RESUMEN
Anti-CD22 moxetumomab pasudotox achieved 46% complete remissions (CRs) in previously reported phase 1 testing in relapsed/refractory hairy cell leukemia (HCL; n = 28). The importance of minimal residual disease (MRD) after CR in HCL is unknown. A 21-patient extension cohort received 50 µg/kg every other day for 3 doses in 4-week cycles. These patients plus 12 previously reported at this upper dose level received 143 cycles without dose-limiting toxicity. The combined 33-patient cohort achieved 64% CR and 88% overall response rates, with median CR duration of 42.4 months. Of 32 50-µg/kg patients evaluable for MRD by bone marrow aspirate flow cytometry (most stringent assessment), median CR duration was 13.5 (4.9-42.4) months in 9 MRD-positive CRs vs 42.1 (24.0-69.2) months in 11 MRD-negative CRs (P < .001). Among MRD-negative CRs, 10 patients had ongoing CR, 9 without MRD, at end of study. To our knowledge, moxetumomab pasudotox is the only nonchemotherapy regimen that can eliminate MRD in a significant percentage of HCL patients, to enhance CR duration. Repeated dosing, despite early neutralizing antibodies, increased active drug levels without detectable toxicity from immunogenicity. The activity and safety profiles of moxetumomab pasudotox support ongoing phase 3 testing in HCL. This trial was registered at www.clinicaltrials.gov as #NCT00586924.
Asunto(s)
Antineoplásicos/uso terapéutico , Toxinas Bacterianas/uso terapéutico , Exotoxinas/uso terapéutico , Leucemia de Células Pilosas/tratamiento farmacológico , Leucemia de Células Pilosas/patología , Neoplasia Residual/tratamiento farmacológico , Adulto , Anciano , Antineoplásicos/farmacología , Toxinas Bacterianas/farmacología , Ensayos Clínicos Fase I como Asunto , Monitoreo de Drogas , Exotoxinas/farmacología , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
Immunotoxins (antibody-toxin fusion proteins) target surface antigens on cancer cells and kill these cells via toxin-mediated inhibition of protein synthesis. To identify genes controlling this process, an RNAi whole-genome screen (â¼ 22,000 genes at three siRNAs per gene) was conducted via monitoring the cytotoxicity of the mesothelin-directed immunotoxin SS1P. SS1P, a Pseudomonas exotoxin-based immunotoxin, was chosen because it is now in clinical trials and has produced objective tumor regressions in patients. High and low concentrations of SS1P were chosen to allow for the identification of both mitigators and sensitizers. As expected, silencing known essential genes in the immunotoxin pathway, such as mesothelin, furin, KDEL receptor 2, or members of the diphthamide pathway, protected cells. Of greater interest was the observation that many RNAi targets increased immunotoxin sensitivity, indicating that these gene products normally contribute to inefficiencies in the killing pathway. Of the top sensitizers, many genes encode proteins that locate to either the endoplasmic reticulum (ER) or Golgi and are annotated as part of the secretory system. Genes related to the ER-associated degradation system were not among high-ranking mitigator or sensitizer candidates. However, the p97 inhibitor eeyarestatin 1 enhanced immunotoxin killing. Our results highlight potential targets for chemical intervention that could increase immunotoxin killing of cancer cells and enhance our understanding of toxin trafficking.
Asunto(s)
Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Inmunotoxinas/farmacología , Interferencia de ARN , Animales , HumanosRESUMEN
Abstract: Cell deletion approaches to pain directed at either the primary nociceptive afferents or second-order neurons are highly effective analgesic manipulations. Second-order spinal neurons expressing the neurokinin 1 (NK1) receptor are required for the perception of many types of pain. To delete NK1+ neurons for the purpose of pain control, we generated a toxinpeptide conjugate using DTNB-derivatized (Cys0) substance P (SP) and a N-terminally truncated Pseudomonas exotoxin (PE35) that retains the endosome-release and ADP-ribosylation enzymatic domains but with only one free sulfhydryl side chain for conjugation. This allowed generation of a one-to-one product linked by a disulfide bond (SP-PE35). In vitro, Chinese hamster ovary cells stably transfected with the NK1 receptor exhibited specific cytotoxicity when exposed to SP-PE35 (IC50 = 5 × 10−11 M), whereas the conjugate was nontoxic to NK2 and NK3 receptor-bearing cell lines. In vivo studies showed that, after infusion into the spinal subarachnoid space, the toxin was extremely effective in deleting NK1 receptor-expressing cells from the dorsal horn of the spinal cord. The specific cell deletion robustly attenuated thermal and mechanical pain sensations and inflammatory hyperalgesia but did not affect motoric capabilities. NK1 receptor cell deletion and antinociception occurred without obvious lesion of nonreceptor-expressing cells or apparent reorganization of primary afferent innervation. These data demonstrate the extraordinary selectivity and broad-spectrum antinociceptive efficacy of this ligand-directed protein therapeutic acting via receptor-mediated endocytosis. The loss of multiple pain modalities including heat and mechanical pinch, transduced by different populations of primary afferents, shows that spinal NK1 receptor-expressing neurons are critical points of convergence in the nociceptive transmission circuit. We further suggest that therapeutic end points can be effectively and safely achieved when SP-PE35 is locally infused, thereby producing a regionally defined analgesia.
Asunto(s)
Exotoxinas/farmacología , Neuronas/metabolismo , Pseudomonas/metabolismo , Receptores de Neuroquinina-1/metabolismo , Animales , Axones/metabolismo , Células CHO , Cricetulus , Hiperalgesia/metabolismo , Dolor/metabolismo , Manejo del Dolor , Sustancia P/metabolismoRESUMEN
In this issue of Blood, Frankel et al describe a novel treatment of blastic plasmacytoid dendritic cell neoplasm (BPDCN) using an engineered version of diphtheria toxin that is targeted to malignant cells via a fusion with interleukin (IL)3 (see panel A).
Asunto(s)
Células Dendríticas/patología , Neoplasias Hematológicas/patología , Neoplasias Hematológicas/terapia , Receptores de Interleucina-3/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/uso terapéutico , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/terapia , Humanos , MasculinoRESUMEN
Unconjugated monoclonal antibodies that target hematopoietic differentiation antigens have been developed to treat hematologic malignancies. Although some of these have activity against chronic lymphocytic leukemia and hairy cell leukemia, in general, monoclonal antibodies have limited efficacy as single agents in the treatment of leukemia. To increase their potency, the binding domains of monoclonal antibodies can be attached to protein toxins. Such compounds, termed immunotoxins, are delivered to the interior of leukemia cells based on antibody specificity for cell surface target antigens. Recombinant immunotoxins have been shown to be highly cytotoxic to leukemic blasts in vitro, in xenograft model systems, and in early-phase clinical trials in humans. These agents will likely play an increasing role in the treatment of leukemia.
Asunto(s)
Inmunotoxinas/uso terapéutico , Leucemia/terapia , Animales , Antígenos CD19/metabolismo , Ensayos Clínicos como Asunto , Humanos , Inmunoterapia/métodos , Inmunotoxinas/química , Leucemia/inmunología , Modelos Moleculares , Terapia Molecular Dirigida , Lectina 2 Similar a Ig de Unión al Ácido Siálico/antagonistas & inhibidoresRESUMEN
Immunogenicity remains the "Achilles' heel" of protein-based therapeutics. Anti-drug Abs produced in response to protein therapeutics can severely limit both the safety and efficacy of this expanding class of agent. In this article, we report that monotherapy of mice with tofacitinib (the JAK inhibitor) quells Ab responses to an immunotoxin derived from the bacterial protein Pseudomonas exotoxin A, as well as to the model Ag keyhole limpet hemocyanin. Thousand-fold reductions in IgG1 titers to both Ags were observed 21 d post immunization. In fact, suppression was evident for all IgG isotypes and IgM. A reduction in IgG3 production was also noted with a thymus-independent type II Ag. Mechanistic investigations revealed that tofacitinib treatment led to reduced numbers of CD127+ pro-B cells. Furthermore, we observed fewer germinal center B cells and the impaired formation of germinal centers of mice treated with tofacitinib. Because normal Ig levels were still present during tofacitinib treatment, this agent specifically reduced anti-drug Abs, thus preserving the potential efficacy of biological therapeutics, including those used as cancer therapeutics.
Asunto(s)
ADP Ribosa Transferasas/farmacología , Formación de Anticuerpos/efectos de los fármacos , Toxinas Bacterianas/farmacología , Exotoxinas/farmacología , Hemocianinas/farmacología , Inmunotoxinas/farmacología , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Pirroles/farmacología , Factores de Virulencia/farmacología , Animales , Linfocitos B/inmunología , Femenino , Centro Germinal/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Quinasas Janus/antagonistas & inhibidores , Quinasas Janus/inmunología , Ratones , Ratones Endogámicos BALB C , Exotoxina A de Pseudomonas aeruginosaRESUMEN
Immune targeting of B-cell malignancies using chimeric antigen receptors (CARs) is a promising new approach, but critical factors impacting CAR efficacy remain unclear. To test the suitability of targeting CD22 on precursor B-cell acute lymphoblastic leukemia (BCP-ALL), lymphoblasts from 111 patients with BCP-ALL were assayed for CD22 expression and all were found to be CD22-positive, with median CD22 expression levels of 3500 sites/cell. Three distinct binding domains targeting CD22 were fused to various TCR signaling domains ± an IgG heavy chain constant domain (CH2CH3) to create a series of vector constructs suitable to delineate optimal CAR configuration. CARs derived from the m971 anti-CD22 mAb, which targets a proximal CD22 epitope demonstrated superior antileukemic activity compared with those incorporating other binding domains, and addition of a 4-1BB signaling domain to CD28.CD3 constructs diminished potency, whereas increasing affinity of the anti-CD22 binding motif, and extending the CD22 binding domain away from the membrane via CH2CH3 had no effect. We conclude that second-generation m971 mAb-derived anti-CD22 CARs are promising novel therapeutics that should be tested in BCP-ALL.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Receptores de Antígenos de Linfocitos T/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos T/inmunología , Lectina 2 Similar a Ig de Unión al Ácido Siálico/antagonistas & inhibidores , Lectina 2 Similar a Ig de Unión al Ácido Siálico/inmunología , Animales , Línea Celular Tumoral , Citocinas/metabolismo , Citotoxicidad Inmunológica , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas Recombinantes de Fusión/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Rationally designed anticancer agents that target cell-surface antigens or receptors represent a promising approach for treating cancer patients. However, antibodies that bind these targets are often, by themselves, non-cytotoxic. By attaching potent toxins we can dramatically improve the clinical utility of some anti-tumour antibodies. Here we describe the construction and clinical utility of several recombinant immunotoxins; each of which is composed of antibody Fv fragments fused to powerful bacterial toxins. Results from clinical trials indicate that recombinant immunotoxins and similar agents that are designed to combine antibody selectivity with toxin cell-killing potency will be useful additions to cancer therapy.
Asunto(s)
Antineoplásicos/uso terapéutico , Inmunoterapia , Inmunotoxinas/uso terapéutico , Neoplasias/terapia , Animales , Toxinas Bacterianas/inmunología , Toxinas Bacterianas/uso terapéutico , Humanos , Región Variable de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/uso terapéutico , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/uso terapéuticoRESUMEN
HA22 is a recombinant immunotoxin composed of an anti-CD22 Fv fused to a portion of Pseudomonas exotoxin A. HA22 produced a high rate of complete remissions in drug-resistant hairy cell leukemia and has a lower response rate in pediatric acute lymphoblastic leukemia (ALL). To understand why patients with ALL have poorer responses, we isolated an ALL cell line that is resistant to killing by HA22. The resistance is unstable; without HA22 the cells revert to HA22 sensitivity in 4 mo. We showed that in the resistant cell line, HA22 is unable to ADP ribosylate and inactivate elongation factor-2 (EF2), owing to a low level of DPH4 mRNA and protein, which prevents diphthamide biosynthesis and renders EF2 refractory to HA22. Analysis of the promoter region of the DPH4 gene shows that the CpG island was hypomethylated in the HA22-sensitive cells, heavily methylated in the resistant cells, and reverted to low methylation in the revertant cells. Our data show that immunotoxin resistance is associated with reversible CpG island methylation and silencing of DPH4 gene transcription. Incubation of sensitive cells with the methylation inhibitor 5-azacytidine prevented the emergence of resistant cells, suggesting that this agent in combination with HA22 may be useful in the treatment of some cases of ALL.
Asunto(s)
Toxinas Bacterianas/farmacología , Metilación de ADN , Exotoxinas/farmacología , Proteínas del Choque Térmico HSP40/genética , Inmunotoxinas/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Regiones Promotoras Genéticas , Azacitidina/farmacología , Secuencia de Bases , Línea Celular Tumoral , Islas de CpG , Metilación de ADN/efectos de los fármacos , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Humanos , Datos de Secuencia Molecular , Factor 2 de Elongación Peptídica/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Lectina 2 Similar a Ig de Unión al Ácido Siálico/inmunologíaRESUMEN
Synergistic killing was achieved when Small Cell Lung Cancer (SCLC) cell lines were incubated with ABT-263 and an immunotoxin directed to the transferrin receptor. SCLC lines are variably sensitive to the BH-3 only peptide mimetic, ABT-263. To determine their sensitivity to toxin-based reagents, we incubated four representative SCLC lines with a model Pseudomonas exotoxin-based immunotoxin directed to the transferrin receptor. Remarkably in 4-of-4 lines, there was little evidence of immunotoxin-mediated cytotoxicity despite near complete inhibition of protein synthesis. However, when combinations of ABT-263 and immunotoxin were added to the ABT-263-resistant cell lines (H196 and H69AR), there was synergistic killing as evidenced by increased activation of caspase 3/7, annexin V staining, and loss of cell integrity. Synergistic killing was evident at 6 hr and correlated with loss of Mcl-1. This synergy was also noted when the closely related compound ABT-737 was combined with the same immunotoxin. To establish that the synergy seen in tissue culture could be achieved in vivo, H69AR cells were grown as tumors in nude mice and shown to be susceptible to the killing action of an immunotoxin-ABT-737 combination but not to either agent alone. When immunotoxin-ABT combinations were added to ABT-263-sensitive lines (H146 and H1417), killing was additive. Our data support combination approaches for treating ABT-263-resistant SCLC with ABT-263 and a second agent that provides synergistic killing action.
Asunto(s)
Compuestos de Anilina/uso terapéutico , Compuestos de Bifenilo/farmacología , Inmunotoxinas/uso terapéutico , Nitrofenoles/farmacología , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Compuestos de Anilina/farmacología , Animales , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Inmunotoxinas/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Receptores de Transferrina/inmunologíaRESUMEN
Delivery of cancer therapeutics to non-specific sites decreases treatment efficacy while increasing toxicity. In ovarian cancer, overexpression of the cell surface marker HER2, which several therapeutics target, relates to poor prognosis. We recently reported the assembly of biocompatible bacterial spore-like particles, termed "SSHELs." Here, we modify SSHELs with an affibody directed against HER2 and load them with the chemotherapeutic agent doxorubicin. Drug-loaded SSHELs reduce tumor growth and increase survival with lower toxicity in a mouse tumor xenograft model compared with free drug and with liposomal doxorubicin by preferentially accumulating in the tumor mass. Target cells actively internalize and then traffic bound SSHELs to acidic compartments, whereupon the cargo is released to the cytosol in a pH-dependent manner. We propose that SSHELs represent a versatile strategy for targeted drug delivery, especially in cancer settings.
Asunto(s)
Neoplasias , Esporas Bacterianas , Ratones , Humanos , Animales , Esporas Bacterianas/metabolismo , Sistemas de Liberación de Medicamentos , Membrana Celular/metabolismo , Neoplasias/metabolismo , Proteínas Bacterianas/metabolismo , Bacillus subtilis/metabolismoRESUMEN
The tumor microenvironment (TME) influences tumor growth, metastatic spread and response to treatment. Often immunosuppression, mediated by the TME, impairs a beneficial response. The complexity of the tumor composition challenges our abilities to design new and more effective therapies. Going forward we will need to 'manage' the content and or functionality of the TME to improve treatment outcomes. Currently, several different kinds of treatments are available to patients with cancer: there are the traditional approaches of chemotherapy, radiation and surgery; there are targeted agents that inhibit kinases associated with oncogenic pathways; there are monoclonal antibodies that target surface antigens often delivering toxic payloads or cells and finally there are antibodies and biologics that seek to overcome the immunosuppression caused by elements within the TME. How each of these therapies interact with the TME is currently under intense and widespread investigation. In this review we describe how the TME and its immunosuppressive components can influence both tumor progression and response to treatment focusing on three particular tumor types, classic Hodgkin Lymphoma (cHL), Pancreatic Ductal Adenocarcinoma (PDAC) and Glioblastoma Multiforme (GBM). And, finally, we offer five approaches to manipulate or manage the TME to improve outcomes for cancer patients.
Asunto(s)
Antineoplásicos Inmunológicos , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Microambiente Tumoral , Neoplasias Pancreáticas/patología , Carcinoma Ductal Pancreático/patología , Inmunoterapia , Antineoplásicos Inmunológicos/uso terapéuticoRESUMEN
Immunotoxins based on Pseudomonas exotoxin A (PE) are promising anticancer agents that combine a variable fragment (Fv) from an antibody to a tumor-associated antigen with a 38-kDa fragment of PE (PE38). The intoxication pathway of PE immunotoxins involves receptor-mediated internalization and trafficking through endosomes/lysosomes, during which the immunotoxin undergoes important proteolytic processing steps but must otherwise remain intact for eventual transport to the cytosol. We have investigated the proteolytic susceptibility of PE38 immunotoxins to lysosomal proteases and found that cleavage clusters within a limited segment of PE38. We subsequently generated mutants containing deletions in this region using HA22, an anti-CD22 Fv-PE38 immunotoxin currently undergoing clinical trials for B-cell malignancies. One mutant, HA22-LR, lacks all identified cleavage sites, is resistant to lysosomal degradation, and retains excellent biologic activity. HA22-LR killed chronic lymphocytic leukemia cells more potently and uniformly than HA22, suggesting that lysosomal protease digestion may limit immunotoxin efficacy unless the susceptible domain is eliminated. Remarkably, mice tolerated doses of HA22-LR at least 10-fold higher than lethal doses of HA22, and these higher doses exhibited markedly enhanced antitumor activity. We conclude that HA22-LR advances the therapeutic efficacy of HA22 by using an approach that may be applicable to other PE-based immunotoxins.
Asunto(s)
ADP Ribosa Transferasas/farmacología , Anticuerpos Monoclonales/farmacología , Toxinas Bacterianas/farmacología , Exotoxinas/farmacología , Región Variable de Inmunoglobulina/farmacología , Inmunotoxinas/farmacología , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Lectina 2 Similar a Ig de Unión al Ácido Siálico , Factores de Virulencia/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , ADP Ribosa Transferasas/efectos adversos , ADP Ribosa Transferasas/genética , ADP Ribosa Transferasas/farmacocinética , Animales , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/farmacocinética , Toxinas Bacterianas/efectos adversos , Toxinas Bacterianas/genética , Toxinas Bacterianas/farmacocinética , Ensayos Clínicos como Asunto , Endosomas/metabolismo , Exotoxinas/efectos adversos , Exotoxinas/genética , Exotoxinas/farmacocinética , Femenino , Humanos , Región Variable de Inmunoglobulina/efectos adversos , Región Variable de Inmunoglobulina/genética , Inmunotoxinas/efectos adversos , Inmunotoxinas/genética , Inmunotoxinas/farmacocinética , Leucemia Linfocítica Crónica de Células B/metabolismo , Lisosomas/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Mutación , Factores de Virulencia/efectos adversos , Factores de Virulencia/genética , Factores de Virulencia/farmacocinética , Exotoxina A de Pseudomonas aeruginosaRESUMEN
Attention-deficit/hyperactivity disorder is a problematic diagnosis in the context of aeromedical certification. Certain characteristics of the disorder such as impaired attention potentially affect the safe conduct of flying. Pharmacological treatment with stimulants also has issues surrounding short half-lives and effects on the recognition of fatigue. This article gives a broad overview of the issues involved and provides certification guidelines as adopted in the Australian Civil Aviation Safety Authority which may be helpful if adopted by other certification bodies.
Asunto(s)
Medicina Aeroespacial , Trastorno por Déficit de Atención con Hiperactividad/diagnóstico , Trastorno por Déficit de Atención con Hiperactividad/tratamiento farmacológico , Certificación , Toma de Decisiones , Accidentes , Estimulantes del Sistema Nervioso Central/farmacocinética , Comorbilidad , Diagnóstico Diferencial , Manual Diagnóstico y Estadístico de los Trastornos Mentales , Guías como Asunto , Humanos , RiesgoRESUMEN
Immunotoxins are antibody-toxin fusion proteins under development as cancer therapeutics. In early clinical trials, immunotoxins constructed with domains II and III of Pseudomonas exotoxin (termed PE38), have produced a high rate of complete remissions in Hairy Cell Leukemia and objective responses in other malignancies. Cholera exotoxin (also known as cholix toxin) has a very similar three-dimensional structure to Pseudomonas exotoxin (PE) and when domains II and III of each are compared at the primary sequence level, they are 36% identical and 50% similar. Here we report on the construction and activity of an immunotoxin made with domains II and III of cholera exotoxin (here termed CET40). In cell viability assays, the CET40 immunotoxin was equipotent to tenfold less active compared to a PE-based immunotoxin made with the same single-chain Fv. A major limitation of toxin-based immunotoxins is the development of neutralizing antibodies to the toxin portion of the immunotoxin. Because of structure and sequence similarities, we evaluated a CET40 immunotoxin for the presence of PE-related epitopes. In western blots, three-of-three anti-PE antibody preparations failed to react with the CET40 immunotoxin. More importantly, in neutralization studies neither these antibodies nor those from patients with neutralizing titers to PE38, neutralized the CET40-immunotoxin. We propose that the use of modular components such as antibody Fvs and toxin domains will allow a greater flexibility in how these agents are designed and deployed including the sequential administration of a second immunotoxin after patients have developed neutralizing antibodies to the first.
Asunto(s)
Toxina del Cólera/inmunología , Inmunotoxinas/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Secuencia de Bases , Western Blotting , Toxina del Cólera/genética , Reacciones Cruzadas , Exotoxinas/inmunología , Humanos , Inmunotoxinas/genética , Datos de Secuencia Molecular , Neoplasias/sangre , Neoplasias/inmunología , Pseudomonas/inmunología , Receptores de Transferrina/inmunología , Proteínas Recombinantes de Fusión/genética , Anticuerpos de Cadena Única/inmunologíaRESUMEN
Cancer cells frequently upregulate surface receptors that promote growth and survival. These receptors constitute valid targets for intervention. One strategy involves the delivery of toxic payloads with the goal of killing those cancer cells with high receptor levels. Delivery can be accomplished by attaching a toxic payload to either a receptor-binding antibody or a receptor-binding ligand. Generally, the cell-binding domain of the toxin is replaced with a ligand or antibody that dictates a new binding specificity. The advantage of this "immunotoxin" approach lies in the potency of these chimeric molecules for killing cancer cells. However, receptor expression on normal tissue represents a significant obstacle to therapeutic intervention.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Inmunotoxinas/inmunología , Neoplasias/inmunología , Receptores de Superficie Celular/inmunología , Toxinas Biológicas/inmunología , Animales , Anticuerpos Monoclonales/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Humanos , Inmunotoxinas/metabolismo , Inmunotoxinas/uso terapéutico , Ligandos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Unión Proteica , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/metabolismo , Toxinas Biológicas/metabolismoRESUMEN
Low density lipoprotein receptor-related protein 1, (LRP1) is a large multifunctional receptor that binds more than 25 physiologic ligands. In addition, it functions as the surface receptor for several Rhinoviruses, HIV-tat and Pseudomonas exotoxin (PE). We report that the expression of PE within mammalian cells can serve as a probe of LRP1 maturation and functionality. To avoid cell killing, an enzymatically inactive form of the toxin (PEDelta553) was expressed. A permanent cell line (termed CY301) was established whereby PEDelta553 was expressed continually into the ER of CHO cells. CY301 cells were 100-fold resistant to exogenously added active PE but exhibited no cross-resistance to other toxins. Our studies indicate that PEDelta553 bound to immature LRP1 in the ER, prevented its maturation to the cell surface and thereby produced a toxin resistant phenotype. By confocal microscopy, cell-associated PEDelta553 was localized to the ER and co-localized with LRP1. Further characterization of CY301 cells indicated that RAP, the chaperone that aids in LRP1 folding, was released to the growth media. Thus the intracellular expression of PEDelta553 appears to be a valuable probe of LRP1 maturation and trafficking.
Asunto(s)
Exotoxinas/genética , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/antagonistas & inhibidores , Mutación , Pseudomonas/química , Animales , Sitios de Unión/genética , Células CHO , Cricetinae , Cricetulus , Retículo Endoplásmico/metabolismo , Expresión Génica , Ligandos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Técnicas de Sonda Molecular , Proteínas Mutantes , Unión Proteica , Transporte de ProteínasRESUMEN
Pseudomonas aeruginosa is an opportunistic pathogen that produces several virulence factors, among them Pseudomonas Exotoxin A (PE). Previously, low-density lipoprotein receptor-related protein 1 (LRP 1) was shown to be the primary receptor for PE. In this report, we show that a close family member, LRP 1B, can also function as a receptor.
Asunto(s)
Proteínas Bacterianas/metabolismo , Inhibidores de la Síntesis de la Proteína/metabolismo , Receptores de LDL/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/toxicidad , Células CHO , Cricetinae , Cricetulus , Electroforesis en Gel de Poliacrilamida , Técnicas de Transferencia de Gen , Membranas Artificiales , Mutación/genética , Polivinilos , Unión Proteica/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/toxicidad , Pruebas de ToxicidadRESUMEN
PURPOSE: To conduct a phase I trial of recombinant immunotoxin BL22, an anti-CD22 Fv fragment fused to truncated Pseudomonas exotoxin. PATIENTS AND METHODS: Forty-six pretreated patients with CD22+ non-Hodgkin's lymphoma (NHL; n = 4), chronic lymphocytic leukemia (CLL; n = 11), and hairy cell leukemia (HCL; n = 31) received 265 cycles at 3 to 50 microg/Kg every other day x 3 doses. RESULTS: BL22 was active in HCL, with 19 complete remissions (CRs; 61%) and six partial responses (PRs; 19%) in 31 patients. Of 19 CRs, 11 were achieved after one cycle and eight after two to 14 cycles. All 25 responders benefited clinically with one cycle. The CR rate was 86% in patients enrolled at > or = 40 microg/Kg every other day x 3, and 41% at lower doses (P = .011). The median duration for CR was 36 months (range, 5 to 66 months), and eight patients remain in CR at 45 months (range, 29 to 66 months). Lower but significant activity occurred in CLL. Neutralizing antibodies occurred in 11 (24%) of 46 patients (all HCL). A reversible hemolytic uremic syndrome requiring plasmapheresis was observed in one patient with NHL during cycle 1 and in four patients with HCL during cycle 2 or 3. The maximum-tolerated dose (MTD) evaluated at cycle 1 was 40 microg/Kg IV. QOD x 3. The most common toxicities at 30 to 50 microg/Kg every other day x 3 included hypoalbuminemia, transaminase elevations, fatigue, and edema. CONCLUSION: BL22 was well tolerated and highly effective in HCL, even after one cycle. Phase II testing is underway to define the efficacy with one cycle and to study safety when additional cycles are needed for optimal response.
Asunto(s)
Inmunotoxinas/uso terapéutico , Leucemia de Células Pilosas/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Linfoma no Hodgkin/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Enterotoxinas , Femenino , Estudios de Seguimiento , Humanos , Infusiones Intravenosas , Leucemia de Células Pilosas/diagnóstico , Leucemia de Células Pilosas/mortalidad , Leucemia Linfocítica Crónica de Células B/diagnóstico , Leucemia Linfocítica Crónica de Células B/mortalidad , Linfoma no Hodgkin/diagnóstico , Linfoma no Hodgkin/mortalidad , Masculino , Dosis Máxima Tolerada , Persona de Mediana Edad , Medición de Riesgo , Índice de Severidad de la Enfermedad , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
The intersection of small molecular weight drugs and antibody-based therapeutics is rarely studied in large scale. Both types of agents are currently part of the cancer armamentarium. However, very little is known about how to combine them in optimal ways. Immunotoxins are antibody-toxin gene fusion proteins engineered to target cancer cells via antibody binding to surface antigens. For fusion proteins derived from Pseudomonas exotoxin (PE), potency relies on the enzymatic domain of the toxin which catalyzes the ADP-ribosylation of EF2 causing inhibition of protein synthesis leading to cell death. Candidate immunotoxins have demonstrated clear value in clinical trials but generally have not been curative as single agents. Therefore we undertook three screens to discover effective combinations that could act synergistically. From the MIPE-3 library of compounds we identified various enhancers of immunotoxin action and at least one major class of inhibitor. Follow-up experiments confirmed the screening data and suggested that immunotoxins when administered with everolimus or nilotinib exhibit favorable combinatory activity and would be candidates for preclinical development. Mechanistic studies revealed that everolimus-immunotoxin combinations acted synergistically on elements of the protein synthetic machinery, including S61 kinase and 4E-BP1 of the mTORC1 pathway. Conversely, PARP inhibitors antagonized immunotoxins and also blocked the toxicity due to native ADP-ribosylating toxins. Thus, our goal of investigating a chemical library was justified based on the identification of several approved compounds that could be developed preclinically as 'enhancers' and at least one class of mitigator to be avoided.