RESUMEN
In this chapter we discuss vaccines to protect against the highly toxic plant-derived toxin, ricin. Due to its prevalence, ease of use, and stability it has been used in sporadic incidents of espionage. There is also concern that it will be used as an agent of bioterrorism. As a result there has been a great deal of interest in developing a safe vaccine or antidote to protect humans, and in particular soldiers and first responders. Although multiple types of vaccines have been tested, at this time two recombinant vaccines are the leading candidates for the national vaccine stockpile. In terms of passive post-exposure protection, monoclonal neutralizing antibodies that passively protect animals are also under development. These vaccines and antibodies are discussed in the context of the toxicity and structure of ricin.
Asunto(s)
Antitoxinas , Sustancias para la Guerra Química , Ricina/antagonistas & inhibidores , Vacunas , Animales , Anticuerpos/inmunología , Anticuerpos/uso terapéutico , Sustancias para la Guerra Química/química , Sustancias para la Guerra Química/toxicidad , Humanos , Profilaxis Posexposición , Ricina/química , Ricina/toxicidad , Vacunas SintéticasRESUMEN
RiVax is a recombinant protein that is currently under clinical development as part of a human vaccine to protect against ricin poisoning. RiVax includes ricin A-chain (RTA) residues 1-267 with two intentional amino-acid substitutions, V76M and Y80A, aimed at reducing toxicity. Here, the crystal structure of RiVax was solved to 2.1â Å resolution and it was shown that it is superposable with that of the ricin toxin A-chain from Ricinus communis with a root-mean-square deviation of 0.6â Å over 258 C(α) atoms. The RiVax structure is also compared with the recently determined structure of another potential ricin-vaccine immunogen, RTA 1-33/44-198 R48C/T77C. Finally, the locations and solvent-exposure of two toxin-neutralizing B-cell epitopes were examined and it was found that these epitopes are within or near regions predicted to be involved in catalysis. The results demonstrate the composition of the RiVax clinical material and will guide ongoing protein-engineering strategies to develop improved immunogens.
Asunto(s)
Vacunas/química , Cristalografía por Rayos X , Epítopos de Linfocito B/química , Humanos , Proteínas Recombinantes/químicaRESUMEN
Vascular leak syndrome is a major and often dose-limiting side effect of immunotoxins and cytokines. We postulated that this syndrome is initiated by damage to vascular endothelial cells. Our earlier studies identified a three-amino acid motif that is shared by toxins, ribosome-inactivating proteins, and interleukin-2, all of which cause this problem. We have now generated a panel of recombinant ricin A chains with mutations in this sequence or in amino acids flanking it in the three-dimensional structure. These have been evaluated alone and as immunotoxins for activity, ability to induce pulmonary vascular leak in mice, pharmacokinetics, and activity in tumor-xenografted mice. One mutant was comparable to the ricin A chain used before in all respects except that it did not cause vascular leak at the same dose and, when used as an immunotoxin, was more effective in xenografted SCID mice.
Asunto(s)
Síndrome de Fuga Capilar/tratamiento farmacológico , Endotelio/efectos de los fármacos , Ingeniería Genética/métodos , Inmunotoxinas/genética , Ricina/administración & dosificación , Secuencias de Aminoácidos , Animales , Síndrome de Fuga Capilar/inducido químicamente , Inmunotoxinas/administración & dosificación , Inmunotoxinas/metabolismo , Inmunotoxinas/toxicidad , Dosificación Letal Mediana , Pulmón/irrigación sanguínea , Pulmón/efectos de los fármacos , Linfoma/tratamiento farmacológico , Ratones , Ratones SCID , Mutagénesis Sitio-Dirigida , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ricina/genética , Ricina/metabolismo , Ricina/toxicidadRESUMEN
The purpose of this study was to prepare a tetravalent anti-human CD22 recombinant antibody with improved antitumor activity and a half life longer than that of its divalent counterpart. We compared the ability of tetravalent vs. divalent antibody to associate/dissociate to/from CD22-positive Daudi cells, to interact with murine and human Fcgamma receptors (FcgammaR), to bind human complement component C1q, to inhibit the growth of tumor cells, to diffuse into various tissues, to be internalized by Daudi cells, to react with human neonatal Fc receptors (FcRn), and to persist in the circulation of normal mice. As compared to the murine or chimeric divalent antibodies, the chimeric tetravalent counterpart has a longer half life in mice. It also has an affinity for FcRns that is identical to that of human IgG. The tetravalent antibody has increased antitumor activity in vitro and completely conserved effector functions (binding to FcgammaR-positive cells and to C1q) in vitro. Despite its 33% higher molecular weight, it penetrates mouse tissues as well as its divalent antibody counterpart. Based on the improved in vitro performance and pharmacokinetics of the tetravalent antibody it will now be tested for its antitumor activity in vivo.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/farmacocinética , Antineoplásicos/farmacología , Antineoplásicos/farmacocinética , Lectina 2 Similar a Ig de Unión al Ácido Siálico/inmunología , Animales , Anticuerpos Monoclonales/química , Antineoplásicos/química , Supervivencia Celular/efectos de los fármacos , Complemento C1q/inmunología , Femenino , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Inmunoglobulina G/inmunología , Ratones , Ratones Transgénicos , Receptores Fc/genética , Receptores Fc/inmunología , Receptores de IgG/inmunología , Células U937RESUMEN
We demonstrate that a peptoid composed of five monomers and attached via a maleimide linker to a carrier protein elicits anti-peptoid, anti-linker and anti-carrier antibodies in rabbits. Specific anti-peptoid antibodies were affinity purified and used to reproducibly retrieve three specific peptoid-coupled beads from 20,000 irrelevant peptoid-beads using magnetic screening.
RESUMEN
PURPOSE: The purpose of this study was to prepare chimeric antihuman CD22 tetravalent monoclonal antibodies (MAbs) with high functional affinity, long persistence in the circulation, increased antitumor activity, and conserved effector function in vitro. EXPERIMENTAL DESIGN: We investigated the association/dissociation rates of these tetravalent antibodies using CD22(+) Daudi lymphoma cells. We then tested their ability to interact with Fc receptors on a human cell line (U937), to mediate antibody-dependent cellular cytotoxicity with human natural killer cells, to bind human C1q, to inhibit the in vitro growth of CD22 Daudi cells, and to persist in the circulation. RESULTS: The rate of dissociation of the tetravalent MAbs versus the divalent antibody was considerably slower. These tetravalent MAbs inhibited the in vitro proliferation of CD22 Daudi cells at a concentration that was at least 100-fold lower than that of the divalent murine antibody. The tetravalent MAbs containing both the CH2 and CH3 domains and a chimeric recombinant divalent antibody bound similarly to Fc receptor, C1q, and mediate antibody-dependent cellular cytotoxicity equally well with human natural killer cells. The persistence in the circulation of chimeric tetravalent MAbs was considerably longer than that of chemical homodimers. CONCLUSIONS: The tetravalent anti-CD22 MAbs with intact Fc regions should make effective therapeutic agents for B-cell tumors.
Asunto(s)
Anticuerpos/química , Antígenos CD/química , Antígenos de Diferenciación de Linfocitos B/química , Moléculas de Adhesión Celular/química , Lectinas/química , Neoplasias/terapia , Proteínas Recombinantes/química , Animales , Anticuerpos Monoclonales/química , Antineoplásicos/farmacología , Área Bajo la Curva , División Celular , Complemento C1q/química , Dimerización , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Humanos , Células Asesinas Naturales/metabolismo , Linfoma/metabolismo , Ratones , Unión Proteica , Estructura Terciaria de Proteína , Lectina 2 Similar a Ig de Unión al Ácido Siálico , Temperatura , Factores de Tiempo , Células U937RESUMEN
There is no FDA-approved vaccine for the potent plant toxin ricin. We have developed a recombinant ricin vaccine, RiVax. Without adjuvant it is safe and immunogenic in mice, rabbits, and humans. Based on our studies in mice, we now report the results of a small clinical trial with Alhydrogel-adsorbed RiVax.
Asunto(s)
Proyectos Piloto , Ricina/inmunología , Vacunas , Adsorción , Adulto , Hidróxido de Aluminio , Anticuerpos/inmunología , Formación de Anticuerpos , Femenino , Humanos , Masculino , Ricina/antagonistas & inhibidores , Vacunas/administración & dosificación , Vacunas/efectos adversos , Vacunas/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/efectos adversos , Vacunas Sintéticas/inmunología , Adulto JovenRESUMEN
Ricin toxin (RT) is derived from castor beans, produced by the plant Ricinus communis. RT and its toxic A chain (RTA) have been used therapeutically to arm ligands that target disease-causing cells. In most cases these ligands are cell-binding monoclonal antibodies (MAbs). These ligand-toxin conjugates or immunotoxins (ITs) have shown success in clinical trials [1]. Ricin is also of concern in biodefense and has been classified by the CDC as a Class B biothreat. Virtually all reports of RT poisoning have been due to ingestion of castor beans, since they grow abundantly throughout the world and are readily available. RT is easily purified and stable, and is not difficult to weaponize. RT must be considered during any "white powder" incident and there have been documented cases of its use in espionage [2,3]. The clinical syndrome resulting from ricin intoxication is dependent upon the route of exposure. Countermeasures to prevent ricin poisoning are being developed and their use will depend upon whether military or civilian populations are at risk of exposure. In this review we will discuss ricin toxin, its cellular mode of action, the clinical syndromes that occur following exposure and the development of pre- and post-exposure approaches to prevent of intoxication.
Asunto(s)
Sustancias para la Guerra Química/envenenamiento , Ricina/envenenamiento , Animales , Humanos , Intoxicación/prevención & control , VacunaciónRESUMEN
Ricin is a CDC level B biothreat. Our recombinant ricin A chain vaccine (RiVax) contains two mutations, rendering it non-toxic at high doses. Frozen or alum formulations of RiVax protected mice against ricin administered by injection, gavage or aerosol. Without alum, RiVax was safe and immunogenic in rabbits and human volunteers. For military use, the predominant target group, it would be optimal not to require a cold chain for transport and storage. We have now developed a lyophilized formulation and demonstrated stability and efficacy for at least 1 year stored refrigerated or at room temperature administered with or without alum.
Asunto(s)
Almacenaje de Medicamentos , Liofilización , Vacunas/inmunología , Animales , Antitoxinas/sangre , Estabilidad de Medicamentos , Femenino , Inyecciones Intramusculares , Ratones , Intoxicación/prevención & control , Subunidades de Proteína , Ricina/toxicidad , Análisis de Supervivencia , Vacunas/administración & dosificaciónRESUMEN
Ricin toxin is a CDC level B biothreat. We have developed a ricin vaccine, RiVax, which is a recombinant mutant of ricin A chain. RiVax is safe, immunogenic and protective in mice when administered intramuscularly (IM). We have now attempted to increase the utility and immunogenicity of RiVax by administering it intradermally (ID) with or without alum. Without alum, Rivax administered by the ID and IM routes was equally immunogenic and protective. With alum, ID vaccinations were more immunogenic and protective against both systemic and mucosal challenge with ricin and superior in protecting animals from ricin-induced lung damage.
Asunto(s)
Membrana Mucosa/inmunología , Ricina/administración & dosificación , Vacunación/métodos , Vacunas de Subunidad/inmunología , Vacunas/administración & dosificación , Adyuvantes Inmunológicos/administración & dosificación , Compuestos de Alumbre/administración & dosificación , Animales , Anticuerpos/sangre , Formación de Anticuerpos , Femenino , Inyecciones Intradérmicas , Inyecciones Intramusculares , Pulmón/fisiopatología , Ratones , Vacunas de Subunidad/administración & dosificaciónRESUMEN
Ricin is a plant toxin that is a CDC level B biothreat. Our recombinant ricin A chain vaccine (RiVax), which contains mutations in both known toxic sites, has no residual toxicity at doses at least 800 times the immunogenic dose. RiVax without adjuvant given intramuscularly (i.m.) protected mice against intraperitoneally administered ricin. Furthermore the vaccine without alum was safe and immunogenic in human volunteers. Here we describe the development of gavage and aerosol ricin challenge models in mice and demonstrate that i.m. vaccination protects mice against ricin delivered by either route. Also RiVax protects against aerosol-induced lung damage as determined by histology and lung function tests.
Asunto(s)
Ricina/antagonistas & inhibidores , Vacunas Sintéticas/administración & dosificación , Administración por Inhalación , Administración Oral , Aerosoles , Animales , Bioterrorismo , Genes de Plantas , Humanos , Inyecciones Intramusculares , Intestinos/efectos de los fármacos , Intestinos/patología , Dosificación Letal Mediana , Pulmón/efectos de los fármacos , Pulmón/patología , Pulmón/fisiopatología , Ratones , Mutación , Plantas Tóxicas/genética , Ricina/genética , Ricina/inmunología , Ricina/toxicidad , Ricinus/genética , VacunasRESUMEN
UV3 is a monoclonal antibody that recognizes human CD54 (intercellular adhesion molecule-1), and it was generated for the therapy of human multiple myeloma. In a severe combined immunodeficient (SCID) xenograft model of human multiple myeloma, UV3 significantly prolonged the survival of mice with either early or advanced stages of disease. However, the mechanism by which UV3 exerted its antitumor effect remained unknown. As reported previously UV3 could mediate antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity in vitro. F(ab)'2 fragments of UV3 had therapeutic efficacy in vivo, suggesting that effector functions were not critical. The purpose of this study was to further define the importance of the Fc portion of UV3 for its antitumor activity in vivo. To this end, we examined the effect of an "ultrapure" preparation of UV3 F(ab)'2 to treat SCID mice xenografted with either ARH-77 cells, a human multiple myeloma cell line, or Daudi cells, a human Burkitt's lymphoma cell line. In addition, we evaluated different doses of UV3 immunoglobulin G (IgG) in these mice to determine the minimum amount of IgG that would produce a therapeutic effect. Data obtained from this study suggest that (1) the Fc portion of UV3 is critical for its antitumor activity in vivo, (2) low levels of UV3 IgG in a preparation of F(ab)'2 fragments account for all of its in vivo activity in multiple myeloma and most of its activity in lymphoma, and (3) UV3 IgG significantly prolongs the survival of SCID/ARH-77 mice as well as SCID/Daudi mice.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Linfoma de Burkitt/tratamiento farmacológico , Fragmentos Fc de Inmunoglobulinas/inmunología , Molécula 1 de Adhesión Intercelular/inmunología , Mieloma Múltiple/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/inmunología , Linfoma de Burkitt/inmunología , Linfoma de Burkitt/mortalidad , Línea Celular Tumoral , Proliferación Celular , Humanos , Inmunoglobulina G/inmunología , Técnicas In Vitro , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Mieloma Múltiple/inmunología , Trasplante de Neoplasias , Trasplante HeterólogoRESUMEN
Ricin, a highly potent toxin produced by castor beans, is classified by the Centers for Disease Control and Prevention as a level B biothreat because it is easily produced, readily available, and highly stable. There have been >750 cases of documented ricin intoxication in humans. There is no approved vaccine for ricin. Ricin contains a lectin-binding B chain and a ribotoxic A chain (RTA). In addition to its ribotoxic site, we have identified a separate site on RTA that is responsible for inducing vascular leak syndrome (VLS) in humans. We have generated a recombinant RTA with two amino acid substitutions that disrupt its ribotoxic site (Y80A) and its VLS-inducing site (V76M). This mutant recombinant RTA (named RiVax) was expressed and produced in Escherichia coli and purified. When RiVax was injected i.m. into mice it protected them against a ricin challenge of 10 LD50s. Preclinical studies in both mice and rabbits demonstrated that RiVax was safe. Based on these results, we have now conducted a pilot clinical trial in humans under an investigational new drug application submitted to the Food and Drug Administration. In this study, three groups of five normal volunteers were injected three times at monthly intervals with 10, 33, or 100 mug of RiVax. The vaccine was safe and elicited ricin-neutralizing Abs in one of five individuals in the low-dose group, four of five in the intermediate-dose group, and five of five in the high-dose group. These results justify further development of the vaccine.
Asunto(s)
Bioterrorismo , Ricina/inmunología , Ricina/envenenamiento , Vacunas Sintéticas/administración & dosificación , Adolescente , Adulto , Sustitución de Aminoácidos , Anticuerpos/sangre , Femenino , Humanos , Masculino , Proyectos Piloto , Conformación Proteica , Ricina/química , Vacunación , Vacunas , Vacunas Sintéticas/efectos adversos , Vacunas Sintéticas/inmunologíaRESUMEN
Ricin toxin is a plant-derived ribosome inactivating protein (RIP) of extraordinary toxicity. Vaccination using ricin toxoid or its A chain (RTA) is protective in animals but both vaccines have two potential toxicities, RIP and vascular leak syndrome (VLS). Previously we described three recombinant RTA constructs from which both toxicities were eliminated by site-specific mutations. One mutant, V76M/Y80A, RiVax, has now been further characterized for immunogenicity and toxicity in animals. We have found that RiVax is safe at doses of at least 8 mg in mice, 800-fold higher than the protective dose, and induces neutralizing antibodies in both mice and rabbits.
Asunto(s)
Ricina/inmunología , Vacunas Sintéticas/inmunología , Animales , Anticuerpos/sangre , Anticuerpos Monoclonales/inmunología , Femenino , Sueros Inmunes/inmunología , Masculino , Ratones , Conejos , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/toxicidad , Vacunas , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/toxicidadRESUMEN
Immunization of mice with the idiotype (Id) immunoglobulin from the murine B cell lymphoma, BCL1, before inoculating tumor cells can induce tumor dormancy. In this model, the tumor cells grow for a short period of time and then regress. The mice live for months or years with approximately 1 million tumor cells in their spleens. Some mice relapse due to decreases in the anti-Id antibody titers or the development of mutations in the residual tumor cells which render them refractory to negative signaling by the anti-Id antibody. In this study we determined whether we could eliminate the residual dormant cells by using a DNA vaccine against the Id or by immunomodulation of T-cell subsets in vivo. Our results demonstrate that dormancy can be maintained by further immunizations with either the BCL1 Id protein or DNA vaccine encoding its single-chain Fv fragment. We also found that a cytotoxic T-cell response was not induced by either in vivo administration of vaccine alone or by the vaccine plus interleukin-2. In addition the injection of anti-cytotoxic T-lymphocyte-associate antigen did not prolong dormancy. Finally, the in vivo administration of anti-CD25 to deplete regulatory T cells did not prolong dormancy. Dormancy in this model is dependent primarily upon anti-Id antibodies, our results suggest that other strategies to target residual dormant BCL1 cells are warranted. They also suggest that the elimination of dormant tumor may represent a greater challenge than the elimination of primary tumors.
Asunto(s)
Ciclina D1/uso terapéutico , Idiotipos de Inmunoglobulinas/uso terapéutico , Linfoma de Células B/terapia , Vacunas de ADN/uso terapéutico , Animales , Antígenos CD , Antígenos de Diferenciación/inmunología , Antígeno CTLA-4 , Línea Celular , Idiotipos de Inmunoglobulinas/inmunología , Inmunoterapia Activa , Interleucina-2/farmacología , Linfoma de Células B/inmunología , Linfoma de Células B/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Receptores de Interleucina-2/inmunología , Bazo/citología , Linfocitos T Reguladores/inmunologíaRESUMEN
Despite new treatment options, including autologous and allogeneic stem cell transplants, multiple myeloma remains an incurable disease. The authors developed and characterized a murine anti-human ICAM-1 (CD54) monoclonal antibody, UV3, which is highly effective in SCID mice with advanced human myeloma xenografts (SCID/ARH-77). To improve the effector functions and pharmacokinetic parameters and to reduce its immunogenicity in humans, the authors engineered this monoclonal antibody into a mouse/human IgG1kappa chimeric (c) antibody, cUV3. Following coexpression and purification of the genetically spliced heavy and light chain constructs, the authors compared cUV3 and UV3 in various in vitro assays, including relative cell-binding affinities and effector functions, namely antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. The authors compared their in vivo retention times and biodistribution patterns in normal mice. In each assay, the authors found that cUV3 was essentially equivalent to UV3. Finally, these antibodies were tested in a SCID/ARH-77 model of advanced myeloma, with daily treatments of 4 mug/g for 4 consecutive days commencing 14 days after tumor cell inoculation. cUV3 was at least as effective as UV3; 40% and 20% of the mice, respectively, were cured, with no sign of disease at day 150. The authors intend to evaluate the efficacy of cUV3 further in SCID/ARH-77 mice using other doses and dosing schedules to try to improve the cure rate. Eventually, they hope to test the efficacy of cUV3 in patients with multiple myeloma.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Molécula 1 de Adhesión Intercelular/inmunología , Mieloma Múltiple/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacocinética , Afinidad de Anticuerpos , Citotoxicidad Celular Dependiente de Anticuerpos , Antineoplásicos/inmunología , Antineoplásicos/farmacocinética , Humanos , Ratones , Ratones SCID , Mieloma Múltiple/inmunología , Mieloma Múltiple/mortalidad , Trasplante de Neoplasias , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Ricin toxin (RT) is a plant-derived toxin of extraordinary toxicity; a single molecule successfully internalized into the cytoplasm of a cell is lethal for that cell. An estimated dose of 1-10 microg/kg is lethal to humans, making aerosolized ricin a potential agent for bioterrorism. Vaccination against ricin using either denatured toxin or its modified A chain subunit (RTA) has been successful in experimental animals but both vaccines have potential toxicities. Recombinant (r) RTA has not been evaluated as a vaccine. However, the advantage of such a vaccine is that these potential toxicities can be deleted by appropriate mutations. In this study we have generated three mutants and shown that two lack toxicity as compared to the wild type rRTA. These mutants induce protective humoral immune responses in mice. One or both should be considered for use in humans.