RESUMEN
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
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
Traditional cancer chemotherapy is often accompanied by systemic toxicity to the patient. Monoclonal antibodies against antigens on cancer cells offer an alternative tumor-selective treatment approach. However, most monoclonal antibodies are not sufficiently potent to be therapeutically active on their own. Antibody-drug conjugates (ADCs) use antibodies to deliver a potent cytotoxic compound selectively to tumor cells, thus improving the therapeutic index of chemotherapeutic agents. The recent approval of two ADCs, brentuximab vedotin and ado-trastuzumab emtansine, for cancer treatment has spurred tremendous research interest in this field. This Review touches upon the early efforts in the field, and describes how the lessons learned from the first-generation ADCs have led to improvements in every aspect of this technology, i.e., the antibody, the cytotoxic compound, and the linker connecting them, leading to the current successes. The design of ADCs currently in clinical development, and results from mechanistic studies and preclinical and clinical evaluation are discussed. Emerging technologies that seek to further advance this exciting area of research are also discussed.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Neoplasias/tratamiento farmacológico , HumanosRESUMEN
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
Antibody-drug conjugates (ADCs) that incorporate the exatecan derivative DXd in their payload are showing promising clinical results in solid tumor indications. The payload has an F-ring that also contains a second chiral center, both of which complicate its synthesis and derivatization. Here we report on new camptothecin-ADCs that do not have an F-ring in their payloads yet behave similarly to DXd-bearing conjugates in vitro and in vivo. This simplification allows easier derivatization of camptothecin A and B rings for structure-activity relationship studies and payload optimization. ADCs having different degrees of bystander killing and the ability to release hydroxyl or thiol-bearing metabolites following peptide linker cleavage were investigated.
RESUMEN
A new type of antibody-drug conjugate (ADC) has been prepared that contains a sulfur-bearing maytansinoid attached to an antibody via a highly stable tripeptide linker. Once internalized by cells, proteases in catabolic vesicles cleave the peptide of the ADC's linker causing self-immolation that releases a thiol-bearing metabolite, which is then S-methylated. Conjugates were prepared with peptide linkers containing only alanyl residues, which were all l isomers or had a single d residue in one of the three positions. A d-alanyl residue in the linker did not significantly impair a conjugate's cytotoxicity or bystander killing unless it was directly attached to the immolative moiety. Increasing the number of methylene units in the maytansinoid side chain of a conjugate did not typically affect an ADC's cytotoxicity to targeted cells but did increase bystander killing activity. ADCs with the highest in vitro bystander killing were then evaluated in vivo in mice, where they displayed improved efficacy compared to previously described types of maytansinoid conjugates.
RESUMEN
Antibody-drug conjugates are targeted anticancer agents consisting of a cytotoxic drug covalently linked to a monoclonal antibody for tumor antigen-specific activity. Once bound to the target cell-surface antigen, the conjugate must be processed to release an active form of the drug, which can reach its intracellular target. Here, we used both biological and biochemical methods to better define this process for antibody-maytansinoid conjugates. In particular, we examined the metabolic fate in cells of huC242-maytansinoid conjugates containing either a disulfide linker (huC242-SPDB-DM4) or a thioether linker (huC242-SMCC-DM1). Using cell cycle analysis combined with lysosomal inhibitors, we showed that lysosomal processing is required for the activity of antibody-maytansinoid conjugates, irrespective of the linker. We also identified and characterized the released maytansinoid molecules from these conjugates, and measured their rate of release compared with the kinetics of cell cycle arrest. Both conjugates are efficiently degraded in lysosomes to yield metabolites consisting of the intact maytansinoid drug and linker attached to lysine. The lysine adduct is the sole metabolite from the thioether-linked conjugate. However, the lysine metabolite generated from the disulfide-linked conjugate is reduced and S-methylated to yield the lipophilic and potently cytotoxic metabolite, S-methyl-DM4. These findings provide insight into the mechanism of action of antibody-maytansinoid conjugates in general, and more specifically, identify a biochemical mechanism that may account for the significantly enhanced antitumor efficacy observed with disulfide-linked conjugates.
Asunto(s)
Antineoplásicos Fitogénicos/farmacocinética , Neoplasias del Colon/tratamiento farmacológico , Inmunotoxinas/farmacocinética , Maitansina/análogos & derivados , Animales , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Ciclo Celular/efectos de los fármacos , Neoplasias del Colon/inmunología , Neoplasias del Colon/metabolismo , Reactivos de Enlaces Cruzados/química , Reactivos de Enlaces Cruzados/farmacocinética , Reactivos de Enlaces Cruzados/farmacología , Disulfuros/química , Disulfuros/farmacocinética , Disulfuros/farmacología , Células HT29 , Humanos , Inmunotoxinas/química , Inmunotoxinas/farmacología , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Maitansina/química , Maitansina/farmacocinética , Ratones , Ratones SCID , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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
A triglycyl peptide linker (CX) was designed for use in antibody -: drug conjugates (ADC), aiming to provide efficient release and lysosomal efflux of cytotoxic catabolites within targeted cancer cells. ADCs comprising anti-epithelial cell adhesion molecule (anti-EpCAM) and anti-EGFR antibodies with maytansinoid payloads were prepared using CX or a noncleavable SMCC linker (CX and SMCC ADCs). The in vitro cytotoxic activities of CX and SMCC ADCs were similar for several cancer cell lines; however, the CX ADC was more active (5-100-fold lower IC50) than the SMCC ADC in other cell lines, including a multidrug-resistant line. Both CX and SMCC ADCs showed comparable MTDs and pharmacokinetics in CD-1 mice. In Calu-3 tumor xenografts, antitumor efficacy was observed with the anti-EpCAM CX ADC at a 5-fold lower dose than the corresponding SMCC ADC in vivo Similarly, the anti-EGFR CX ADC showed improved antitumor activity over the respective SMCC conjugate in HSC-2 and H1975 tumor models; however, both exhibited similar activity against FaDu xenografts. Mechanistically, in contrast with the charged lysine-linked catabolite of SMCC ADC, a significant fraction of the carboxylic acid catabolite of CX ADC could be uncharged in the acidic lysosomes, and thus diffuse out readily into the cytosol. Upon release from tumor cells, CX catabolites are charged at extracellular pH and do not penetrate and kill neighboring cells, similar to the SMCC catabolite. Overall, these data suggest that CX represents a promising linker option for the development of ADCs with improved therapeutic properties. Mol Cancer Ther; 15(6); 1311-20. ©2016 AACR.
Asunto(s)
Molécula de Adhesión Celular Epitelial/antagonistas & inhibidores , Receptores ErbB/antagonistas & inhibidores , Inmunoconjugados/administración & dosificación , Maitansina/química , Neoplasias/tratamiento farmacológico , Péptidos/síntesis química , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Inmunoconjugados/química , Inmunoconjugados/farmacocinética , Inmunoconjugados/farmacología , Dosis Máxima Tolerada , Ratones , Ratones SCID , Péptidos/química , Péptidos/farmacocinética , 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
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.