RESUMEN
FLT3 (FMS-like tyrosine kinase 3), expressed on the surface of acute myeloid leukemia (AML) blasts, is a promising AML target, given its role in the development and progression of leukemia, and its limited expression in tissues outside the hematopoietic system. Small molecule FLT3 kinase inhibitors have been developed, but despite having clinical efficacy, they are effective only on a subset of patients and associated with high risk of relapse. A durable therapy that can target a wider population of AML patients is needed. Here, we developed an anti-FLT3-CD3 immunoglobulin G (IgG)-based bispecific antibody (7370) with a high affinity for FLT3 and a long half-life, to target FLT3-expressing AML blasts, irrespective of FLT3 mutational status. We demonstrated that 7370 has picomolar potency against AML cell lines in vitro and in vivo. 7370 was also capable of activating T cells from AML patients, redirecting their cytotoxic activity against autologous blasts at low effector-to-target (E:T) ratio. Additionally, under our dosing regimen, 7370 was well tolerated and exhibited potent efficacy in cynomolgus monkeys by inducing complete but reversible depletion of peripheral FLT3+ dendritic cells (DCs) and bone marrow FLT3+ stem cells and progenitors. Overall, our results support further clinical development of 7370 to broadly target AML patients.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , Antineoplásicos Inmunológicos/farmacología , Complejo CD3/antagonistas & inhibidores , Hematopoyesis/efectos de los fármacos , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Animales , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/uso terapéutico , Antineoplásicos Inmunológicos/química , Antineoplásicos Inmunológicos/uso terapéutico , Médula Ósea/efectos de los fármacos , Médula Ósea/metabolismo , Médula Ósea/patología , Complejo CD3/química , Línea Celular Tumoral , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Humanos , Inmunoglobulina G/farmacología , Inmunofenotipificación , Leucemia Mieloide Aguda , Depleción Linfocítica , Macaca fascicularis , Ratones , Modelos Moleculares , Dominios Proteicos/efectos de los fármacos , Relación Estructura-Actividad , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa 3 Similar a fms/químicaRESUMEN
Antibody-drug conjugates (ADCs) are promising therapies for haematological cancers. Historically, their therapeutic benefit is due to ADC targeting of lineage-restricted antigens. The C-X-C motif chemokine receptor 4 (CXCR4) is attractive for targeted therapy of haematological cancers, given its expression in multiple tumour types and role in cancer "homing" to bone marrow. However, CXCR4 is also expressed in haematopoietic cells and other normal tissues, raising safety challenges to the development of anti-CXCR4 ADCs for cancer treatment. Here, we designed the first anti-CXCR4 ADC with favourable therapeutic index, effective in xenografts of haematopoietic cancers resistant to standard of care and anti-CXCR4 antibodies. We screened multiple ADC configurations, by varying type of linker-payload, drug-to-antibody ratio (DAR), affinity and Fc format. The optimal ADC bears a non-cleavable linker, auristatin as payload at DAR = 4 and a low affinity antibody with effector-reduced Fc. Contrary to other drugs targeting CXCR4, anti-CXCR4 ADCs effectively eliminated cancer cells as monotherapy, while minimizing leucocytosis. The optimal ADC selectively eliminated CXCR4+ cancer cells in solid tumours, but showed limited toxicity to normal CXCR4+ tissues, sparing haematopoietic stem cells and progenitors. Our work provides proof-of-concept that through empirical ADC design, it is possible to target proteins with broad normal tissue expression.
Asunto(s)
Antineoplásicos Inmunológicos , Diseño de Fármacos , Inmunoconjugados , Receptores CXCR4/inmunología , Animales , Antineoplásicos Inmunológicos/administración & dosificación , Antineoplásicos Inmunológicos/efectos adversos , Antineoplásicos Inmunológicos/síntesis química , Antineoplásicos Inmunológicos/química , Células CHO , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Células Cultivadas , Cricetinae , Cricetulus , Desoxicitidina/administración & dosificación , Desoxicitidina/efectos adversos , Desoxicitidina/análogos & derivados , Femenino , Humanos , Inmunoconjugados/administración & dosificación , Inmunoconjugados/efectos adversos , Inmunoconjugados/química , Fragmentos Fab de Inmunoglobulinas/administración & dosificación , Fragmentos Fab de Inmunoglobulinas/efectos adversos , Fragmentos Fab de Inmunoglobulinas/química , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Ratones Transgénicos , Modelos Moleculares , Estructura Terciaria de Proteína , Receptores CXCR4/antagonistas & inhibidores , Resultado del Tratamiento , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
This manuscript aims to provide insights and updates on emerging technologies from a throughput and multiplexing perspective and to update readers on changes in previously reported technologies. The technologies discussed range from nascent (ultrasensitive Cira, Intellicyt®, Dynaxi and Captsure™) to the more established (Ella and SQIDlite™). For the nascent technologies, there was an emphasis on user interviews and reviews, where available, to help provide an unbiased view to our readers. For the Ella, a review of published user data as well as author and other user experiences are summarized. Due to their emergent nature, all the technologies described are applicable in the early drug development stage, may require an upfront investment of capital and may not perform as expected.
Asunto(s)
Citocinas/sangre , Drogas en Investigación/análisis , Inmunoensayo , Técnicas Analíticas Microfluídicas , Anticuerpos Monoclonales/química , Automatización de Laboratorios , Humanos , Límite de DetecciónRESUMEN
An in-depth evaluation of the Quanterix© Simoa™ platform was undertaken by scientists from the AAPS Emerging Technologies Focus Group to determine the overall performance of the technology as well as provide guidance to future users. In order to test the platform in a non-GLP bioanalytical setting, a cross-site evaluation of the Quanterix IL-6 biomarker kit was performed. Parameters tested during this evaluation included sensitivity, accuracy and precision, and parallelism in human serum from normal individuals. The results demonstrated improved sensitivity compared to the claimed sensitivity of other commercially available IL-6 kits and showed excellent site-to-site reproducibility. Observed issues included difficulties with system reliability and a lack of parallelism and specificity in a subset of samples. Overall, these results demonstrate that while there are challenges to the Simoa platform this technology offers automation capabilities and excellent sensitivity that enhance bioanalysis especially of low-abundance analytes.
Asunto(s)
Interleucina-6/sangre , Juego de Reactivos para Diagnóstico , Humanos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
There are many sources of analytical variability in ligand binding assays (LBA). One strategy to reduce variability has been duplicate analyses. With recent advances in LBA technologies, it is conceivable that singlet analysis is possible. We retrospectively evaluated singlet analysis using Gyrolab data. Relative precision of duplicates compared to singlets was evaluated using 60 datasets from toxicokinetic (TK) or pharmacokinetic (PK) studies which contained over 23,000 replicate pairs composed of standards, quality control (QC), and animal samples measured with 23 different bioanalytical assays. The comparison was first done with standard curve and QCs followed by PK parameters (i.e., Cmax and AUC). Statistical analyses were performed on combined duplicate versus singlets using a concordance correlation coefficient (CCC), a measurement used to assess agreement. Variance component analyses were conducted on PK estimates to assess the relative analytical and biological variability. Overall, 97.5% of replicate pairs had a %CV of <11% and 50% of the results had a %CV of ≤1.38%. There was no observable bias in concentration comparing the first replicate with the second (CCC of 0.99746 and accuracy value of 1). The comparison of AUC and Cmax showed no observable difference between singlet and duplicate (CCC for AUC and Cmax >0.99999). Analysis of variance indicated an AUC inter-subject variability 35.3-fold greater than replicate variability and 8.5-fold greater for Cmax. Running replicates from the same sample will not significantly reduce variation or change PK parameters. These analyses indicated the majority of variance was inter-subject and supported the use of a singlet strategy.