/metabolismo , Glicosilación , Humanos , Técnicas para Inmunoenzimas , Mucinas/inmunologíaRESUMEN
A human MUC1-transfected mouse mammary adenocarcinoma cell line (GZHI) was used to develop both subcutaneous and intravenous tumor models. A vaccine formulation comprised of a 24 mer (human MUC1) synthetic peptide encapsulated with monophosphoryl lipid A adjuvant (MPLA) in multilamellar liposomes was tested for immunogenicity and anti-tumor activity. A low dose of the human MUC1 peptide (5 microg) administered in liposomes provided excellent protection of mice in both tumor challenge models. The protective antitumor activity mediated by the liposome formulation correlated with anti-MUC1-specific T-cell proliferation, gamma-interferon (IFN-gamma) production and IgG2a anti-MUC1 antibodies, suggesting a type 1 (T1) T-cell response. In contrast, lack of protection in mice immunized with negative control vaccines correlated with IgG1 anti-MUCI antibody formation, low or no anti-MUC1 IgG2a and low antigen-specific T-cell proliferation, consistent with a type 2 (T2) T-cell response to the tumor.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Mucina-1/inmunología , Neoplasias Experimentales/prevención & control , Secuencia de Aminoácidos , Animales , Vacunas contra el Cáncer/administración & dosificación , Femenino , Humanos , Inmunización , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Liposomas , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mucina-1/administración & dosificaciónRESUMEN
A number of adenocarcinomas abundantly express and secrete underglycosylated MUC1 mucin. Underglycosylation exposes tandem repeat peptide sequences on cancer-associated MUC1 mucin that are normally cryptic. High levels of MUC1 mucin are correlated with a poor prognosis and immunosuppression in adenocarcinoma patients. In this report we show that cancer-associated MUC1 mucin, affinity-purified from ascites fluids of cancer patients, and synthetic tandem repeats of MUC1 mucin core peptide can suppress human T-cell proliferative responses. This MUC1 mucin-induced suppression of T-cell responses can be reversed by the addition of exogenous IL-2 or anti-CD28 monoclonal antibody. These results are consistent with other studies showing that lymphocytes present in the vicinity of tumor cells are anergic and can be reactivated with exogenous interleukin-2. Overcoming MUC1 mucin-induced immunosuppression with IL-2 combined with active specific immunotherapy might be an effective immunotherapeutic strategy against human adenocarcinomas.
Asunto(s)
Antígenos CD28/fisiología , Interleucina-2/farmacología , Activación de Linfocitos/efectos de los fármacos , Mucina-1/farmacología , Linfocitos T/inmunología , Anticuerpos Monoclonales/farmacología , Antígenos CD/inmunología , Antígenos CD/fisiología , Apoptosis , Líquido Ascítico , Antígenos CD28/inmunología , Células Cultivadas , Cromatografía de Afinidad , Técnicas de Cocultivo , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Interferón gamma/biosíntesis , Prueba de Cultivo Mixto de Linfocitos , Mitomicina/farmacología , Mucina-1/aislamiento & purificación , Neoplasias Ováricas/inmunología , Linfocitos T/citología , Linfocitos T/efectos de los fármacosRESUMEN
Sixteen research groups participated in the ISOBM TD-4 Workshop in which the reactivity and specificity of 56 monoclonal antibodies against the MUC1 mucin was investigated using a diverse panel of target antigens and MUC1 mucin-related synthetic peptides and glycopeptides. The majority of antibodies (34/56) defined epitopes located within the 20-amino acid tandem repeat sequence of the MUC1 mucin protein core. Of the remaining 22 antibodies, there was evidence for the involvement of carbohydrate residues in the epitopes for 16 antibodies. There was no obvious relationship between the type of immunogen and the specificity of each antibody. Synthetic peptides and glycopeptides were analyzed for their reactivity with each antibody either by assay of direct binding (e.g. by ELISA or BiaCore) or by determining the capacity of synthetic ligands to inhibit antibody binding interactions. There was good concordance between the research groups in identifying antibodies reactive with peptide epitopes within the MUC1 protein core. Epitope mapping tests were performed using the Pepscan analysis for antibody reactivity against overlapping synthetic peptides, and results were largely consistent between research groups. The dominant feature of epitopes within the MUC1 protein core was the presence, in full or part, of the hydrophilic sequence of PDTRAPAP. Carbohydrate epitopes were less easily characterized and the most useful reagents in this respect were defined oligosaccharides, rather than purified mucin preparations enriched in particular carbohydrate moieties. It was evident that carbohydrate residues were involved in many epitopes, by regulating epitope accessibility or masking determinants, or by stabilizing preferred conformations of peptide epitopes within the MUC1 protein core. Overall, the studies, highlight concordance between groups rather than exposing inconsistencies which gives added confidence to the results of analyses of the specificity of antimucin monoclonal antibodies.
Asunto(s)
Anticuerpos Monoclonales/análisis , Mucina-1/inmunología , Secuencia de Aminoácidos , Animales , Afinidad de Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Epítopos Inmunodominantes/inmunología , Masculino , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunologíaRESUMEN
A panel of 56 MAbs submitted to the ISOBM TD-4 (MUC1) Workshop were analysed in two systems. These systems were designed to screen for peptide type 1 core O-glycan-related reactivities. Using synthetic MUC1 mucin-related peptides and glycopeptides, the panel of MAbs were tested for relative binding affinities to type 1 core O-glycan-substituted MUC1 structures. These studies utilized a competitive binding format with a native human adenocarcinoma-derived mucin as a solid phase. This system allows for analysis of the type 1 core glycoform subspecificity of each MAb. The second approach taken in parallel, utilized MCF-7 (BrCa) and OVCAR (OVCa) cell lines which were grown in the presence or absence of phenyl-N-acetylgalactosaminide (p-gal), a blocker of mucin O-linked glycosylation. These cells were analysed by FACS to examine the role these same glycan substitutions play with regard to either the diagnostic or therapeutic application of these MAbs. By FACS analysis there was a consistent increased 'epitope exposure' for peptide-specific MAbs binding in the presence of p-gal. In addition, a single MAb (TD-4 #150) is interpreted to react with a type 1 core O-glycan, probably with Tn, TF or STn specificity.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Sitios de Unión de Anticuerpos/inmunología , Glicoproteínas/inmunología , Mucina-1/inmunología , Fragmentos de Péptidos/inmunología , Polisacáridos/inmunología , Secuencia de Aminoácidos , Neoplasias de la Mama/inmunología , Mapeo Epitopo , Femenino , Citometría de Flujo , Humanos , Datos de Secuencia Molecular , Neoplasias Ováricas/inmunología , Células Tumorales CultivadasRESUMEN
The levels of mRNA corresponding to the MUC1, MUC2, MUC5AC, MUC5B, and MUC6 genes were determined in 19 human colon adenocarcinoma cell lines by the reverse transcriptase-polymerase chain reaction method using specific primers in an attempt to correlate to the levels of cell surface carbohydrate epitopes. All 19 cell lines expressed MUC1 and MUC5B mRNA, whereas MUC2, MUC5AC, or MUC6 mRNA were only detected in 8, 3, or 2 of 19 cell lines, respectively. Sialyl Lewis a carbohydrates, identified by the monoclonal antibody (mAb) CA19-9, and sialyl Lewis X carbohydrates. identified by mAb KM93, were observed, with most of the cell lines expressing multiple mucin core polypeptide genes but with few cell lines expressing only MUC1 and MUC5B. Sialyl Tn epitopes identified by mAb B195.3R11 and by mAb TKH-2 were strongly expressed on both of two MUC6-positive cells, whereas only a small portion of MUC6-negative cells expressed these epitopes. Strict correlation between mucin gene expression and any carbohydrate epitopes examined was not observed.
Asunto(s)
Neoplasias del Colon/metabolismo , Epítopos/biosíntesis , Mucinas/biosíntesis , Antígenos de Carbohidratos Asociados a Tumores/biosíntesis , Antígenos de Carbohidratos Asociados a Tumores/genética , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Neoplasias del Colon/genética , Neoplasias del Colon/inmunología , Epítopos/genética , Epítopos/inmunología , Expresión Génica , Humanos , Mucinas/genética , Mucinas/inmunología , Células Tumorales CultivadasRESUMEN
Antigen-specific MHC class II- and class I-restricted helper and cytotoxic T cell responses are important anti-cancer immune responses. MUC1 mucin is a potentially important target for immunotherapy because of its high expression on most human adenocarcinomas. MUC1 peptide-specific type 1 T cell responses were generated in vitro using human peripheral blood lymphocytes (PBL), incubated with liposomes containing synthetic MUC1 lipopeptide antigen. Only two weekly stimulations with the liposomal MUC1 formulation led to the generation of potent anti-MUC1-specific T cell proliferation as well as class I-restricted cytotoxic responses. Thus the use of PBL pulsed with liposome-encapsulated antigen provides an effective approach of rapidly generating effective antigen-presenting cell (APC) function as well as antigen specific T cells in vitro. It may be feasible to use this technology for the rapid and effective generation of APC and/or T cells as cellular vaccines for adenocarcinomas.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T/inmunología , Activación de Linfocitos/inmunología , Mucina-1 , Oligopéptidos/inmunología , Fragmentos de Péptidos , Secuencia de Aminoácidos , Animales , Anticuerpos Bloqueadores/farmacología , Anticuerpos Monoclonales/farmacología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Células Cultivadas , Citocinas/biosíntesis , Humanos , Lipoproteínas/inmunología , Lipoproteínas/farmacología , Liposomas/inmunología , Ratones , Datos de Secuencia Molecular , Oligopéptidos/farmacologíaRESUMEN
The fine specificities of MAbs generated using novel synthetic clustered STn and Tn glycopeptides as immunogens were compared with the anti-TAG-72 antibodies B72.3 and CC49. Hapten inhibition experiments demonstrated the specificity of several of the MAbs for STn and Tn expressed on ovine submaxillary mucin and tumor derived MUC-1 mucin. Amongst the STn specific MAbs only the B195.3 MAb shows absolute dependence on the presence of sialic acid and specificity to the simple disaccharide NANAA alpha2-6-GalNAc. Identification of tumor associated carbohydrate epitopes in cluster and monomer configurations are possible using MAbs detecting the defined structure specificities described herein.
Asunto(s)
Anticuerpos Monoclonales , Antígenos de Carbohidratos Asociados a Tumores/análisis , Animales , Especificidad de Anticuerpos , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Líquido Ascítico/química , Biomarcadores de Tumor , Neoplasias de la Mama/química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Ensayo de Inmunoadsorción Enzimática , Epítopos/análisis , Femenino , Glicopéptidos/análisis , Glicopéptidos/síntesis química , Glicosilación , Haptenos , Ratones , Datos de Secuencia Molecular , Mucina-1/biosíntesis , Mucina-1/química , Mucinas/biosíntesis , Mucinas/química , Ácido N-Acetilneuramínico/análisis , Radioinmunoensayo , Ovinos , Glándula Submandibular/metabolismoRESUMEN
The TRUQUANT BR radioimmunoassay, which uses monoclonal antibody B27.29 to quantitate CA27.29 mucin antigen (MUC-1 gene product) in serum, has recently received Food and Drug Administration approval for predicting recurrent breast cancer in patients with stage II and III disease. The purpose of this study was to determine whether the new radioimmunoassay for serum MUC-1 has prognostic significance for patients with metastatic adenocarcinoma receiving active specific immunotherapy (ASI). Using 40 U/ml as the upper limit of "normal," patients with metastatic breast and ovarian cancer with a preimmunotherapy serum CA27.29 mucin > 40 U/ml (CA27.29 Hi patients) had a poorer survival than CA27.29 Lo patients (< or = 40 U/ml) after ASI. There was no significant correlation between preimmunotherapy CA27.29 serum levels and measurable tumor burden. The preimmunotherapy CA27.29 serum level was a predictor of poor survival of metastatic colorectal and pancreatic cancer patients independent of other prognostic factors. There seemed to be two populations of pancreatic cancer patients, separated at 60 U/ml serum CA27.29 (CA27.29 Hi versus Lo patients). A CA27.29 serum level of 22 U/ml separated patients with CA27.29 Hi vs. Lo colorectal cancer. Patients with CA27.29 Lo colorectal and pancreatic cancer survived longer after ASI compared with patients with CA27.29 Hi colorectal and pancreatic cancer, respectively. We suggest that various CA27.29 serum levels define poor prognosis patients (CA27.29 Hi secretors) versus good prognosis patients (CA27.29 Lo secretors) for different cancer types.
Asunto(s)
Adenocarcinoma/terapia , Neoplasias de la Mama/terapia , Inmunoterapia , Mucina-1/sangre , Neoplasias Ováricas/terapia , Adenocarcinoma/inmunología , Adenocarcinoma/mortalidad , Adenocarcinoma/secundario , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/mortalidad , Neoplasias Colorrectales/terapia , Femenino , Humanos , Tolerancia Inmunológica , Inmunoglobulina G/biosíntesis , Inmunohistoquímica , Hibridación de Ácido Nucleico , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/mortalidad , Neoplasias Pancreáticas/terapia , Pronóstico , Modelos de Riesgos Proporcionales , Tasa de SupervivenciaRESUMEN
By direct or indirect immunofluorescence using FITC as a fluorochrome, 0 to 2% of the T cells isolated from peripheral blood of normal healthy individuals was found to be CD30+. Using indirect immunofluorescence and phycoerythrin-labeled Ab, higher percentages of CD30+ T cells (3-31%) were repeatedly found in the peripheral blood of normal healthy donors. The majority (85-90%) of CD30+ T cells obtained from PBLs of normal healthy donors were found in the CD8+ cell population. Following FACS sorting of the T cell populations into CD30+ and CD30- subpopulations, approximately 85 to 90% CD30+ T cells were CD8+, whereas the CD30- T cells were CD4+. Upon activation, the sorted CD30+ T cells produced both IFN-gamma and IL-4. In contrast to previous reports, these results demonstrate that CD30+CD8+ T cells are present in significant numbers in the PBLs of normal healthy individuals.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Antígeno Ki-1/metabolismo , Anticuerpos/farmacología , Complejo CD3 , Linfocitos T CD8-positivos/clasificación , Citometría de Flujo , Fluoresceína-5-Isotiocianato , Técnica del Anticuerpo Fluorescente Directa , Técnica del Anticuerpo Fluorescente Indirecta , Colorantes Fluorescentes , Humanos , Técnicas In Vitro , Interferón gamma/biosíntesis , Interleucina-2/farmacología , Interleucina-4/biosíntesis , Linfocitos/inmunología , Ficoeritrina , Subgrupos de Linfocitos T/inmunologíaRESUMEN
Studies in animal models of mouse mammary carcinoma have shown that ovine submaxillary mucin, which carries multiple sialyl-Tn (STn) epitopes, is effective in stimulating an immune response and inhibiting tumour growth. In similar studies using carbohydrate antigens, pretreatment with low-dose cyclophosphamide has been shown to be important in modulating the immune response to antigen possibly by inhibiting suppresser T-cell activity. In a clinical trial assessing the efficacy and toxicity of synthetic STn, patients with metastatic breast cancer were randomised to receive 100 micrograms STn linked to keyhole limpet haemocyanin (KLH) with DETOX-B adjuvant given by subcutaneous injection at weeks 0, 2, 5 and 9 with or without low-dose cyclophosphamide (CTX, 300 mg m-2) pretreatment, 3 days before the start of immunotherapy. Patients with responding or stable disease after the first four injections were eligible to receive STn-KLH at 4 week intervals. The main toxicity noted was the development of subcutaneous granulomata at injection sites. Of 23 patients randomised, 18 received four injections, 5 patients having developed progressive disease during the initial 12 week period. Two minor responses were noted in the 18 patients who received four active specific immunotherapy (ASI) injections and a further five patients had stable disease. Six patients continued ASI at 4 week intervals and a partial response was noted in a patient who had previously had stable disease. All patients developed IgG and IgM responses to sialyl-Tn and levels of IgM antibodies were significantly higher in those patients who were pretreated with CTX. Measurable tumour responses have been recorded following ASI with STn-KLH plus DETOX and the immunomodulatory properties of low-dose CTX have been confirmed.
Asunto(s)
Adyuvantes Inmunológicos/uso terapéutico , Antígenos de Carbohidratos Asociados a Tumores/uso terapéutico , Neoplasias de la Mama/terapia , Ciclofosfamida/uso terapéutico , Hemocianinas/uso terapéutico , Inmunoconjugados/uso terapéutico , Inmunoterapia Activa , Adulto , Anciano , Anticuerpos Antineoplásicos/biosíntesis , Anticuerpos Antineoplásicos/sangre , Neoplasias de la Mama/sangre , Neoplasias de la Mama/tratamiento farmacológico , Quimioterapia Adyuvante , Terapia Combinada , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/sangre , Inmunoglobulina M/biosíntesis , Inmunoglobulina M/sangre , Persona de Mediana Edad , Sensibilidad y EspecificidadRESUMEN
The MUC 1 mucin is expressed on normal breast epithelium and in 90% of breast cancers. We report here that tumor-associated MUC1 is a ligand for intercellular adhesion molecule 1 (ICAM-1). Antibodies to ICAM-1 and to MUC1 inhibited adhesion of human and transfected mouse MUC1-positive cell lines to human umbilical vein endothelial cell monolayers and immobilized recombinant human ICAM-1-immunoglobulin fusion protein. Purified MUC1 pretreatment of recombinant human ICAM-1 was an equally effective inhibitor of adhesion. The interaction between MUC1 and ICAM-1 may be critical to the process of bloodborne metastases in breast cancer.