RESUMEN
Despite the growing use of visible-light photochemistry in both chemistry and biology, no general low-heat photoreactor for use across these different disciplines exists. Herein, we describe the design and use of a standardized photoreactor for visible-light-driven activation and photocatalytic chemical transformations. Using this single benchtop photoreactor, we performed photoredox reactions across multiple visible light wavelengths, a high-throughput photocatalytic cross-coupling reaction, and inâ vitro labeling of proteins and live cells. Given the success of this reactor in all tested applications, we envision that this multi-use photoreactor will be widely used in biology, chemical biology, and medicinal chemistry settings.
Asunto(s)
Biotina/análisis , Luz , Fotobiorreactores , Tiramina/química , Catálisis , Línea Celular Tumoral , Diseño de Equipo , Humanos , Estructura Molecular , Procesos Fotoquímicos , Tiramina/análogos & derivados , Tiramina/síntesis químicaRESUMEN
Antibody-drug conjugates are an emerging class of cancer therapeutics constructed from monoclonal antibodies conjugated with small molecule effectors. First-generation molecules of this class often employed heterogeneous conjugation chemistry, but many site-specifically conjugated ADCs have been described recently. Here, we undertake a systematic comparison of ADCs made with the same antibody and the same macrocyclic maytansinoid effector but conjugated either heterogeneously at lysine residues or site-specifically at cysteine residues. Characterization of these ADCs in vitro reveals generally similar properties, including a similar catabolite profile, a key element in making a meaningful comparison of conjugation chemistries. In a mouse model of cervical cancer, the lysine-conjugated ADC affords greater efficacy on a molar payload basis. Rather than making general conclusions about ADCs conjugated by a particular chemistry, we interpret these results as highlighting the complexity of ADCs and the interplay between payload class, linker chemistry, target antigen, and other variables that determine efficacy in a given setting.
Asunto(s)
Anticuerpos Monoclonales/química , Cisteína/química , Inmunoconjugados/farmacocinética , Inmunoconjugados/uso terapéutico , Lisina/química , Maitansina/inmunología , Neoplasias del Cuello Uterino/tratamiento farmacológico , Animales , Supervivencia Celular/efectos de los fármacos , Femenino , Células HeLa , Humanos , Inmunoconjugados/administración & dosificación , Inyecciones Intravenosas , Ratones , Ratones SCID , Resultado del Tratamiento , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Bioanalysis of antibody-drug conjugates (ADCs) is challenging due to the complex, heterogeneous nature of their structures and their complicated catabolism. To fully describe the pharmacokinetics (PK) of an ADC, several analytes are commonly quantified, including total antibody, conjugate, and payload. Among them, conjugate is the most challenging to measure, because it requires detection of both small and large molecules as one entity. Existing approaches to quantify the conjugated species of ADCs involve a ligand binding assay (LBA) for conjugated antibody or hybrid LBA/liquid chromatography/tandem mass spectrometry (LC/MS/MS) for quantitation of conjugated drug. In our current work for a protein-drug conjugate (PDC) using the Centyrin scaffold, a similar concept to ADCs but with smaller protein size, an alternative method to quantify the conjugate by using a surrogate peptide approach, was utilized. The His-tagged proteins were isolated from biological samples using immobilized metal affinity chromatography (IMAC), followed by trypsin digestion. The tryptic peptide containing the linker attached to the payload was used as a surrogate of the conjugate and monitored by LC/MS/MS analysis. During method development and its application, we found that hydrolysis of the succinimide ring of the linker was ubiquitous, taking place at many stages during the lifetime of the PDC including in the initial drug product, in vivo in circulation in the animals, and ex vivo during the trypsin digestion step of the sample preparation. We have shown that hydrolysis during trypsin digestion is concentration-independent and consistent during the work flow-therefore, having no impact on assay performance. However, for samples that have undergone extensive hydrolysis prior to trypsin digestion, significant bias could be introduced if only the non-hydrolyzed form is considered in the quantitation. Therefore, it is important to incorporate succinimide hydrolysis products in the quantitation method in order to provide an accurate estimation of the total conjugate level. More importantly, the LC/MS/MS-based method described here provides a useful tool to quantitatively evaluate succinimide hydrolysis of ADCs in vivo, which has been previously reported to have significant impact on their stability, exposure, and efficacy.
Asunto(s)
Inmunoconjugados/análisis , Succinimidas/química , Animales , Cromatografía Liquida , Hidrólisis , Ratones , Ratones Endogámicos BALB C , Estructura Molecular , Espectrometría de Masas en TándemRESUMEN
Antibody-drug conjugates (ADCs) have become a widely investigated modality for cancer therapy, in part due to the clinical findings with ado-trastuzumab emtansine (Kadcyla). Ado-trastuzumab emtansine utilizes the Ab-SMCC-DM1 format, in which the thiol-functionalized maytansinoid cytotoxic agent, DM1, is linked to the antibody (Ab) via the maleimide moiety of the heterobifunctional SMCC linker. The pharmacokinetic (PK) data for ado-trastuzumab emtansine point to a faster clearance for the ADC than for total antibody. Cytotoxic agent release in plasma has been reported with nonmaytansinoid, cysteine-linked ADCs via thiol-maleimide exchange, for example, brentuximab vedotin. For Ab-SMCC-DM1 ADCs, however, the main catabolite reported is lysine-SMCC-DM1, the expected product of intracellular antibody proteolysis. To understand these observations better, we conducted a series of studies to examine the stability of the thiol-maleimide linkage, utilizing the EGFR-targeting conjugate, J2898A-SMCC-DM1, and comparing it with a control ADC made with a noncleavable linker that lacked a thiol-maleimide adduct (J2898A-(CH2)3-DM). We employed radiolabeled ADCs to directly measure both the antibody and the ADC components in plasma. The PK properties of the conjugated antibody moiety of the two conjugates, J2898A-SMCC-DM1 and J2898A-(CH2)3-DM (each with an average of 3.0 to 3.4 maytansinoid molecules per antibody), appear to be similar to that of the unconjugated antibody. Clearance values of the intact conjugates were slightly faster than those of the Ab components. Furthermore, J2898A-SMCC-DM1 clears slightly faster than J2898A-(CH2)3-DM, suggesting that there is a fraction of maytansinoid loss from the SMCC-DM1 ADC, possibly through a thiol-maleimide dependent mechanism. Experiments on ex vivo stability confirm that some loss of maytansinoid from Ab-SMCC-DM1 conjugates can occur via thiol elimination, but at a slower rate than the corresponding rate of loss reported for thiol-maleimide links formed at thiols derived by reduction of endogenous cysteine residues in antibodies, consistent with expected differences in thiol-maleimide stability related to thiol pKa. These findings inform the design strategy for future ADCs.
Asunto(s)
Inmunoconjugados/química , Inmunoconjugados/farmacocinética , Lisina/química , Maleimidas/química , Maitansina/química , Animales , Estabilidad de Medicamentos , Ratones , Relación Estructura-ActividadRESUMEN
Antibody anilino maytansinoid conjugates (AaMCs) have been prepared in which a maytansinoid bearing an aniline group was linked through the aniline amine to a dipeptide, which in turn was covalently attached to a desired monoclonal antibody. Several such conjugates were prepared utilizing different dipeptides in the linkage including Gly-Gly, l-Val-l-Cit, and all four stereoisomers of the Ala-Ala dipeptide. The properties of AaMCs could be altered by the choice of dipeptide in the linker. Each of the AaMCs, except the AaMC bearing a d-Ala-d-Ala peptide linker, displayed more bystander killing in vitro than maytansinoid ADCs that utilize disulfide linkers. In mouse models, the anti-CanAg AaMC bearing a d-Ala-l-Ala dipeptide in the linker was shown to be more efficacious against heterogeneous HT-29 xenografts than maytansinoid ADCs that utilize disulfide linkers, while both types of the conjugates displayed similar tolerabilities.
Asunto(s)
Compuestos de Anilina/química , Antineoplásicos Fitogénicos/química , Inmunoconjugados/química , Maitansina/química , Compuestos de Anilina/farmacocinética , Compuestos de Anilina/uso terapéutico , Animales , Antineoplásicos Fitogénicos/farmacocinética , Antineoplásicos Fitogénicos/uso terapéutico , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Inmunoconjugados/farmacocinética , Inmunoconjugados/uso terapéutico , Maitansina/farmacocinética , Maitansina/uso terapéutico , Ratones , Neoplasias/tratamiento farmacológicoRESUMEN
Several antibody-maytansinoid conjugates (AMCs) are in clinical trials for the treatment of various cancers. Each of these conjugates can be metabolized by tumor cells to give cytotoxic maytansinoid metabolites that can kill targeted cells. In preclinical studies in mice, the cytotoxic metabolites initially formed in vivo are further processed in the mouse liver to give several oxidized metabolic species. In this work, the primary AMC metabolites were synthesized and incubated with human liver microsomes (HLMs) to determine if human liver would likely give the same metabolites as those formed in mouse liver. The results of these HLM metabolism studies as well as the subsequent syntheses of the resulting HLM oxidation products are presented. Syntheses of the minor impurities formed during the conjugation of AMCs were also conducted to determine their cytotoxicities and to establish how these impurities would be metabolized by HLM.
Asunto(s)
Inmunoconjugados/química , Inmunoconjugados/metabolismo , Maitansina/química , Animales , Línea Celular Tumoral , Humanos , Espectroscopía de Resonancia Magnética , Ratones , Microsomas Hepáticos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Antibody-maytansinoid conjugates (AMCs) are targeted chemotherapeutic agents consisting of a potent microtubule-depolymerizing maytansinoid (DM1 or DM4) attached to lysine residues of a monoclonal antibody (mAb) using an uncleavable thioether linker or a stable disulfide linker. Most of the administered dose of an antibody-based therapeutic is slowly catabolized by the liver and other tissues of the reticuloendothelial system. Maytansinoids released from an AMC during this catabolic process could potentially be a source of toxicity. To investigate this, we isolated and identified liver metabolites in mice for three different [(3)H]AMCs with structures similar to those currently undergoing evaluation in the clinic. We then synthesized each metabolite to confirm the identification and assessed their cytotoxic potencies when added extracellularly. We found that the uncleavable mAb-SMCC-[(3)H]DM1 conjugate was degraded to a single major maytansinoid metabolite, lysine-SMCC-[(3)H]DM1, that was nearly 50-fold less cytotoxic than maytansine. The two disulfide-linked conjugates, mAb-SPP-[(3)H]DM1 and mAb-SPDB-[(3)H]DM4, were also found to be catabolized to the analogous lysine-linked maytansinoid metabolites. However, subsequent reduction, S-methylation, and NADPH-dependent oxidation steps in the liver yielded the corresponding S-methyl sulfoxide and S-methyl sulfone derivatives. The cytotoxic potencies of the oxidized maytansinoids toward several human carcinoma cell lines were found to be 5- to 50-fold less potent than maytansine. Our results suggest that liver plays an important role in the detoxification of both cleavable and uncleavable AMCs.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Diseño de Fármacos , Hígado/metabolismo , Maitansina/metabolismo , Animales , Anticuerpos Monoclonales/química , Femenino , Hígado/química , Maitansina/análogos & derivados , Maitansina/química , Ratones , Ratones Endogámicos , Estructura MolecularRESUMEN
Antibody-drug conjugates (ADCs) are designed to eradicate cancer cells that express the target antigen on their cell surface. A key component of an ADC is the linker that covalently connects the cytotoxic agent to the antibody. Several antibody-maytansinoid conjugates prepared with disulfide-based linkers such as those targeting the CanAg antigen have been shown to display more activity in preclinical mouse xenograft models than corresponding conjugates prepared with uncleavable thioether-based linkers. To investigate how the linker influences delivery and activation of antibody-maytansinoid conjugates, we isolated and characterized the [(3)H]maytansinoids from CanAg-positive tumor tissues following a single intravenous administration of 300 microg/kg (based on maytansinoid dose) of anti-CanAg antibody (huC242)-(3)H-maytansinoid conjugates prepared with cleavable disulfide linkers and an uncleavable thioether linker. We identified three target-dependent tumor metabolites of the disulfide-linked huC242-SPDB-DM4, namely, lysine-N(epsilon)-SPDB-DM4, DM4, and S-methyl-DM4. We found similar metabolites for the less hindered disulfide-linked huC242-SPP-DM1 conjugate with the exception that no S-methyl-DM1 was detected. The sole metabolite of the uncleavable thioether-linked huC242-SMCC-DM1 was lysine-N(epsilon)-SMCC-DM1. The AUC for the metabolites of huC242-SMCC-DM1 at the tumor over 7 d was about 2-fold greater than the corresponding AUC for the metabolites of the disulfide-linked conjugates. The lipophilic metabolites of the disulfide-linked conjugates were found to be nearly 1000 times more cytotoxic than the more hydrophilic lysine-N(epsilon)-linker-maytansinoids in cell-based viability assays when added extracellularly. The cell killing properties associated with the lipophilic metabolites of the disulfide-linked conjugates (DM4 and S-methyl-DM4, and DM1) provide an explanation for the superior in vivo efficacy that is often observed with antibody-maytansinoid conjugates prepared with disulfide-based linkers in xenograft mouse models.
Asunto(s)
Anticuerpos/metabolismo , Disulfuros/química , Inmunoconjugados/metabolismo , Inmunoconjugados/uso terapéutico , Maitansina/metabolismo , Neoplasias/metabolismo , Sulfuros/química , Animales , Anticuerpos/química , Anticuerpos/inmunología , Anticuerpos/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Inmunoconjugados/química , Inmunoconjugados/inmunología , Maitansina/química , Maitansina/inmunología , Maitansina/uso terapéutico , Ratones , Ratones SCID , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
AIM: Alternative scaffold proteins have emerged as novel platforms for development of therapeutic applications. One such application is in protein-drug conjugates (PDCs), which are analogous to antibody-drug conjugates. METHODOLOGY: Liquid chromatography-mass spectrometry methods for quantitation of total protein, conjugate and free payload for a PDC based on Centyrin scaffold were developed. Tryptic peptides generated from a region of the Centyrin that does not contain a conjugation site, and another that has the conjugation site with the linker-payload attached were used as surrogates of the total and conjugated Centyrin, respectively. CONCLUSION: The methods were successfully applied to analysis of samples from mice to quantify the plasma and tissue concentrations. This same workflow can potentially be applied to other PDCs and site-specific antibody-drug conjugates.
Asunto(s)
Péptidos/química , Péptidos/farmacocinética , Preparaciones Farmacéuticas/química , Tenascina/química , Tenascina/farmacocinética , Animales , Cromatografía Liquida/métodos , Humanos , Ratones , Ratones Endogámicos BALB C , Péptidos/sangre , Preparaciones Farmacéuticas/sangre , Farmacocinética , Dominios Proteicos , Espectrometría de Masas en Tándem/métodos , Tenascina/sangre , Flujo de TrabajoRESUMEN
Maytansine, a highly cytotoxic natural product, failed as an anticancer agent in human clinical trials because of unacceptable systemic toxicity. The potent cell killing ability of maytansine can be used in a targeted delivery approach for the selective destruction of cancer cells. A series of new maytansinoids, bearing a disulfide or thiol substituent were synthesized. The chain length of the ester side chain and the degree of steric hindrance on the carbon atom bearing the thiol substituent were varied. Several of these maytansinoids were found to be even more potent in vitro than maytansine. The targeted delivery of these maytansinoids, using monoclonal antibodies, resulted in a high, specific killing of the targeted cells in vitro and remarkable antitumor activity in vivo.
Asunto(s)
Antineoplásicos/síntesis química , Maitansina/análogos & derivados , Maitansina/síntesis química , Animales , Anticuerpos Monoclonales/química , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Disulfuros/síntesis química , Disulfuros/química , Disulfuros/farmacología , Portadores de Fármacos , Sistemas de Liberación de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Maitansina/química , Maitansina/farmacología , Ratones , Ratones SCID , Trasplante de Neoplasias , Relación Estructura-Actividad , Compuestos de Sulfhidrilo/síntesis química , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/farmacología , Trasplante HeterólogoRESUMEN
Taxoids bearing methyldisulfanyl(alkanoyl) groups for taxoid-antibody immunoconjugates were designed, synthesized and their activities evaluated. A highly cytotoxic C-10 methyldisulfanylpropanoyl taxoid was conjugated to monoclonal antibodies recognizing the epidermal growth factor receptor (EGFR) expressed in human squamous cancers. These conjugates were shown to possess remarkable target-specific antitumor activity in vivo against EGFR-expressing A431 tumor xenografts in severe combined immune deficiency mice, resulting in complete inhibition of tumor growth in all the treated mice.
Asunto(s)
Anticuerpos Monoclonales/química , Antineoplásicos/síntesis química , Inmunoconjugados/química , Paclitaxel/análogos & derivados , Paclitaxel/química , Profármacos/síntesis química , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Receptores ErbB/inmunología , Humanos , Inmunoconjugados/farmacología , Ratones , Ratones SCID , Profármacos/química , Profármacos/farmacología , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The synthesis and biological evaluation of hydrophilic heterobifunctional cross-linkers for conjugation of antibodies with highly cytotoxic agents are described. These linkers contain either a negatively charged sulfonate group or a hydrophilic, noncharged PEG group in addition to an amine-reactive N-hydroxysuccinimide (NHS) ester and sulfhydryl reactive termini. These hydrophilic linkers enable conjugation of hydrophobic organic molecule drugs, such as a maytansinoid, at a higher drug/antibody ratio (DAR) than hydrophobic SPDB and SMCC linkers used earlier without triggering aggregation or loss of affinity of the resulting conjugate. Antibody-maytansinoid conjugates (AMCs) bearing these sulfonate- or PEG-containing hydrophilic linkers were, depending on the nature of the targeted cells, equally to more cytotoxic to antigen-positive cells and equally to less cytotoxic to antigen-negative cells than conjugates made with SPDB or SMCC linkers and thus typically displayed a wider selectivity window, particularly against multidrug resistant (MDR) cancer cell lines in vitro and tumor xenograft models in vivo.
Asunto(s)
Anticuerpos/química , Inmunoconjugados/química , Maitansina/química , Animales , Química Farmacéutica/métodos , Diseño de Fármacos , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Ratones , Modelos Químicos , Trasplante de Neoplasias , Polietilenglicoles/química , Succinimidas/química , Sulfonas/químicaRESUMEN
Maytansine is a potent microtubule-targeted compound that induces mitotic arrest and kills tumor cells at subnanomolar concentrations. However, its side effects and lack of tumor specificity have prevented successful clinical use. Recently, antibody-conjugated maytansine derivatives have been developed to overcome these drawbacks. Several conjugates show promising early clinical results. We evaluated the effects on microtubule polymerization and dynamic instability of maytansine and two cellular metabolites (S-methyl-DM1 and S-methyl-DM4) of antibody-maytansinoid conjugates that are potent in cells at picomolar levels and that are active in tumor-bearing mice. Although S-methyl-DM1 and S-methyl-DM4 inhibited polymerization more weakly than maytansine, at 100 nmol/L they suppressed dynamic instability more strongly than maytansine (by 84% and 73%, respectively, compared with 45% for maytansine). However, unlike maytansine, S-methyl-DM1 and S-methyl-DM4 induced tubulin aggregates detectable by electron microscopy at concentrations ≥2 µmol/L, with S-methyl-DM4 showing more extensive aggregate formation than S-methyl-DM1. Both maytansine and S-methyl-DM1 bound to tubulin with similar K(D) values (0.86 ± 0.2 and 0.93 ± 0.2 µmol/L, respectively). Tritiated S-methyl-DM1 bound to 37 high-affinity sites per microtubule (K(D), 0.1 ± 0.05 µmol/L). Thus, S-methyl-DM1 binds to high-affinity sites on microtubules 20-fold more strongly than vinblastine. The high-affinity binding is likely at microtubule ends and is responsible for suppression of microtubule dynamic instability. Also, at higher concentrations, S-methyl-DM1 showed low-affinity binding either to a larger number of sites on microtubules or to sedimentable tubulin aggregates. Overall, the maytansine derivatives that result from cellular metabolism of the antibody conjugates are themselves potent microtubule poisons, interacting with microtubules as effectively as or more effectively than the parent molecule.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Inmunoconjugados/inmunología , Maitansina/farmacología , Microtúbulos/efectos de los fármacos , Animales , Antineoplásicos Fitogénicos/inmunología , Antineoplásicos Fitogénicos/metabolismo , Sitios de Unión , Bovinos , Maitansina/inmunología , Maitansina/metabolismo , Ratones , Microtúbulos/metabolismo , Polímeros , Solubilidad , Tubulina (Proteína)/metabolismoRESUMEN
Conjugation of cytotoxic compounds to antibodies that bind to cancer-specific antigens makes these drugs selective in killing cancer cells. However, many of the compounds used in such antibody-drug conjugates (ADC) are substrates for the multidrug transporter MDR1. To evade the MDR1-mediated resistance, we conjugated the highly cytotoxic maytansinoid DM1 to antibodies via the maleimidyl-based hydrophilic linker PEG(4)Mal. Following uptake into target cells, conjugates made with the PEG(4)Mal linker were processed to a cytotoxic metabolite that was retained by MDR1-expressing cells better than a metabolite of similar conjugates prepared with the nonpolar linker N-succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC). In accord, PEG(4)Mal-linked conjugates were more potent in killing MDR1-expressing cells in culture. In addition, PEG(4)Mal-linked conjugates were markedly more effective in eradicating MDR1-expressing human xenograft tumors than SMCC-linked conjugates while being tolerated similarly, thus showing an improved therapeutic index. This study points the way to the development of ADCs that bypass multidrug resistance.