RESUMEN
With the ultimate goal of developing a therapeutic cancer vaccine, we encapsulated the Her-2/neu peptide p369-377 in poly(lactide-co-glycolide) microspheres. This formulation was found to effectively elicit CD8+ cytotoxic T cell (CTL) responses in an HLA-A*0201 transgenic mouse model. In contrast, immunization with either peptide alone or peptide formulated in incomplete Freund's adjuvant (IFA) failed to elicit such CTL responses. Responses induced by the peptide-microsphere formulation were found to peak at approximately 6 weeks post-immunization, and were enhanced by delivering increased doses of peptide and with repeated administrations over time. Co-administration of the peptide-microspheres with adjuvants, including granulocyte-macrophage colony stimulating factor, MPL adjuvant and select synthetic Toll-Like Receptor 4 ligands, the aminoalkyl glucosaminide-4 phosphates, significantly augmented CTL responses. These studies provide important guidance for the design of human clinical trials of microsphere vaccines in terms of optimal peptide-microsphere formulation, vaccination regimen, vaccine dose, and adjuvant selection.
Asunto(s)
Adyuvantes Inmunológicos , Vacunas contra el Cáncer/síntesis química , Vacunas contra el Cáncer/inmunología , Citotoxicidad Inmunológica , Microesferas , Fragmentos de Péptidos/inmunología , Receptor ErbB-2/inmunología , Linfocitos T Citotóxicos/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Vacunas contra el Cáncer/administración & dosificación , Células Cultivadas , Femenino , Humanos , Ratones , Ratones Transgénicos , Fragmentos de Péptidos/administración & dosificación , Poliglactina 910/administración & dosificación , Receptor ErbB-2/administración & dosificación , Bazo/citología , Bazo/inmunologíaRESUMEN
Among clinicians, initial awareness of the Wilms' tumor gene was limited mostly to pediatric oncologists. Almost a decade ago, overexpression of Wilms' tumor 1 (WT1) was observed in adult acute leukemia. Subsequent studies indicated that WT1 overexpression occurs in most cases of acute myelogenous leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia (CML), and myelodysplastic syndrome (MDS). Limited tissue expression of WT1 in adults suggests that WT1 can be a target for leukemia/MDS therapy. WT1 expression in stem/progenitor cells remains unsettled. However, lack of progenitor cell suppression by WT1 antisense or WT1-specific cytotoxic T cells provide some assurance that WT1 expression in progenitor cells is minimal or absent. Immunotherapy-based WT1 approaches are furthest along in preclinical development. WT1-specific cytotoxic lymphocytes can be generated from normals and leukemic patients. In mice, WT1 vaccines elicit specific immune responses without evidence of tissue damage. In this paper, we review studies validating the immunogenicity of WT1 and propose that leukemia and MDS may be a good clinical model to test the efficacy of a WT1 vaccine.
Asunto(s)
Inmunoterapia/métodos , Leucemia Mielógena Crónica BCR-ABL Positiva/terapia , Síndromes Mielodisplásicos/terapia , Proteínas WT1/antagonistas & inhibidores , Enfermedad Aguda , Vacunas contra el Cáncer , Humanos , Inmunoterapia Adoptiva , Proteínas WT1/análisis , Proteínas WT1/inmunologíaRESUMEN
WT1 is an oncogenic protein expressed by the Wilms' tumor gene and overexpressed in the majority of acute myelogenous leukemias (AMLs) and chronic myelogenous leukemias (CMLs). The current study analyzed the sera of patients with AML and CML for the presence of antibodies to full-length and truncated WT1 proteins. Sixteen of 63 patients (25%) with AML had serum antibodies reactive with WT1/full-length protein. Serum antibodies from all 16 were also reactive with WT1/NH2-terminal protein. By marked contrast, only 2 had reactivity to WT1/COOH-terminal protein. Thus, the level of immunological tolerance to the COOH terminus may be higher than to the NH2 terminus. The WT1/COOH-terminal protein contains four zinc finger domains with homology to other self-proteins. By implication, these homologies may be related to the increased immunological tolerance. Results in patients with CML were similar with antibodies reactive to WT1/full-length protein detectable in serum of 15 of 81 patients (19%). Antibodies reactive with WT1/NH2-terminal protein were present in the serum of all 15, whereas antibodies reactive with WT1/COOH-terminal protein were present in only 3. By contrast to results in leukemia patients, antibodies reactive with WT1/full-length protein were detected in only 2 of 96 normal individuals. The greater incidence of antibody in leukemia patients provides strong evidence that immunization to the WT1 protein occurred as a result of patients bearing malignancy that expresses WT1. These data provide further stimulus to test therapeutic vaccines directed against WT1 with increased expectation that the vaccines will be able to elicit and/or boost an immune response to WT1.
Asunto(s)
Anticuerpos/sangre , Proteínas de Unión al ADN/inmunología , Leucemia/sangre , Leucemia/inmunología , Factores de Transcripción/inmunología , Adulto , ADN Complementario/metabolismo , Ensayo de Inmunoadsorción Enzimática , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/sangre , Leucemia Mielógena Crónica BCR-ABL Positiva/inmunología , Leucemia Mielógena Crónica BCR-ABL Positiva/mortalidad , Leucemia Mieloide Aguda/sangre , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/mortalidad , Proteínas Recombinantes/metabolismo , Proteínas WT1RESUMEN
The present study utilizes an in vivo murine tumor expressing human Her-2/neu to evaluate potential Her-2/neu vaccines consisting of either full length or various subunits of Her-2/neu delivered in either protein or plasmid DNA form. Our results demonstrate that protective immunity against Her-2/neu-expressing tumor challenge can be achieved by vaccination with plasmid DNA encoding either full length or subunits of Her-2/neu. Partial protective immunity was also observed following vaccination with the intracellular domain (ICD), but not extracellular domain (ECD), protein subunit of Her-2/neu. The mechanism of protection elicited by plasmid DNA vaccination appeared to be exclusively CD4 dependent, whereas the protection observed with ICD protein vaccination required both CD4 and CD8 T cells.
Asunto(s)
Vacunas contra el Cáncer/farmacología , Neoplasias Experimentales/prevención & control , Receptor ErbB-2/inmunología , Vacunas de ADN/farmacología , Animales , Linfocitos T CD4-Positivos/inmunología , Vacunas contra el Cáncer/genética , Femenino , Genes erbB-2 , Ratones , Ratones Endogámicos C57BL , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Subunidades de Proteína , Receptor ErbB-2/química , Receptor ErbB-2/genética , Timoma/inmunología , Timoma/patología , Timoma/terapia , Neoplasias del Timo/inmunología , Neoplasias del Timo/patología , Neoplasias del Timo/terapia , Células Tumorales Cultivadas , Vacunas de ADN/genéticaRESUMEN
The Wilms' tumor (WT1) gene participates in leukemogenesis and is overexpressed in most types of leukemia in humans. WT1 is also detectable in many types of lung, thyroid, breast, testicular, and ovarian cancers and melanoma in humans. Initial studies evaluated whether immune responses to murine WT1 can be elicited in mice. Murine and human WT1 are similar. Thus, mouse models might lead to resolution of many of the critical issues for developing WT1 vaccines. C57/BL6 (B6) mice were injected with synthetic peptides from the natural sequence of WT1 containing motifs for binding to major histocompatibility (MHC) class II molecules. Immunization induced helper T-cell responses specific for the immunizing WT1 peptides and antibody responses specific for WT1 protein. Screening of multiple murine cancer cell lines identified 2 murine cancers, TRAMP-C and BLKSV40, that "naturally" overexpress WT1. Immunization with MHC class I binding peptides induced WT1 peptide-specific cytotoxic T-lymphocyte (CTL) that specifically lysed TRAMP-C and BLKSV40. WT1 specificity of lysis was confirmed by cold target inhibition. No toxicity was noted by histopathologic evaluation in the WT1 peptide-immunized animals. WT1 peptide immunization did not show any effect on TRAMP-C tumor growth in vivo. Immunization of B6 mice to syngeneic TRAMP-C elicited WT1-specific antibody, demonstrating that WT1 can be immunogenic in the context of cancer cells. To evaluate whether WT1 might be similarly immunogenic in humans, serum from patients with leukemia was evaluated for pre-existing antibody responses. Western blot analyses showed WT1-specific antibodies directed against the N-terminus portion of the WT1 protein in the sera of 3 of 18 patients with acute myeloid leukemia (AML). (Blood. 2000;96:1480-1489)
Asunto(s)
Proteínas de Unión al ADN/inmunología , Leucemia Mieloide/inmunología , Factores de Transcripción/inmunología , Enfermedad Aguda , Animales , Anticuerpos/inmunología , Vacunas contra el Cáncer/inmunología , Femenino , Genes del Tumor de Wilms , Humanos , Ratones , Ratones Endogámicos C57BL , Péptidos/inmunología , Subgrupos de Linfocitos T/inmunología , Células Tumorales Cultivadas , Proteínas WT1RESUMEN
Immature dendritic cells (DC) take up, process and present protein antigens; mature DC are specialized for stimulating primary T cell responses with increased expression of MHC class II and co-stimulatory molecules, but are incapable of processing and presenting soluble protein. The current study examined whether maturation of DC is triggered by T cell recognition of antigens presented by immature DC. Human DC derived from CD34+ progenitor cells by culture with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-6 (IL-6) in serum-free medium could prime naive CD4+ T cells to keyhole limpet hemocyanin (KLH) and ovalbumin (OVA). The cultured DC retained the ability to prime T cells to native protein for at least 15 days. To test for changes in DC function after participation in an immune response, DC were co-cultured with either allogeneic or autologous CD4+ T cells. DC co-cultured with autologous T cells retained the ability to prime T cells to intact protein antigens. By contrast, DC which had previously stimulated an allogeneic T cell response lost ability to prime T cells to soluble proteins. However, such <
Asunto(s)
Presentación de Antígeno , Células Dendríticas/inmunología , Antígenos de Histocompatibilidad Clase II/análisis , Presentación de Antígeno/inmunología , Antígeno B7-1/análisis , Células de la Médula Ósea , Linfocitos T CD4-Positivos/inmunología , Antígenos CD40/análisis , Comunicación Celular/inmunología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Células Cultivadas , Técnicas de Cocultivo , Medio de Cultivo Libre de Suero , Células Dendríticas/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Hemocianinas/inmunología , Humanos , Molécula 1 de Adhesión Intercelular/análisis , Interferón gamma/farmacología , Interleucina-6/farmacología , Ovalbúmina/inmunología , Factores de Tiempo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
OBJECTIVES/HYPOTHESIS: To discover unique genes specific for squamous cell carcinoma of the head and neck for eventual development as tumor markers and vaccine candidates. STUDY DESIGN: Molecular biological analysis of fresh-frozen head and neck squamous cell cancer (HNSCC). METHODS: A subtractive library was made from two HNSCC and six normal tissues using a polymerase chain reaction (PCR)-based approach. Genes from this library were PCR amplified and placed on a microarray glass slide. RNA was prepared or obtained from 16 fresh-frozen HNSCC and 22 normal tissue sources. Fluorescent probes were made from the polyA+ RNA derived from the tumor and normal tissues. The probes were hybridized to the glass slides and excited by a tuneable laser. One hundred seven of the genes showing the highest differential fluorescence value between tumor and normal tissue were identified by sequence analysis. RESULTS: Thirteen independent genes were found to be overexpressed in tumor tissues. Of these, nine were previously known: keratins K6 and K16, laminin-5, plakophilin-1, matrix metalloproteinase-2 (MMP), vascular endothelial growth factor, connexin 26, 14-3-3 sigma, and CaN19. The level of polyA+ RNA of these genes in the tumors was significantly different from the levels in normal tissue (P < .05). Four previously unidentified genes were also discovered to have increased expression in tumor tissue. Comparing the total tumor group (n = 16) to the normal group (n = 22), only one of these genes showed significant overexpression. CONCLUSION: We report the identification of nine known genes that are significantly overexpressed in HNSCC as compared to normal tissue using subtractive and microarray technology. In addition, we present four previously unidentified genes that are overexpressed in a subset of tumors. These genes will be developed as tumor markers and vaccine candidates.
Asunto(s)
Carcinoma de Células Escamosas/genética , Regulación Neoplásica de la Expresión Génica/genética , Neoplasias de Cabeza y Cuello/genética , Tirosina 3-Monooxigenasa , Proteínas 14-3-3 , Adolescente , Adulto , Anciano , Biomarcadores de Tumor/genética , Vacunas contra el Cáncer/genética , Moléculas de Adhesión Celular/genética , Factores Quimiotácticos/genética , Conexina 26 , Conexinas/genética , Sondas de ADN , ADN Complementario/genética , Factores de Crecimiento Endotelial/genética , Biblioteca de Genes , Humanos , Hibridación Fluorescente in Situ , Queratinas/genética , Rayos Láser , Linfocinas/genética , Metaloproteinasa 2 de la Matriz/genética , Persona de Mediana Edad , Biología Molecular , Placofilinas , Reacción en Cadena de la Polimerasa , Proteínas/genética , ARN Mensajero/genética , ARN Neoplásico/genética , Proteínas S100/genética , Análisis de Secuencia de ADN , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular , KalininaRESUMEN
CD34(+) hematopoietic stem cells from normal individuals and from patients with chronic myelogenous leukemia can be induced to differentiate into dendritic cells (DC). The aim of the current study was to determine whether acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) cells could be induced to differentiate into DC. CD34(+) AML-M2 cells with chromosome 7 monosomy were cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNFalpha), and interleukin-4 (IL-4). After 3 weeks of culture, 35% of the AML-M2 cells showed DC morphology and phenotype. The DC phenotype was defined as upmodulation of the costimulatory molecules CD80 and CD86 and the expression of CD1a or CD83. The leukemic nature of the DC was validated by detection of chromosome 7 monosomy in sorted DC populations by fluorescence in situ hybridization (FISH). CD34(+) leukemic cells from 2 B-ALL patients with the Philadelphia chromosome were similarly cultured, but in the presence of CD40-ligand and IL-4. After 4 days of culture, more than 58% of the ALL cells showed DC morphology and phenotype. The leukemic nature of the DC was validated by detection of the bcr-abl fusion gene in sorted DC populations by FISH. In functional studies, the leukemic DC were highly superior to the parental leukemic blasts for inducing allogeneic T-cell responses. Thus, CD34(+) AML and ALL cells can be induced to differentiate into leukemic DC with morphologic, phenotypic, and functional similarities to normal DC.
Asunto(s)
Células Madre Hematopoyéticas/inmunología , Leucemia Mieloide Aguda/sangre , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangre , Antígenos CD/sangre , Antígenos CD34/sangre , Diferenciación Celular , Células Cultivadas , Cromosomas Humanos Par 7 , Células Dendríticas/inmunología , Células Dendríticas/patología , Citometría de Flujo , Genotipo , Células Madre Hematopoyéticas/patología , Humanos , Inmunofenotipificación , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/patología , Monosomía , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Factores de TiempoRESUMEN
HER-2/neu is a "self" tumor antigen that is overexpressed in 15-30% of human adenocarcinomas. Vaccine strategies directed against HER-2/neu and other self tumor antigens require development of methods to overcome immune tolerance to self-proteins. In rats, rat neu peptide vaccines have been shown to be an effective way of circumventing tolerance to rat neu protein and generating rat neu-specific immunity. The present report validates that a similar peptide-based vaccine formulation is effective for inducing T-cell immunity to HER-2/neu protein in humans with breast and ovarian cancer. The vaccine formulation included groups of peptides derived from the HER-2/neu extracellular domain (ECD) or intracellular domain (ICD) mixed with granulocyte macrophage colony stimulating factor as an adjuvant. These peptides were 15-18 amino acids in length and designed to elicit a CD4 T helper-specific immune response. Patients underwent intradermal immunization once a month for a total of two to six immunizations. To date, all of the patients immunized with HER-2/neu peptides developed HER-2/neu peptide-specific T-cell responses. The majority of patients (six of eight) also developed HER-2/neu protein-specific responses. Responses to HER-2/neu protein occurred with epitope spreading. Immune T cells elicited by vaccination were shown to migrate outside the peripheral circulation by virtue of generating delayed type hypersensitivity responses distant from the vaccine site, which indicated the potential ability to traffic to the site of tumor. The use of peptide-based vaccines may be a simple, yet effective, vaccine strategy for immunizing humans to oncogenic self-proteins.
Asunto(s)
Neoplasias de la Mama/inmunología , Vacunas contra el Cáncer/inmunología , Neoplasias Ováricas/inmunología , Fragmentos de Péptidos/inmunología , Receptor ErbB-2/inmunología , Adyuvantes Inmunológicos/uso terapéutico , Neoplasias de la Mama/terapia , Linfocitos T CD4-Positivos/inmunología , Relación Dosis-Respuesta a Droga , Epítopos/inmunología , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/uso terapéutico , Humanos , Hipersensibilidad Tardía/inmunología , Inmunidad Celular/inmunología , Neoplasias Ováricas/terapiaRESUMEN
We have investigated a murine model of acute lung injury caused by i.v. administration of a T cell clone (CD4+, Th1 phenotype) that recognizes Ly5, a polymorphic cell surface glycoprotein expressed on hemopoietic cells. Alloreactive cloned T cells, specific for host Ly5 Ag, cause a mononuclear cell pulmonary vasculitis and interstitial pneumonitis. In further studies of the cellular mechanisms involved in this model, we found that mature host T cells or B cells are not required, since lung injury was comparable in transgenic host mice that lack these cells (RAG-1 knockout). Cloned T cells labeled in vitro with bromodeoxyuridine were localized in inflammation foci in lung, but the majority of cells in the foci were not labeled. Using transgenic mice that constitutively express lacZ, we determined that the mononuclear cell vasculitis is of host cell origin. Alveolar macrophages (AM) from T cell-treated mice spontaneously secreted TNF-alpha in culture, whereas TNF-alpha was not detected in AM cultures from control mice. TNF-alpha production in response to LPS stimulation was significantly higher in AM cultures derived from T cell-treated mice than in those from control mice. Challenge with sublethal doses of LPS resulted in 50% mortality in T cell-treated mice and was associated with augmented AM TNF-alpha production and protein in bronchoalveolar lavage fluid. We conclude that immune activation of T cells of the Th1 phenotype can initiate lung injury characterized by a host-derived mononuclear cell inflammation and activation of AM.
Asunto(s)
Leucocitos Mononucleares/patología , Pulmón/inmunología , Pulmón/patología , Activación de Linfocitos/inmunología , Activación de Macrófagos/inmunología , Macrófagos Alveolares/inmunología , Células TH1/inmunología , Traslado Adoptivo , Animales , Linfocitos B/patología , Bromodesoxiuridina/metabolismo , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/inmunología , Células Clonales , Inmunidad Innata , Inmunohistoquímica , Inflamación/inmunología , Inflamación/patología , Interferón gamma/metabolismo , Isoantígenos/inmunología , Antígenos Comunes de Leucocito/genética , Leucocitos Mononucleares/inmunología , Lipopolisacáridos/toxicidad , Pulmón/química , Linfopenia/genética , Linfopenia/inmunología , Linfopenia/terapia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas/metabolismo , Linfocitos T/patología , Linfocitos T/trasplante , Células TH1/química , Células TH1/trasplante , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The aim of the current study was to determine whether immunization with synthetic peptides corresponding to the joining region segment of p210 bcr-abl chimeric protein can elicit CD8+ cytotoxic T lymphocytes (CTLs) capable of specifically lysing leukemia cells. BALB/c mice were immunized with peptides identical to the joining region segment of p210 bcr-abl protein. Class I major histocompatibility complex (MHC)-restricted bcr-abl peptide-specific CD8+ CTLs were elicited. The CTL clones were H-2 Kd restricted and specifically recognized a nonamer peptide of the combined sequence of bcr-abl amino acids but neither bcr nor abl amino acid sequence alone. Despite specificity and substantial lytic potential against syngeneic cell line incubated with exogenously supplied peptides, the bcr-abl peptide-specific CTLs failed to lyse syngeneic murine leukemia cells expressing human p210 bcr-abl protein containing the same bcr-abl joining region peptide sequence. Similarly, the bcr-abl peptide-specific CTLs did not lyse human bcr-abl-positive chronic myelogenous leukemia cells expressing murine class I MHC antigen (i.e., K562 cells infected with vaccinia virus expressing H-2 Kd). The appropriateness of the joining region segment of bcr-abl protein to serve as a T cell target depends upon whether that segment is presented by class I MHC in a concentration high enough to stimulate CTLs. The current experiments using murine peptide-specific CTLs could not establish that the joining region of bcr-abl protein is processed and presented by class I MHC antigen-processing pathway, but the possibility was not ruled out. Alternative models and/or strategies are necessary.
Asunto(s)
Proteínas de Fusión bcr-abl/inmunología , Leucemia/terapia , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Animales , Femenino , Antígenos H-2/fisiología , Humanos , Inmunización , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Células Tumorales CultivadasRESUMEN
To study the character of graft-vs-host disease (GVHD) induced by T cells specific for hemopoietic cells, T cells specific for a polymorphic segment of CD45 were transferred into CD45 congenic mice. C57BL/6 mice that express the CD45b allele were immunized with a 13 mer peptide representing the polymorphic segment (p257-268) of CD45a protein. Conversely, C57BL/6 mice congenic for CD45a were immunized with the CD45b peptide. CD4+ T cells specific for allelic CD45 peptides were elicited. Importantly, T cells specific for CD45 peptides proliferated specifically and vigorously in response to spleen cells expressing the appropriate polymorphic CD45 protein. T cells specific for CD45 induced a substantial graft-vs-host response (GVHR) with predominant early pulmonary vasculitis and later more widespread interstitial mononuclear cell infiltration and alveolitis. No GVHR was induced in bone marrow chimeras expressing only donor hemopoietic cells. Thus, donor T cell recognition of host hemopoietic cells is sufficient to elicit GVHR, but the classical skin, liver, and gut manifestations of GVHD were not observed. The CD45-specific T cells used secreted Th1 cytokines, but without detectable soluble IL-2. Studies using CD45-specific T cells with different effector functions might allow further dissection of donor cell requirements for GVHD syndromes.
Asunto(s)
Enfermedad Injerto contra Huésped/inmunología , Antígenos Comunes de Leucocito/inmunología , Enfermedades Pulmonares/inmunología , Fragmentos de Péptidos/inmunología , Polimorfismo Genético , Subgrupos de Linfocitos T/inmunología , Vasculitis/inmunología , Animales , Comunicación Celular/inmunología , Línea Celular , Movimiento Celular/inmunología , Células Clonales , Epítopos de Linfocito T/inmunología , Femenino , Enfermedad Injerto contra Huésped/patología , Células Madre Hematopoyéticas/inmunología , Inyecciones Intravenosas , Antígenos Comunes de Leucocito/biosíntesis , Antígenos Comunes de Leucocito/genética , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/patología , Hígado/patología , Enfermedades Pulmonares/patología , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fragmentos de Péptidos/genética , Fibrosis Pulmonar/inmunología , Fibrosis Pulmonar/patología , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Subgrupos de Linfocitos T/trasplante , Vasculitis/patologíaRESUMEN
Many newly defined tumour antigens are 'self' proteins. Immunizing cancer patients against these antigens may be difficult due to tolerance. The HER-2/neu oncogenic protein is such a 'self' tumour antigen. Rat neu is homologous with human HER-2/neu and provides a model system for studying vaccination strategies. Rats are tolerant to rat neu. Vaccination with this 'self' protein elicits no detectable immune response. The current studies evaluated whether tolerance to rat neu can be circumvented by immunizing with the highly homologous foreign human HER-2/neu protein. Rats were immunized with human HER-2/neu intracellular domain (hICD) protein that is 92% homologous to rat neu ICD. Animals immunized with hICD developed significant antibody and T-cell responses that were specific for both human HER-2/neu and rat neu. Neu-specific antibodies were present in titres of greater than 1:200,000. Analysis of the specificity of the antibody response using synthetic peptides demonstrated substantial reactivity to an epitope with 100% homology between rat and human protein. Significant T-cell responses (stimulation index > 10) to hICD and rat neu protein (stimulation index > 4) were detected. The T cells also responded to both human and rat ICD. The results imply that immunization with foreign proteins, which are highly homologous to 'self' tumour antigens, may be an effective vaccine strategy for 'self' tumour antigens.
Asunto(s)
Antígenos de Neoplasias/inmunología , Inmunización , Proteínas de Neoplasias/inmunología , Receptor ErbB-2/inmunología , Autotolerancia , Animales , Anticuerpos Antineoplásicos/biosíntesis , Especificidad de Anticuerpos , Humanos , Activación de Linfocitos , Ratas , Ratas Endogámicas F344 , Proteínas Recombinantes/inmunología , Especificidad de la Especie , Linfocitos T/inmunología , Células Tumorales Cultivadas/inmunologíaRESUMEN
Vaccine studies using whole tumor cells or heterogeneous mixtures of tumor antigens provide intriguing evidence that cancer vaccines might be effective. Now it is possible to test vaccines composed of well-characterized proteins and peptides. Testing vaccine formulations composed of known and defined antigens will allow a more precise determination as to why vaccines work when they work, and why they do not work when they fail. The demonstration that human malignancy is immunogenic and the definition of human tumor antigens has set the stage for a new generation of cancer vaccines directly targeting immunogenic cancer-related peptides and proteins. Many newly defined tumor antigens are self proteins. As an example, screening existent immunity in human melanoma has identified responses to nonmutated self proteins: MAGE, MART, gp100, and tyrosinase. Tolerance to self antigens now emerges as a possible mechanism of tumor immune escape. A new puzzle has emerged for tumor immunologists to solve; how to harness immunity to "self" tumor antigens for cancer therapeutics.
Asunto(s)
Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/inmunología , Neoplasias/prevención & control , Receptor ErbB-2/inmunología , Vacunas Sintéticas/inmunología , Ensayos Clínicos como Asunto , HumanosRESUMEN
PURPOSE: To evaluate HER-2/neu-specific antibody immunity in patients with breast cancer, to determine the rate of occurrence of serum antibodies to HER-2/neu in patients with breast cancer, and to relate the presence of specific immunity to overexpression of HER-2/neu protein in primary tumor. METHODS: The antibody response to HER-2/neu protein was analyzed in 107 newly diagnosed breast cancer patients. Sera was analyzed for the presence of HER-2/neu-specific antibodies with a capture enzyme-linked immunosorbent assay (ELISA) and verified by Western blot. Sera from 200 volunteer blood donors was used as a control population. RESULTS: The presence of antibodies to HER-2/neu correlated with the presence of breast cancer. HER-2/neu antibodies at titers of > or = 1:100 were detected in 12 of 107 (11%) breast cancer patients versus none of 200 (0%) normal controls (P < .01). The presence of antibodies to HER-2/neu also correlated to overexpression of HER-2/neu protein in the patient's primary tumor. Nine of 44 (20%) patients with HER-2/neu-positive tumors had HER-2/neu-specific antibodies, whereas three of 63 (5%) patients with HER-2/neu-negative tumors had antibodies (P = .03). The antibody responses could be substantial. Titers of greater than 1:5,000 were detected in five of 107 (5%). CONCLUSION: The presence of HER-2/neu antibodies in breast cancer patients and the correlation with HER-2/neu-positive cancer implies that immunity to HER-2/neu develops as a result of exposure of patients to HER-2/neu protein expressed by their own cancer. These findings should stimulate further studies to develop the detection of immunity to oncogenic proteins as tumor markers, as well as the development and testing of vaccine strategies to induce and augment immunity to HER-2/neu for the treatment of breast cancer or prevention of recurrent disease.
Asunto(s)
Anticuerpos Antineoplásicos/sangre , Neoplasias de la Mama/inmunología , Receptor ErbB-2/inmunología , Adulto , Anciano , Neoplasias de la Mama/sangre , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Receptor ErbB-2/sangre , Células Tumorales Cultivadas/inmunologíaRESUMEN
The long term goal of this study is to develop autoimmune prostatitis as a therapy for prostate cancer. An immune attack capable of destroying normal prostate epithelial cells should also destroy malignant prostate tissue and provide therapeutic benefit in cancer patients. The current study was initiated to identify antigenic targets for experimental autoimmune prostatitis on the assumption that such proteins might also be suitable targets for immunotherapy of prostate cancer. Male Lewis rats were immunized with syngeneic prostate homogenates, and the immune sera were used to screen prostate proteins for immunoreactivity by Western blot analysis. The dominant protein recognized by the immune sera was purified by ion exchange chromatography and reverse phase HPLC. Microsequence analysis of two polypeptide components of this immunodominant protein demonstrated N-terminal sequences identical with two of the three component chains of rat prostatic steroid-binding protein (PSBP). T cell responses to PSBP were also detected in rats immunized with prostate homogenate. Immunizing male rats with purified PSBP induced vigorous Ab and T cell responses. Significant prostate inflammation was observed in some rats immunized with PSBP. Adoptive transfer of T cells immune to PSBP induced rapid and severe destructive autoimmune prostatitis. These results demonstrate that PSBP is a major target Ag of experimental autoimmune prostatitis in a rat model and may serve as a target Ag for vaccine and T cell therapy against prostate cancer.
Asunto(s)
Autoantígenos/inmunología , Enfermedades Autoinmunes/inmunología , Prostatitis/inmunología , Proteínas/inmunología , Esteroides/metabolismo , Animales , Autoantígenos/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Inmunización , Inmunoterapia , Masculino , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/terapia , Proteínas/metabolismo , Ratas , Ratas Endogámicas LewRESUMEN
The bispecific monoclonal antibody (bsmAb) 2B1, targeting the extracellular domain of c-erbB-2, the protein product of the HER-2/neu proto-ocogene, and Fc gamma RIII (CD16), expressed by human natural killer cells, neutrophils and differentiated monocytes, mediates the specific cytotoxic activity of these effector cells to tumor cells. A group of 24 patients with c-erbB-2-overexpressing tumors were treated with intravenously administered 2B1 in a phase I clinical trial and followed after treatment to evaluate the diversity and extent of the 2B1-induced humoral immune responses. As expected, 17 of 24 patients developed human anti-(murine Ig) antibodies (HAMA) to whole 2B1 IgG in a range from 100 ng/ml to more than 50000 ng/ml; 10 of these patients (42%) had strong (at least 1000 ng/ml) HAMA responses, some of which were still detectable at day 191. These responses were usually associated with similar reactivity to the F(ab')2 fragments of the parental antibodies 520C9 (anti-c-erbB-2) and 3G8 (anti-CD16). We sought evidence of an idiotypic cascade induction, indicating a prolonged specific treatment-induced effect on at least one selected target of 2B1. Using competition-based enzyme-linked immunosorbent assays, specific anti-idiotypic antibodies (Ab2) were detectable against 520C9 in 11 patients and against 3G8 in 13 patients. Peak anti-idiotypic antibodies generally occurred 3-5 weeks from treatment initiation, with a downward trend thereafter. There was a statistically significant correlation among the induction of significant HAMA responses, anti-idiotypic antibody production and the development of antibodies to c-erbB-2. The anti-c-erbB-2 responses, which were distinct from anti-anti-idiotypic (Ab3) antibodies, were detected in the post-treatment sera of 6/16 patients examined. No obvious correlation could be made between the development of humoral immune responses, the dose received, and the clinical response. Future investigation involving 2B1 therapy will concentrate on investigating an association of these humoral responses to any c-erbB-2-specific cellular responses. Manipulations of 2B1 therapy effects that augment immunity to c-erbB-2 could provide additional avenues for immunotherapy with this and other bispecific antibodies.
Asunto(s)
Adenocarcinoma/prevención & control , Anticuerpos Antiidiotipos/biosíntesis , Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Especificidad de Anticuerpos , Receptor ErbB-2/inmunología , Vacunación , Adenocarcinoma/secundario , Citotoxicidad Inmunológica , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Receptores de IgG/inmunologíaRESUMEN
In animals and in humans, T-cell therapy can cure advanced disseminated leukemia that would otherwise be fatal. The therapeutic effect of immune T cells is quantitative. As the dose of effector T cells is increased, survival is proportionately increased. Therefore, effective T-cell therapy is predicated on the ability to procure large numbers of immune effector T cells. By using cultured T cells, the number of immune T cells can be increased in vivo substantially above the level achievable by vaccination. The survival of cultured T cells in vivo is dependent upon both the culture conditions used and the therapeutic regimens employed. Under appropriate conditions, cultured T cells can proliferate in vivo in response to stimulation by antigen, distribute widely and survive long term to provide effector function and immunologic memory. Given that T cells recognize peptides, the need for immunization with tumor can be circumvented by immunization with peptide. Peptide-specific T cells and the progeny of single T-cell clones can provide the necessary cellular functions to eradicate disseminated murine leukemia. The ability of cloned T cells to similarly provide substantial measurable immunity in humans has been validated in clinical trials. By priming with peptides and by using established culture conditions, T-cell therapy can now be directed against virtually any antigen within the host T-cell repertoire. The major remaining question to be answered is which proteins and which peptides are the most suitable targets for T-cell therapy trials.
Asunto(s)
Inmunoterapia Adoptiva/métodos , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/terapia , Linfocitos T/trasplante , Animales , Células Cultivadas , HumanosRESUMEN
The Her-2/neu protooncogene is associated with malignant transformation and aggressive disease. Because of its overexpression in tumor cells and because it has been shown to be immunogenic, this protein represents an excellent target for T-cell immunotherapy. By identifying potential HLA-A2.1-binding peptides from the Her-2/neu sequence, peptides were selected as candidate T-cell epitopes. The immunogenicity of each peptide was evaluated by priming double transgenic mice expressing both the human (hu) CD8 and HLA-A2.1 molecules with synthetic peptides corresponding to these sequences. Because of the lack of interaction between murine CD8 and HLA-A2.1, expression of huCD8 on murine cells facilitates recognition of HLA molecules on human tumor cell lines. This led to the identification of two peptides that elicit an A2-restricted CTL response, one of which has not been previously identified. Both peptide-specific CTL populations were able to specifically lyse A2.1 and Her-2/neu expressing human tumor cells originating from a variety of tissues, demonstrating the utility of this murine model in identifying peptides presented by human cells. However, several Her-2/neu peptides previously reported to be immunogenic for human CTL were found not to be immunogenic in transgenic mice. The basis for these discrepancies is discussed.
Asunto(s)
Antígenos CD8/metabolismo , Epítopos/genética , Antígeno HLA-A2/genética , Receptor ErbB-2/genética , Receptor ErbB-2/inmunología , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Animales , Femenino , Expresión Génica , Humanos , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Oligopéptidos/genética , Oligopéptidos/inmunología , Células Tumorales CultivadasRESUMEN
The HER-2/neu oncogenic protein is a tumor antigen. Some patients with cancer have a preexistent immune response directed against the HER-2/neu protein. Effective cancer vaccines targeting HER-2/neu will be able to boost this immunity to potentially therapeutic levels. In addition, HER-2/neu-directed monoclonal antibody therapy has been effective in eradicating malignancy in animal models and has shown benefit in the treatment of human HER-2/neu-overexpressing cancers. This review outlines studies that define HER-2/neu-specific immunity in patients with cancer and overviews the current vaccine strategies for generating or augmenting neu-specific immunity. The potential problems associated with eliciting HER-2/neu-specific immunity are addressed, including the question of precipitating autoimmune toxicity against this "self" -protein and the mechanisms of immunological escape that may play a role in preventing effective function of the HER-2/neu-specific immune response. Finally, antibody-based HER-2/neu-directed therapies are overviewed. HER-2/neu is a prototype antigen for groups investigating innovative modifications of monoclonal antibody technology, and cutting edge therapies targeting this antigen are being contemplated for clinical use in the treatment of human malignancy. Immune-based treatments designed to target the HER-2/neu oncogenic protein will soon give the clinical oncologist new therapeutic weapons, directed against a biologically relevant tumor-related protein, with which to fight cancer.