RESUMO
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.
RESUMO
Conjugates of the anti-CanAg humanized monoclonal antibody huC242 with the microtubule-formation inhibitor DM1 (a maytansinoid), or with the DNA alkylator DC1 (a CC1065 analogue), have been evaluated for their ability to eradicate mixed cell populations formed from CanAg-positive and CanAg-negative cells in culture and in xenograft tumors in mice. We found that in culture, conjugates of either drug killed not only the target antigen-positive cells but also the neighboring antigen-negative cells. Furthermore, we showed that, in vivo, these conjugates were effective in eradicating tumors containing both antigen-positive and antigen-negative cells. The presence of antigen-positive cells was required for this killing of bystander cells. This target cell-activated killing of bystander cells was dependent on the nature of the linker between the antibody and the drug. Conjugates linked via a reducible disulfide bond were capable of exerting the bystander effect whereas equally potent conjugates linked via a nonreducible thioether bond were not. Our data offer a rationale for developing optimally constructed antibody-drug conjugates for treating tumors that express the target antigen either in a homogeneous or heterogeneous manner.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Imunoconjugados/farmacologia , Maitansina/análogos & derivados , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Neoplasias do Colo/imunologia , Reagentes de Ligações Cruzadas/química , Feminino , Células HT29 , Humanos , Imunoconjugados/química , Imunoconjugados/imunologia , Maitansina/química , Maitansina/farmacologia , Camundongos , Camundongos SCID , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Antineoplásicos Fitogênicos/farmacocinética , Neoplasias do Colo/tratamento farmacológico , Imunotoxinas/farmacocinética , Maitansina/análogos & derivados , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Neoplasias do Colo/imunologia , Neoplasias do Colo/metabolismo , Reagentes de Ligações Cruzadas/química , Reagentes de Ligações Cruzadas/farmacocinética , Reagentes de Ligações Cruzadas/farmacologia , Dissulfetos/química , Dissulfetos/farmacocinética , Dissulfetos/farmacologia , Células HT29 , Humanos , Imunotoxinas/química , Imunotoxinas/farmacologia , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Maitansina/química , Maitansina/farmacocinética , Camundongos , Camundongos SCID , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Antibody-drug conjugates (ADCs) are designed to specifically bind to and kill cells expressing their target antigens. In addition to the obvious requirement of the presence of the target antigen on the cell surface, several other factors contribute to the sensitivity of target cells to the action of ADCs. These include (i) the rate of internalization of the ADC, (ii) its proteolytic degradation in late endosomes and lysosomes and the subsequent release of cytotoxic drug, and (iii) the intracellular concentration of the released drug. In addition to killing antigen-expressing cells, some ADCs were found to kill bystander cells irrespective of their antigen expression. This review summarizes the current knowledge of the mechanisms of killing of antigen-expressing and bystander cells by antibody-drug conjugates.
Assuntos
Anticorpos Monoclonais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos , Antineoplásicos/farmacologia , Imunoconjugados/farmacologia , Neoplasias/imunologia , Anticorpos Monoclonais/metabolismo , Antígenos de Neoplasias/metabolismo , Antineoplásicos/metabolismo , Humanos , Imunoconjugados/metabolismoRESUMO
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.
Assuntos
Molécula de Adesão da Célula Epitelial/antagonistas & inibidores , Receptores ErbB/antagonistas & inibidores , Imunoconjugados/administração & dosagem , Maitansina/química , Neoplasias/tratamento farmacológico , Peptídeos/síntese química , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Imunoconjugados/química , Imunoconjugados/farmacocinética , Imunoconjugados/farmacologia , Dose Máxima Tolerável , Camundongos , Camundongos SCID , Peptídeos/química , Peptídeos/farmacocinética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The microtubule-targeting maytansinoids accumulate in cells and induce mitotic arrest at 250- to 1000-fold lower concentrations than those required for their association with tubulin or microtubules. To identify the mechanisms of this intracellular accumulation and exceptional cytotoxicity of maytansinoids we studied interaction of a highly cytotoxic maytansinoid, S-methyl DM1 and several other maytansinoids with cells. S-methyl DM1 accumulated inside the cells with a markedly higher apparent affinity than to tubulin or microtubules. The apparent affinities of maytansinoids correlated with their cytotoxicities. The number of intracellular binding sites for S-methyl DM1 in MCF7 cells was comparable to the number of tubulin molecules per cell (~ 4-6 × 10(7) copies). Efflux of 3[H]-S-methyl DM1 from cells was enhanced in the presence of an excess of non-labeled S-methyl DM1, indicating that re-binding of 3 [H]-S-methyl DM1 to intracellular binding sites contributed to its intracellular retention. Liposomes loaded with non-polymerized tubulin recapitulated the apparent high-affinity association of S-methyl DM1 to cells. We propose a model for the intracellular accumulation of maytansinoids in which molecules of the compounds diffuse into a cell and associate with tubulin. Affinities of maytansinoids for individual tubulin molecules are weak, but the high intracellular concentration of tubulin favors, after dissociation of a compound-tubulin complex, their re-binding to a tubulin molecule, or to a tip of a microtubule in the same cell, over their efflux. As a result, a significant fraction of microtubule tips is occupied with a maytansinoid when added to cells at sub-nanomolar concentrations, inducing mitotic arrest and cell death.
Assuntos
Maitansina/metabolismo , Tubulina (Proteína)/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Lipossomos , Maitansina/farmacologia , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Modelos Biológicos , Ligação ProteicaRESUMO
One approach to improving activity of anticancer drugs is to conjugate them to antibodies that recognize tumor-associated, cell-surface antigens. The antibody-drug conjugate concept evolved following major advances, first, in the development of humanized and fully human antibodies; second, in the discoveries of highly cytotoxic compounds ('drugs) linkable to antibodies; and finally, in the optimization of linkers that couple the drug to the antibody and provide sufficient stability of the antibody-drug conjugate in the circulation, optimal activation of the drug in the tumor, and the ability of the activated drug to overcome multidrug resistance. In this article, we will review the considerations for selecting a target antigen, the design of the conjugate, and the pre-clinical and clinical experiences with the current generation of antibody-drug conjugates.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/administração & dosagem , Imunoconjugados/uso terapêutico , Neoplasias/tratamento farmacológico , Animais , Anticorpos Monoclonais/efeitos adversos , Humanos , Imunoconjugados/efeitos adversosRESUMO
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.