RESUMEN
PURPOSE: The first-in-human clinical trial with human bolus intravenous infusion IL15 (rhIL15) was limited by treatment-associated toxicity. Here, we report toxicity, immunomodulation, and clinical activity of rhIL15 administered as a 10-day continuous intravenous infusion (CIV) to patients with cancers in a phase I trial. PATIENTS AND METHODS: Patients received treatment for 10 days with CIV rhIL15 in doses of 0.125, 0.25, 0.5, 1, 2, or 4 µg/kg/day. Correlative laboratory tests included IL15 pharmacokinetic (PK) analyses, and assessment of changes in lymphocyte subset numbers. RESULTS: Twenty-seven patients were treated with rhIL15; 2 µg/kg/day was identified as the MTD. There were eight serious adverse events including two bleeding events, papilledema, uveitis, pneumonitis, duodenal erosions, and two deaths (one due to likely drug-related gastrointestinal ischemia). Evidence of antitumor effects was observed in several patients, but stable disease was the best response noted. Patients in the 2 µg/kg/day group had a 5.8-fold increase in number of circulating CD8+ T cells, 38-fold increase in total NK cells, and 358-fold increase in CD56bright NK cells. Serum IL15 concentrations were markedly lower during the last 3 days of infusion. CONCLUSIONS: This phase I trial identified the MTD for CIV rhIL15 and defined a treatment regimen that produced significant expansions of CD8+ T and NK effector cells in circulation and tumor deposits. This regimen has identified several biological features, including dramatic increases in numbers of NK cells, supporting trials of IL15 with anticancer mAbs to increase antibody-dependent cell-mediated cytotoxicity and anticancer efficacy.
Asunto(s)
Interleucina-15/administración & dosificación , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Subgrupos Linfocitarios/efectos de los fármacos , Subgrupos Linfocitarios/inmunología , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Citocinas/metabolismo , Femenino , Humanos , Inmunohistoquímica , Factores Inmunológicos/administración & dosificación , Factores Inmunológicos/efectos adversos , Factores Inmunológicos/farmacocinética , Inmunomodulación/efectos de los fármacos , Mediadores de Inflamación/metabolismo , Infusiones Intravenosas , Interleucina-15/efectos adversos , Interleucina-15/farmacocinética , Células Asesinas Naturales/metabolismo , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Recuento de Linfocitos , Subgrupos Linfocitarios/metabolismo , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Neoplasias/diagnóstico , Neoplasias/metabolismo , Resultado del TratamientoRESUMEN
Targeted inhibiting insulin-like growth factor 1 is an effective approach for cancer therapy. Insulin-like growth factor binding protein 7 (IGFBP7) is considered as a potential therapeutic protein. However, producing high quality of such non-IgG proteins in mammalian cells is still a challenge in biopharmaceutical development. Here, we report a rapid production process by using transient gene transfection in HEK 293E cells. A set of constructs combining several expression promoters, leader sequences, and 5' un-translated regions were generated and optimized, from which the best vector with expression level at ~50mg/L was selected for production at 2L cell culture scale. Comparison study in downstream purification methods led to development of a scalable, non-affinity chromatography strategy through Super Q, Fast Flow Q, and Heparin columns. The product was characterized in purity (99%), isoelectric point, molecule weight, glycosylation, and stability by using SEC-HPLC, SDS-PAGE, isoelectric focusing and mass spectrometry. The highly purified product shows IGF-1 binding activity and inhibits IGF-1-induced cell proliferation. This process not only provides a remarkable high expression at ~50mg/L and pure glycosylated mammalian rhIGFBP7, also highlights that transient gene expression technology is practical to be used for production and early development of recombinant non-IgG therapeutic proteins.
Asunto(s)
Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/efectos de los fármacos , Regiones no Traducidas 5'/genética , Secuencia de Aminoácidos , Proliferación Celular/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Vectores Genéticos , Glicosilación , Células HEK293 , Humanos , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Datos de Secuencia Molecular , Proteínas Recombinantes/farmacología , TransfecciónRESUMEN
PURPOSE: Interleukin-15 (IL-15) has significant potential in cancer immunotherapy as an activator of antitumor CD8 T and natural killer (NK) cells. The primary objectives of this trial were to determine safety, adverse event profile, dose-limiting toxicity, and maximum-tolerated dose of recombinant human IL-15 (rhIL-15) administered as a daily intravenous bolus infusion for 12 consecutive days in patients with metastatic malignancy. PATIENTS AND METHODS: We performed a first in-human trial of Escherichia coli-produced rhIL-15. Bolus infusions of 3.0, 1.0, and 0.3 µg/kg per day of IL-15 were administered for 12 consecutive days to patients with metastatic malignant melanoma or metastatic renal cell cancer. RESULTS: Flow cytometry of peripheral blood lymphocytes revealed dramatic efflux of NK and memory CD8 T cells from the circulating blood within minutes of IL-15 administration, followed by influx and hyperproliferation yielding 10-fold expansions of NK cells that ultimately returned to baseline. Up to 50-fold increases of serum levels of multiple inflammatory cytokines were observed. Dose-limiting toxicities observed in patients receiving 3.0 and 1.0 µg/kg per day were grade 3 hypotension, thrombocytopenia, and elevations of ALT and AST, resulting in 0.3 µg/kg per day being determined the maximum-tolerated dose. Indications of activity included clearance of lung lesions in two patients. CONCLUSION: IL-15 could be safely administered to patients with metastatic malignancy. IL-15 administration markedly altered homeostasis of lymphocyte subsets in blood, with NK cells and γδ cells most dramatically affected, followed by CD8 memory T cells. To reduce toxicity and increase efficacy, alternative dosing strategies have been initiated, including continuous intravenous infusions and subcutaneous IL-15 administration.
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Linfocitos T CD4-Positivos/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Interleucina-15/uso terapéutico , Células Asesinas Naturales/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Área Bajo la Curva , Linfocitos T CD4-Positivos/metabolismo , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Femenino , Fiebre/inducido químicamente , Humanos , Infusiones Intravenosas , Interleucina-15/efectos adversos , Interleucina-15/genética , Células Asesinas Naturales/metabolismo , Activación de Linfocitos/efectos de los fármacos , Masculino , Tasa de Depuración Metabólica , Persona de Mediana Edad , Náusea/inducido químicamente , Metástasis de la Neoplasia , Neoplasias/inmunología , Neoplasias/metabolismo , Neutropenia/inducido químicamente , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/uso terapéutico , Resultado del Tratamiento , Adulto JovenRESUMEN
In the present study, Hu-Mikß1, a humanized mAb directed at the shared IL-2/IL-15Rß subunit (CD122) was evaluated in patients with T-cell large granular lymphocytic (T-LGL) leukemia. Hu-Mikß1 blocked the trans presentation of IL-15 to T cells expressing IL-2/IL-15Rß and the common γ-chain (CD132), but did not block IL-15 action in cells that expressed the heterotrimeric IL-15 receptor in cis. There was no significant toxicity associated with Hu-Mikß1 administration in patients with T-LGL leukemia, but no major clinical responses were observed. One patient who had previously received murine Mikß1 developed a measurable Ab response to the infused Ab. Nevertheless, the safety profile of this first in-human study of the humanized mAb to IL-2/IL-15Rß (CD122) supports its evaluation in disorders such as refractory celiac disease, in which IL-15 and its receptor have been proposed to play a critical role in the pathogenesis and maintenance of disease activity.
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Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales/administración & dosificación , Subunidad beta del Receptor de Interleucina-2/inmunología , Leucemia Linfocítica Granular Grande/inmunología , Leucemia Linfocítica Granular Grande/terapia , Anciano , Anciano de 80 o más Años , Animales , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales Humanizados/efectos adversos , Anticuerpos Monoclonales Humanizados/inmunología , División Celular/inmunología , Línea Celular Tumoral , Femenino , Humanos , Inyecciones Intravenosas , Subunidad gamma Común de Receptores de Interleucina/inmunología , Interleucina-15/genética , Interleucina-15/inmunología , Subunidad beta del Receptor de Interleucina-2/genética , Macaca fascicularis , Masculino , Ratones , Persona de Mediana Edad , ARN Mensajero/metabolismo , Resultado del TratamientoRESUMEN
Immunotoxins are rationally designed cancer targeting and killing agents. Disulfide stabilized antibody Fv portion-toxin conjugates (dsFv-toxin) are third generation immunotoxins containing only the antibody fragment variable portions and a toxin fused to the V(H) or V(L). Pseudomonas exotoxin fragment (PE-38) is a commonly used toxin in immunotoxin clinical trials. dsFv-toxin purification was previously published, but the recovery was not satisfactory. This report describes the development of a cGMP production process of the dsFv-toxin that incorporated a novel purification method. The method has been successfully applied to the clinical manufacturing of two dsFv-PE38 immunotoxins, MR1-1 targeting EGFRvIII and HA22 targeting CD22. The two subunits, V(L) and V(H) PE-38 were expressed separately in Escherichia coli using recombinant technology. Following cell lysis, inclusion bodies were isolated from the biomass harvested from fermentation in animal source component-free media. The dsFv-toxin was formed after denaturation and refolding, and subsequently purified to homogeneity through ammonium sulfate precipitation, hydrophobic interaction and ion-exchange chromatography steps. It was shown, in a direct comparison experiment using MR1-1 as model protein, that the recovery from the new purification method was improved three times over that from previously published method. The improved recovery was also demonstrated during the clinical production of two dsFv-PE38 immunotoxins-MR1-1 and HA22.
Asunto(s)
Anticuerpos/química , Anticuerpos/aislamiento & purificación , Disulfuros/química , Escherichia coli/metabolismo , Exotoxinas/biosíntesis , Pseudomonas/química , GMP Cíclico/metabolismo , Escherichia coli/genética , Exotoxinas/genéticaRESUMEN
Interleukin 15 (IL-15) has shown remarkable biological properties of promoting NK- and T-cell activation and proliferation, as well as enhancing antitumor immunity of CD8(+) T cells in preclinical models. Here, we report the development of an E. coli cell line to express recombinant human Interleukin-15 (rhIL-15) for clinical manufacturing. Human IL-15 cDNA sequence was inserted into a pET28b plasmid and expressed in several E. coli BL21 strains. Through product quality comparisons among several E. coli strains, including E. coli BL21(DE3), BL21(DE3)pLysS, BLR(DE3)pLysS, and BL21-AI, E. coli BL21-AI was selected for clinical manufacturing. Expression optimization was carried out at shake flask and 20-L fermenter scales, and the product was expressed as inclusion bodies that were solubilized, refolded, and purified to yield active rhIL-15. Stop codons of the expression construct were further investigated after 15-20% of the purified rhIL-15 showed an extraneous peak corresponding to an extra tryptophan residue based on peptide mapping and mass spectrometry analysis. It was determined that the presence of an extra tryptophan was due to a stop codon wobble effect, which could be eliminated by replacing TGA (opal) stop codon with TAA (ochre). As a novel strategy, a simple method of demonstrating lack of tRNA suppressors in the production host cells was developed to validate the cells in this study. The E. coli BL21-AI cells containing the rhIL-15 coding sequence with a triplet stop codon TAATAATGA were banked for further clinical manufacturing.
Asunto(s)
Codón de Terminación , Escherichia coli/genética , Interleucina-15/genética , Ingeniería de Proteínas , Proliferación Celular/efectos de los fármacos , Escherichia coli/metabolismo , Expresión Génica , Humanos , Interleucina-15/metabolismo , Interleucina-15/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
IL-15 promotes activation and maintenance of natural killer (NK) and CD8(+) T effector memory (T(EM)) cells, making it a potential immunotherapeutic agent for the treatment of cancer and immunodeficiency states. Here we report the immunologic effects of 3 different IL-15 dosing strategies in Rhesus macaques. IL-15 at a dose of 20 µg/kg/d administered by continuous intravenous infusion for 10 days resulted in a massive (100-fold) expansion of CD8(+) T(EM) cells in the peripheral blood. In contrast, the administration of 20-40 µg/kg/d of IL-15 by subcutaneous injection resulted in a more modest (10-fold) expansion of CD8(+) T(EM) cells. NK expansion was similar in both the continuous intravenous and daily subcutaneous treatment groups. The observation that IL-15 administered by continuous intravenous infusion is able to induce markedly greater expansions of CD8(+) T(EM) cells than the same dose administered by other routes may have important implications for clinical development of this cytokine.
Asunto(s)
Linfocitos T CD8-positivos/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Memoria Inmunológica/efectos de los fármacos , Interleucina-15/farmacología , Animales , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Relación Dosis-Respuesta a Droga , Humanos , Memoria Inmunológica/inmunología , Infusiones Intravenosas , Inyecciones Subcutáneas , Interleucina-15/administración & dosificación , Interleucina-15/farmacocinética , Células Asesinas Naturales/citología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Macaca mulattaRESUMEN
IL-15 uses the heterotrimeric receptor IL-2/IL-15Rß and the γ chain shared with IL-2 and the cytokine-specific IL-15Rα. Although IL-15 shares actions with IL-2 that include activation of natural killer (NK) and CD8 T cells, IL-15 is not associated with capillary leak syndrome, activation-induced cell death, or with a major effect on the number of functional regulatory T cells. To prepare for human trials to determine whether IL-15 is superior to IL-2 in cancer therapy, recombinant human IL-15 (rhIL-15) was produced under current good manufacturing practices. A safety study in rhesus macaques was performed in 4 groups of 6 animals each that received vehicle diluent control or rhIL-15 at 10, 20, or 50 µg/kg/d IV for 12 days. The major toxicity was grade 3/4 transient neutropenia. Bone marrow examinations demonstrated increased marrow cellularity, including cells of the neutrophil series. Furthermore, neutrophils were observed in sinusoids of enlarged livers and spleens, suggesting that IL-15 mediated neutrophil redistribution from the circulation to tissues. The observation that IL-15 administration was associated with increased numbers of circulating NK and CD8 central and effector-memory T cells, in conjunction with efficacy studies in murine tumor models, supports the use of multiple daily infusions of rhIL-15 in patients with metastatic malignancies.
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Interleucina-15/toxicidad , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/inmunología , Antineoplásicos/farmacocinética , Antineoplásicos/toxicidad , Coagulación Sanguínea/efectos de los fármacos , Médula Ósea/efectos de los fármacos , Médula Ósea/patología , Humanos , Inmunoterapia , Infusiones Intravenosas , Interleucina-15/administración & dosificación , Interleucina-15/inmunología , Interleucina-15/farmacocinética , Hígado/efectos de los fármacos , Hígado/patología , Macaca mulatta , Neoplasias/inmunología , Neoplasias/terapia , Neutropenia/sangre , Neutropenia/inducido químicamente , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/toxicidadRESUMEN
Interleukin-15 (IL-15) is a cytokine with potential therapeutic application in individuals with cancer or immunodeficiency to promote natural killer (NK)- and T-cell activation and proliferation or in vaccination protocols to generate long-lived memory T cells. Here we report that 10-50 µg/kg IL-15 administered intravenously daily for 12 days to rhesus macaques has both short- and long-lasting effects on T-cell homeostasis. Peripheral blood lymphopenia preceded a dramatic expansion of NK cells and memory CD8 T cells in the circulation, particularly a 4-fold expansion of central memory CD8 T cells and a 6-fold expansion of effector memory CD8 T cells. This expansion is a consequence of their activation in multiple tissues. A concomitant inverted CD4/CD8 T-cell ratio was observed throughout the body at day 13, a result of preferential CD8 expansion. Expanded T- and NK-cell populations declined in the blood soon after IL-15 was stopped, suggesting migration to extralymphoid sites. By day 48, homeostasis appears restored throughout the body, with the exception of the maintenance of an inverted CD4/CD8 ratio in lymph nodes. Thus, IL-15 generates a dramatic expansion of short-lived memory CD8 T cells and NK cells in immunocompetent macaques and has long-term effects on the balance of CD4(+) and CD8(+) T cells.
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Interleucina-15/administración & dosificación , Interleucina-15/inmunología , Linfocitos/inmunología , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Proliferación Celular , Citocinas/inmunología , Factores de Transcripción Forkhead/inmunología , Humanos , Memoria Inmunológica , Subunidad alfa del Receptor de Interleucina-2/inmunología , Subunidad alfa del Receptor de Interleucina-7/inmunología , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Activación de Linfocitos , Linfocitos/citología , Macaca mulatta , Células T Asesinas Naturales/citología , Células T Asesinas Naturales/inmunología , Receptores de IgG/inmunología , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunologíaRESUMEN
A colorimetric cell proliferation assay using soluble tetrazolium salt [(CellTiter 96(R) Aqueous One Solution) cell proliferation reagent, containing the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) and an electron coupling reagent phenazine ethosulfate], was optimized and qualified for quantitative determination of IL-15 dependent CTLL-2 cell proliferation activity. An in-house recombinant Human (rHu)IL-15 reference lot was standardized (IU/mg) against an international reference standard. Specificity of the assay for IL-15 was documented by illustrating the ability of neutralizing anti-IL-15 antibodies to block the product specific CTLL-2 cell proliferation and the lack of blocking effect with anti-IL-2 antibodies. Under the defined assay conditions, the linear dose-response concentration range was between 0.04 and 0.17ng/ml of the rHuIL-15 produced in-house and 0.5-3.0IU/ml for the international standard. Statistical analysis of the data was performed with the use of scripts written in the R Statistical Language and Environment utilizing a four-parameter logistic regression fit analysis procedure. The overall variation in the ED(50) values for the in-house reference standard from 55 independent estimates performed over the period of 1year was 12.3% of the average. Excellent intra-plate and within-day/inter-plate consistency was observed for all four parameter estimates in the model. Different preparations of rHuIL-15 showed excellent intra-plate consistency in the parameter estimates corresponding to the lower and upper asymptotes as well as to the 'slope' factor at the mid-point. The ED(50) values showed statistically significant differences for different lots and for control versus stressed samples. Three R-scripts improve data analysis capabilities allowing one to describe assay variations, to draw inferences between data sets from formal statistical tests, and to set up improved assay acceptance criteria based on comparability and consistency in the four parameters of the model. The assay is precise, accurate and robust and can be fully validated. Applications of the assay were established including process development support, release of the rHuIL-15 product for pre-clinical and clinical studies, and for monitoring storage stability.