RESUMEN
Nucleic acid-sensing pathways play critical roles in innate immune activation through the production of type I interferon (IFN-I) and proinflammatory cytokines. These factors are required for effective antitumor immune responses. Pharmacological modulators of the pre-mRNA spliceosome splicing factor 3b subunit 1 (SF3B1) are under clinical investigation as cancer cytotoxic agents. However, potential roles of these agents in aberrant RNA generation and subsequent RNA-sensing pathway activation have not been studied. In this study, we observed that SF3B1 pharmacological modulation using pladienolide B (Plad B) induces production of aberrant RNA species and robust IFN-I responses via engagement of the dsRNA sensor retinoic acid-inducible gene I (RIG-I) and downstream interferon regulatory factor 3. We found that Plad B synergized with canonical RIG-I agonism to induce the IFN-I response. In addition, Plad B induced NF-κB responses and secretion of proinflammatory cytokines and chemokines. Finally, we showed that cancer cells bearing the hotspot SF3B1K700E mutation, which leads to global aberrant splicing, had enhanced IFN-I response to canonical RIG-I agonism. Together, these results demonstrate that pharmacological modulation of SF3B1 in cancer cells can induce an enhanced IFN-I response dependent on RIG-I expression. The study suggests that spliceosome modulation may not only induce direct cancer cell cytotoxicity but also initiate an innate immune response via activation of RNA-sensing pathways.
Asunto(s)
Proteína 58 DEAD Box/metabolismo , Interferón Tipo I/metabolismo , Fosfoproteínas/metabolismo , Precursores del ARN/metabolismo , Factores de Empalme de ARN/metabolismo , Receptores Inmunológicos/metabolismo , Empalmosomas/metabolismo , Células A549 , Sustitución de Aminoácidos , Animales , Proteína 58 DEAD Box/genética , Humanos , Interferón Tipo I/genética , Ratones , Mutación Missense , Fosfoproteínas/genética , Precursores del ARN/genética , Factores de Empalme de ARN/genética , Receptores Inmunológicos/genética , Empalmosomas/genética , Células THP-1RESUMEN
A potent class of DNA-damaging agents, natural product bis-intercalator depsipeptides (NPBIDs), was evaluated as ultrapotent payloads for use in antibody-drug conjugates (ADCs). Detailed investigation of potency (both in cells and via biophysical characterization of DNA binding), chemical tractability, and in vitro and in vivo stability of the compounds in this class eliminated a number of potential candidates, greatly reducing the complexity and resources required for conjugate preparation and evaluation. This effort yielded a potent, stable, and efficacious ADC, PF-06888667, consisting of the bis-intercalator, SW-163D, conjugated via an N-acetyl-lysine-valine-citrulline- p-aminobenzyl alcohol- N, N-dimethylethylenediamine (AcLysValCit-PABC-DMAE) linker to an engineered variant of the anti-Her2 mAb, trastuzumab, catalyzed by transglutaminase.
Asunto(s)
Productos Biológicos/química , Depsipéptidos/química , Inmunoconjugados/química , Sustancias Intercalantes/química , Animales , Antineoplásicos Inmunológicos/química , Línea Celular Tumoral , ADN/química , Depsipéptidos/sangre , Depsipéptidos/farmacocinética , Equinomicina/química , Genes erbB-2 , Semivida , Xenoinjertos , Humanos , Ratones , Trastuzumab/químicaRESUMEN
Novel neolymphostin-based antibody-drug conjugate (ADC) precursors were synthesized either through amide couplings between both cleavable and non-cleavable linkers and neolymphostin derivatives, or through Cu(I)-catalyzed acetylene-azide click cycloadditon between non-cleavable linkers and neolymphostin acetal derivatives. These precursors were site-specifically conjugated to cysteine mutant trastuzumab-A114C to provide neolymphostin-based ADCs. Preliminary in vitro data indicated that the corresponding ADCs were active against HER2-expressing tumor cell lines, thus providing a proof-of-concept for using neolymphostin as ADC-based anticancer agents.
Asunto(s)
Aminoquinolinas/farmacología , Antineoplásicos/farmacología , Inmunoconjugados/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Pirroles/farmacología , Trastuzumab/farmacología , Aminoquinolinas/síntesis química , Antineoplásicos/síntesis química , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Mutación , Inhibidores de las Quinasa Fosfoinosítidos-3/síntesis química , Prueba de Estudio Conceptual , Pirroles/síntesis química , Trastuzumab/genéticaRESUMEN
The objective of this manuscript was to establish in vitro-in vivo correlation (IVIVC) between the in vitro efficacy and in vivo efficacy of antibody drug conjugates (ADCs), using a PK/PD modeling approach. Nineteen different ADCs were used to develop IVIVC. In vitro efficacy of ADCs was evaluated using a kinetic cell cytotoxicity assay. The cytotoxicity data obtained from in vitro studies was characterized using a novel mathematical model, parameter estimates from which were used to derive an in vitro efficacy matrix for each ADC, termed as 'in vitro tumor static concentration' (TSCin vitro). TSCin vitro is a theoretical concentration at continuous exposure of which the number of cells will neither increase nor decrease, compared to the initial cell number in the experiment. The in vivo efficacy of ADCs was evaluated using tumor growth inhibition (TGI) studies performed on human tumor xenograft bearing mice. The TGI data obtained from in vivo studies was characterized using a PK/PD model, parameter estimates from which were used to derive an in vivo efficacy matrix for each ADC, termed as 'in vivo tumor static concentration' (TSCin vivo). TSCin vivo is a theoretical concentration if one were to maintain in the plasma of a tumor bearing mouse, the tumor volume will neither increase nor decrease compared to the initial tumor volume. Comparison of the TSCin vitro and TSCin vivo values from 19 ADCs provided a linear and positive IVIVC. The Spearman's rank correlation coefficient for TSCin vitro and TSCin vivo was found to be 0.82. On average TSCin vivo was found to be ~ 27 times higher than TSCin vitro. The reasonable IVIVC for ADCs suggests that in vitro efficacy data was correctly able to differentiate ADCs for their in vivo efficacy. Thus, IVIVC can be used as a tool to triage ADC molecules in the discovery stage, thereby preventing unnecessary scaling-up of ADCs and waste of time and resources. An ability to predict the concentration of ADC that is efficacious in vivo using the in vitro data can also help in optimizing the experimental design of preclinical efficacy studies. As such, the novel PK/PD modeling method presented here to establish IVIVC for ADCs holds promise, and should be evaluated further using diverse set of cell lines and anticancer agents.
Asunto(s)
Anticuerpos/metabolismo , Anticuerpos/farmacología , Antineoplásicos/farmacología , Antineoplásicos/farmacocinética , Animales , Femenino , Humanos , Inmunoconjugados/farmacocinética , Inmunoconjugados/farmacología , Ratones , Ratones Desnudos , Modelos Biológicos , Neoplasias/tratamiento farmacológico , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
The focus of the antibody-drug conjugate (ADC) field is shifting toward development of site-specific, next-generation ADCs to address the issue of heterogeneity, metabolic instability, conjugatability, and less than ideal therapeutic index associated with the conventional (heterogeneous) ADCs. It is evident from the recent literature that the site of conjugation, the structure of the linker, and the physicochemical properties of the linker-payload all have a significant impact on the safety and efficacy of the resulting ADCs. Screening multiple linker-payloads on multiple sites of an antibody presents a combinatorial problem that necessitates high-throughput conjugation and purification methodology to identify ADCs with the best combination of site and payload. Toward this end, we developed a protein A/L-based solid-phase, site-specific conjugation and purification method that can be used to generate site-specific ADCs in a 96-well plate format. This solid-phase method has been shown to be versatile because of its compatibility with various conjugation functional handles such as maleimides, haloacetamides, copper free click substrates, and transglutaminase substrates. The application of this methodology was further expanded to generate dual labeled, site-specific antibody and Fab conjugates.
Asunto(s)
Anticuerpos/química , Inmunoconjugados/química , Fragmentos Fab de Inmunoglobulinas/químicaRESUMEN
Antibody-drug conjugates (ADC) are currently an active area of research, focused primarily on oncology therapeutics, but also to a limited extent on other areas such as infectious disease. The success of this type of targeted drug delivery is dependent upon many factors, one of which is the performance of the linker in releasing an active drug moiety under the appropriate conditions. As a tool in the development of linker/payload chemistry, we have developed an in vitro method for the identification of payload species released from ADCs in the presence of lysosomal enzymes. This method utilizes commercially available human liver S9 fraction as the source of these enzymes, and this has certain advantages over lysosomal fractions or purified enzymes. This article describes the characterization and performance of this assay with multiple ADCs composed of known and novel linkers and payloads. Additionally, we report the observation of incomplete degradation of mAb protein chains by lysosomal enzymes in vitro, believed to be the first report of this phenomenon involving an ADC therapeutic.
Asunto(s)
Portadores de Fármacos/química , Liberación de Fármacos , Inmunoconjugados/química , Animales , Catepsina B/metabolismo , Línea Celular Tumoral , Portadores de Fármacos/metabolismo , Humanos , Hígado/citología , Lisosomas/enzimología , Espectrometría de Masas , Ratones , RatasRESUMEN
There is a considerable ongoing work to identify new cytotoxic payloads that are appropriate for antibody-based delivery, acting via mechanisms beyond DNA damage and microtubule disruption, highlighting their importance to the field of cancer therapeutics. New modes of action will allow a more diverse set of tumor types to be targeted and will allow for possible mechanisms to evade the drug resistance that will invariably develop to existing payloads. Spliceosome inhibitors are known to be potent antiproliferative agents capable of targeting both actively dividing and quiescent cells. A series of thailanstatin-antibody conjugates were prepared in order to evaluate their potential utility in the treatment of cancer. After exploring a variety of linkers, we found that the most potent antibody-drug conjugates (ADCs) were derived from direct conjugation of the carboxylic acid-containing payload to surface lysines of the antibody (a "linker-less" conjugate). Activity of these lysine conjugates was correlated to drug-loading, a feature not typically observed for other payload classes. The thailanstatin-conjugates were potent in high target expressing cells, including multidrug-resistant lines, and inactive in nontarget expressing cells. Moreover, these ADCs were shown to promote altered splicing products in N87 cells in vitro, consistent with their putative mechanism of action. In addition, the exposure of the ADCs was sufficient to result in excellent potency in a gastric cancer xenograft model at doses as low as 1.5 mg/kg that was superior to the clinically approved ADC T-DM1. The results presented herein therefore open the door to further exploring splicing inhibition as a potential new mode-of-action for novel ADCs.
Asunto(s)
Productos Biológicos/química , Inmunoconjugados/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Ácidos Carboxílicos/química , Línea Celular Tumoral , Transformación Celular Neoplásica , Cisteína/química , Humanos , Inmunoconjugados/farmacocinética , Inmunoconjugados/farmacología , Lisina/química , Maleimidas/química , Ratones , Piranos/química , Distribución TisularRESUMEN
It was by way of total synthesis that the issues concerning the stereostructure of leiodermatolide (1) have recently been solved; with the target now being unambiguously defined, the mission of synthesis changes as to secure a meaningful supply of this exceedingly scarce natural product derived from a deep-sea sponge. To this end, a scalable route of 19 steps (longest linear sequence) has been developed, which features a catalytic asymmetric propargylation of a highly enolizable ß-keto-lactone, a ring closing alkyne metathesis and a modified Stille coupling as the key transformations. Deliberate digression from this robust blueprint brought a first set of analogues into reach, which allowed the lead qualities of 1 to be assessed. The acquired biodata show that 1 is a potent cytotoxin in human tumor cell proliferation assays, distinguished by GI50 values in the ≤3 nM range even for cell lines expressing the Pgp efflux transporter. Studies with human U2OS cells revealed that 1 causes mitotic arrest, micronucleus induction, centrosome amplification and tubulin disruption, even though no evidence for direct tubulin binding has been found in cell-free assays; moreover, the compound does not seem to act through kinase inhibition. Indirect evidence points at centrosome declustering as a possible mechanism of action, which provides a potentially rewarding outlook in that centrosome declustering agents hold promise of being inherently selective for malignant over healthy human tissue.
Asunto(s)
Macrólidos/síntesis química , Macrólidos/farmacología , Macrólidos/química , Estructura MolecularRESUMEN
The stability of the connection between the antibody and the toxin can have a profound impact on ADC safety and efficacy. There has been increasing evidence in recent years that maleimide-based ADCs are prone to payload loss via a retro-Michael type reaction. Herein, we report a mild method for the hydrolysis of the succinimide-thioether ring which results in a "ring-opened" linker. ADCs containing this hydrolyzed succinimide linker show equivalent cytotoxicity, improved in vitro stability, improved PK exposure, and improved efficacy as compared to their nonhydrolyzed counterparts. This method offers a simple way to improve the stability, exposure, and efficacy of maleimide-based ADCs.
Asunto(s)
Inmunotoxinas/química , Succinimidas/química , Sulfuros/química , Animales , Línea Celular Tumoral , Estabilidad de Medicamentos , Humanos , Hidrólisis , Inmunotoxinas/sangre , Inmunotoxinas/uso terapéutico , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Estabilidad ProteicaRESUMEN
Borrelidin (1) is a nitrile-containing bacterially derived polyketide that is a potent inhibitor of bacterial and eukaryotic threonyl-tRNA synthetases. We now report the discovery of borrelidin B (2), a tetrahydro-borrelidin derivative containing an aminomethyl group in place of the nitrile functionality in borrelidin. The discovery of this new metabolite has implications for both the biosynthesis of the nitrile group and the bioactivity of the borrelidin compound class. Screening in the SToPS assay for tRNA synthetase inhibition revealed that the nitrile moiety is essential for activity, while profiling using our in-house image-based cytological profiling assay demonstrated that 2 retains biological activity by causing a mitotic stall, even in the absence of the nitrile motif.
Asunto(s)
Nitrilos/síntesis química , Treonina-ARNt Ligasa/antagonistas & inhibidores , Aminoacil-ARNt Sintetasas/metabolismo , Alcoholes Grasos/química , Alcoholes Grasos/aislamiento & purificación , Alcoholes Grasos/farmacología , Estructura Molecular , Nitrilos/metabolismoRESUMEN
The spliceostatin class of natural products was reported to be potent cytotoxic agents via inhibition of the spliceosome, a key protein complex in the biosynthesis of mature mRNA. As part of an effort to discover novel leads for cancer chemotherapy, we re-examined this class of compounds from several angles, including fermentation of the producing strains, isolation and structure determination of new analogues, and semisynthetic modification. Accordingly, a group of spliceostatins were isolated from a culture broth of Burkholderia sp. FERM BP-3421, and their structures identified by analysis of spectroscopic data. Semisynthesis was performed on the major components 4 and 5 to generate ester and amide derivatives with improved in vitro potency. With their potent activity against tumor cells and unique mode of action, spliceostatins can be considered potential leads for development of cancer drugs.
Asunto(s)
Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Burkholderia/química , Piranos/aislamiento & purificación , Piranos/farmacología , Compuestos de Espiro/aislamiento & purificación , Compuestos de Espiro/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Estructura Molecular , Piranos/síntesis química , Piranos/química , ARN Mensajero/biosíntesis , Compuestos de Espiro/síntesis química , Compuestos de Espiro/química , Relación Estructura-ActividadRESUMEN
Lactimidomycin (1) was described in the literature as an exquisitely potent cell migration inhibitor. Encouraged by this claim, we developed a concise and scalable synthesis of this bipartite glutarimide-macrolide antibiotic, which relies on the power of ring-closing alkyne metathesis (RCAM) for the formation of the unusually strained 12-membered head group. Subsequent deliberate digression from the successful path to 1 also brought the sister compound isomigrastatin (2) as well as a series of non-natural analogues of these macrolides into reach. A careful biological re-evaluation of this compound collection showed 1 and progeny to be potently cytotoxic against a panel of cancer cell lines, even after one day of compound exposure; therefore any potentially specific effects on tumor cell migration were indistinguishable from the acute effect of cell death. No significant cell migration inhibition was observed at sub-toxic doses. Although these findings cannot be reconciled with some reports in the literature, they are in accord with the notion that lactimidomycin is primarily a ribosome-binder able to effectively halt protein biosynthesis at the translation stage.
Asunto(s)
Antibacterianos/síntesis química , Antibacterianos/farmacología , Macrólidos/síntesis química , Macrólidos/farmacología , Piperidonas/síntesis química , Piperidonas/farmacología , Antibacterianos/química , Movimiento Celular/efectos de los fármacos , Femenino , Humanos , Macrólidos/química , Espectroscopía de Resonancia Magnética , Piperidonas/químicaRESUMEN
In this Letter we describe the synthesis and biological evaluation of new benzosuberene analogs with structural modifications on the B-ring. The focus was initially to probe the chemical space around the B-ring C-8 position. This position was readily available for derivatization chemistry using our recently developed new synthesis for this compound class. Furthermore, we describe two new B-ring analogs, one containing a diene and the other a cyclic ether group. Both new analogs show excellent potencies in tumor cell proliferation assays. In addition, we describe molecular modeling studies that provide a binding rationale for reference compound 8 in the colchicine binding site using the known colchicine crystal structure. We also examine whether the cell based potency data obtained with selected new analogs are supported by modeling results.
Asunto(s)
Derivados del Benceno/química , Derivados del Benceno/toxicidad , Moduladores de Tubulina/síntesis química , Moduladores de Tubulina/toxicidad , Animales , Derivados del Benceno/metabolismo , Sitios de Unión , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Colchicina/análogos & derivados , Perros , Humanos , Microsomas Hepáticos/metabolismo , Simulación del Acoplamiento Molecular , Unión Proteica , Estructura Terciaria de Proteína , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/metabolismoRESUMEN
Nanoparticle (NP) technology holds significant promise to mediate targeted drug delivery to specific organs in the body. Understanding the 3D biodistribution of NPs in heterogeneous environments such as the tumor tissue can provide crucial information on efficacy, safety and potential clinical outcomes. Here we present a novel end-to-end workflow, VIOLA, which makes use of tissue clearing methodology in conjunction with high resolution imaging and advanced 3D image processing to quantify the spatiotemporal 3D biodistribution of fluorescently labeled ACCURIN® NPs. Specifically, we investigate the spatiotemporal biodistribution of NPs in three different murine tumor models (CT26, EMT6, and KPC-GEM) of increasing complexity and translational relevance. We have developed new endpoints to characterize NP biodistribution at multiple length scales. Our observations reveal that the macroscale NP biodistribution is spatially heterogeneous and exhibits a gradient with relatively high accumulation at the tumor periphery that progressively decreases towards the tumor core in all the tumor models. Microscale analysis revealed that NP extravasation from blood vessels increases in a time dependent manner and plateaus at 72 h post injection. Volumetric analysis and pharmacokinetic modeling of NP biodistribution in the vicinity of the blood vessels revealed that the local NP density exhibits a distance dependent spatiotemporal biodistribution which provide insights into the dynamics of NP extravasation in the tumor tissue. Our data represents a comprehensive analysis of NP biodistribution at multiple length scales in different tumor models providing unique insights into their spatiotemporal dynamics. Specifically, our results show that NPs exhibit a dynamic equilibrium with macroscale heterogeneity combined with microscale homogeneity.
Asunto(s)
Nanopartículas , Neoplasias , Viola , Animales , Ratones , Distribución Tisular , Sistemas de Liberación de MedicamentosRESUMEN
Extra domain B splice variant of fibronectin (EDB+FN) is an extracellular matrix protein (ECM) deposited by tumor-associated fibroblasts, and is associated with tumor growth, angiogenesis, and invasion. We hypothesized that EDB+FN is a safe and abundant target for therapeutic intervention with an antibody-drug conjugate (ADC). We describe the generation, pharmacology, mechanism of action, and safety profile of an ADC specific for EDB+FN (EDB-ADC). EDB+FN is broadly expressed in the stroma of pancreatic, non-small cell lung (NSCLC), breast, ovarian, head and neck cancers, whereas restricted in normal tissues. In patient-derived xenograft (PDX), cell-line xenograft (CLX), and mouse syngeneic tumor models, EDB-ADC, conjugated to auristatin Aur0101 through site-specific technology, demonstrated potent antitumor growth inhibition. Increased phospho-histone H3, a pharmacodynamic biomarker of response, was observed in tumor cells distal to the target site of tumor ECM after EDB-ADC treatment. EDB-ADC potentiated infiltration of immune cells, including CD3+ T lymphocytes into the tumor, providing rationale for the combination of EDB-ADC with immune checkpoint therapy. EDB-ADC and anti-PD-L1 combination in a syngeneic breast tumor model led to enhanced antitumor activity with sustained tumor regressions. In nonclinical safety studies in nonhuman primates, EDB-ADC had a well-tolerated safety profile without signs of either on-target toxicity or the off-target effects typically observed with ADCs that are conjugated through conventional conjugation methods. These data highlight the potential for EDB-ADC to specifically target the tumor microenvironment, provide robust therapeutic benefits against multiple tumor types, and enhance activity antitumor in combination with checkpoint blockade.
Asunto(s)
Neoplasias de la Mama , Inmunoconjugados , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Fibronectinas/metabolismo , Humanos , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Ratones , Neovascularización Patológica/metabolismo , Microambiente Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
CONTEXT: Mitotic kinase enzymes regulate critical stages of mitosis and are amenable to pharmacological inhibition. Since natural products have been a rich source of antimitotic inhibitors, we postulated that natural products would also provide effective inhibitors of mitotic kinases. OBJECTIVE: To explore unique marine and terrestrial natural product sources for new anticancer drug leads, we screened our natural product extract library for polo-like kinase-1 (Plk1) kinase inhibitors. MATERIALS AND METHODS: Extracts of the lichen Parmotrema sp. (Parmeliaceae) exhibited in vitro inhibitory activity. Bioassay-guided fractionation of the Parmotrema sp. extract led to the isolation of depside inhibitors. RESULTS: A new depside 1 has been isolated from the Sri Lankan lichen Parmotrema sp. along with the known metabolites 2 (ß-collatolic acid) and 3 (ß-alectoronic acid). The structure of depside 1 was elucidated by spectroscopic analysis. The three depsides 1-3 exhibited moderate inhibition of purified recombinant Plk1 kinase with IC50 of 2.8, 0.7, and 1.7 µM, respectively, at 1 µM ATP. Inhibitory activity was also observed at high concentrations of ATP, suggesting the potential for activity in a cellular environment. The depsides were also tested against a panel of 23 other recombinant kinases and were found to possess up to 30-fold selectivity toward Plk1. DISCUSSION AND CONCLUSION: These data suggest that the depsides 1-3 may serve as core structures that can be further explored as potential inhibitors of Plk1 and other kinases.
Asunto(s)
Ascomicetos , Proteínas de Ciclo Celular/antagonistas & inhibidores , Depsidos/farmacología , Líquenes , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/metabolismo , Depsidos/química , Depsidos/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/aislamiento & purificación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Sri Lanka , Quinasa Tipo Polo 1RESUMEN
PURPOSE: Mortality due to acute myeloid leukemia (AML) remains high, and the management of relapsed or refractory AML continues to be therapeutically challenging. The reapproval of Mylotarg, an anti-CD33-calicheamicin antibody-drug conjugate (ADC), has provided a proof of concept for an ADC-based therapeutic for AML. Several other ADCs have since entered clinical development of AML, but have met with limited success. We sought to develop a next-generation ADC for AML with a wide therapeutic index (TI) that overcomes the shortcomings of previous generations of ADCs. EXPERIMENTAL DESIGN: We compared the TI of our novel CD33-targeted ADC platform with other currently available CD33-targeted ADCs in preclinical models of AML. Next, using this next-generation ADC platform, we performed a head-to-head comparison of two attractive AML antigens, CD33 and CD123. RESULTS: Our novel ADC platform offered improved safety and TI when compared with certain currently available ADC platforms in preclinical models of AML. Differentiation between the CD33- and CD123-targeted ADCs was observed in safety studies conducted in cynomolgus monkeys. The CD33-targeted ADC produced severe hematologic toxicity, whereas minimal hematologic toxicity was observed with the CD123-targeted ADC at the same doses and exposures. The improved toxicity profile of an ADC targeting CD123 over CD33 was consistent with the more restricted expression of CD123 in normal tissues. CONCLUSIONS: We optimized all components of ADC design (i.e., leukemia antigen, antibody, and linker-payload) to develop an ADC that has the potential to translate into an effective new therapy against AML.
Asunto(s)
Gemtuzumab/uso terapéutico , Inmunoconjugados/uso terapéutico , Subunidad alfa del Receptor de Interleucina-3/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Lectina 3 Similar a Ig de Unión al Ácido Siálico/antagonistas & inhibidores , Animales , Antineoplásicos Inmunológicos/inmunología , Antineoplásicos Inmunológicos/farmacocinética , Antineoplásicos Inmunológicos/uso terapéutico , Área Bajo la Curva , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Gemtuzumab/inmunología , Gemtuzumab/farmacocinética , Células HL-60 , Humanos , Inmunoconjugados/inmunología , Inmunoconjugados/farmacocinética , Subunidad alfa del Receptor de Interleucina-3/inmunología , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/metabolismo , Macaca fascicularis , Ratones , Lectina 3 Similar a Ig de Unión al Ácido Siálico/inmunología , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
The approval of ado-trastuzumab emtansine (T-DM1) in HER2+ metastatic breast cancer validated HER2 as a target for HER2-specific antibody-drug conjugates (ADC). Despite its demonstrated clinical efficacy, certain inherent properties within T-DM1 hamper this compound from achieving the full potential of targeting HER2-expressing solid tumors with ADCs. Here, we detail the discovery of PF-06804103, an anti-HER2 ADC designed to have a widened therapeutic window compared with T-DM1. We utilized an empirical conjugation site screening campaign to identify the engineered ĸkK183C and K290C residues as those that maximized in vivo ADC stability, efficacy, and safety for a four drug-antibody ratio (DAR) ADC with this linker-payload combination. PF-06804103 incorporates the following novel design elements: (i) a new auristatin payload with optimized pharmacodynamic properties, (ii) a cleavable linker for optimized payload release and enhanced antitumor efficacy, and (iii) an engineered cysteine site-specific conjugation approach that overcomes the traditional safety liabilities of conventional conjugates and generates a homogenous drug product with a DAR of 4. PF-06804103 shows (i) an enhanced efficacy against low HER2-expressing breast, gastric, and lung tumor models, (ii) overcomes in vitro- and in vivo-acquired T-DM1 resistance, and (iii) an improved safety profile by enhancing ADC stability, pharmacokinetic parameters, and reducing off-target toxicities. Herein, we showcase our platform approach in optimizing ADC design, resulting in the generation of the anti-HER2 ADC, PF-06804103. The design elements of identifying novel sites of conjugation employed in this study serve as a platform for developing optimized ADCs against other tumor-specific targets.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Inmunoconjugados/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Gástricas/tratamiento farmacológico , Animales , Neoplasias de la Mama/patología , Femenino , Humanos , Inmunoconjugados/farmacología , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Neoplasias Gástricas/patologíaRESUMEN
Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) that has demonstrated clinical benefit for patients with HER2+ metastatic breast cancer; however, its clinical activity is limited by inherent or acquired drug resistance. The molecular mechanisms that drive clinical resistance to T-DM1, especially in HER2+ tumors, are not well understood. We used HER2+ cell lines to develop models of T-DM1 resistance using a cyclical dosing schema in which cells received T-DM1 in an "on-off" routine until a T-DM1-resistant population was generated. T-DM1-resistant N87 cells (N87-TM) were cross-resistant to a panel of trastuzumab-ADCs (T-ADCs) with non-cleavable-linked auristatins. N87-TM cells do not have a decrease in HER2 protein levels or an increase in drug transporter protein (e.g., MDR1) expression compared with parental N87 cells. Intriguingly, T-ADCs using auristatin payloads attached via an enzymatically cleavable linker overcome T-DM1 resistance in N87-TM cells. Importantly, N87-TM cells implanted into athymic mice formed T-DM1 refractory tumors that remain sensitive to T-ADCs with cleavable-linked auristatin payloads. Comparative proteomic profiling suggested enrichment in proteins that mediate caveolae formation and endocytosis in the N87-TM cells. Indeed, N87-TM cells internalize T-ADCs into intracellular caveolin-1 (CAV1)-positive puncta and alter their trafficking to the lysosome compared with N87 cells. T-DM1 colocalization into intracellular CAV1-positive puncta correlated with reduced response to T-DM1 in a panel of HER2+ cell lines. Together, these data suggest that caveolae-mediated endocytosis of T-DM1 may serve as a novel predictive biomarker for patient response to T-DM1. Mol Cancer Ther; 17(1); 243-53. ©2017 AACR.
Asunto(s)
Antineoplásicos Inmunológicos/uso terapéutico , Endocitosis/efectos de los fármacos , Trastuzumab/uso terapéutico , Animales , Antineoplásicos Inmunológicos/farmacología , Caveolas , Resistencia a Antineoplásicos , Femenino , Humanos , Masculino , Ratones , Trastuzumab/farmacologíaRESUMEN
Resistance to paclitaxel-based therapy is frequently encountered in the clinic. The mechanisms of intrinsic or acquired paclitaxel resistance are not well understood. We sought to characterize the resistance mechanisms that develop upon chronic exposure of a cancer cell line to paclitaxel in the presence of the P-glycoprotein reversal agent, CL-347099. The epidermoid tumor line KB-3-1 was exposed to increasing concentrations of paclitaxel and 5 micromol/L CL-347099 for up to 1 year. Cells grown in 15 nmol/L paclitaxel plus CL-347099 (KB-15-PTX/099) developed 18-fold resistance to paclitaxel and were dependent upon paclitaxel for maximal growth. They grew well and retained resistance to paclitaxel when grown in athymic mice. Cross-resistance (3- to 5-fold) was observed in tissue culture to docetaxel, the novel taxane MAC-321, and epothilone B. Collateral sensitivity (approximately 3-fold) was observed to the depolymerizing agents vinblastine, dolastatin-10, and HTI-286. KB-15-PTX/099-resistant cells did not overexpress P-glycoprotein nor did they have an alteration of [14C]paclitaxel accumulation compared with parental cells. However, a novel point mutation (T to A) resulting in Asp26 to glutamate substitution in class I (M40) beta-tubulin was found. Based on an electron crystallography structure of Zn-stabilized tubulin sheets, the phenyl ring of C-3' NHCO-C6H5 of paclitaxel makes contact with Asp26 of beta-tubulin, suggesting a ligand-induced mutation. Optimized model complexes of paclitaxel, docetaxel, and MAC-321 in beta-tubulin show a novel hydrogen bonding pattern for the glutamate mutant and rationalize the observed resistance profiles. However, a mutation in the paclitaxel binding pocket does not explain the phenotype completely. KB-15-PTX/099 cells have impaired microtubule stability as determined by a reduced percentage of tubulin in microtubules and reflected by less acetylated tubulin. These results suggest that a mutation in tubulin might affect microtubule stability as well as drug binding and contribute to the observed resistance profile.