RESUMO
DNA-targeting indolinobenzodiazepine dimer (IGN) payloads are used in several clinical-stage antibody-drug conjugates. IGN drugs alkylate DNA through the single imine moiety present in the dimer in contrast to the pyrrolobenzodiazepine dimer drugs, such as talirine and tesirine, which contain two imine moieties per dimer and cross-link DNA. This study explored the mechanism of binding of IGN to DNA in cells and to synthetic duplex and hairpin oligonucleotides. New, highly sensitive IGN-DNA binding enzyme-linked immunosorbent assay methods were developed using biotinylated IGN analogues (monoimine, diimine, and diamine IGNs) and digoxigenin-labeled duplex oligonucleotides, which allowed the measurement of drug-DNA adducts in viable cells at concentrations below IC50. Furthermore, the release of free drug from the IGN-DNA adduct upon treatment with nuclease ex vivo was tested under physiological conditions. The monoimine IGN drug formed a highly stable adduct with DNA in cells, with stability similar to that of the diimine drug analogue. Both monoimine and diimine IGN-DNA adducts released free drugs upon DNA cleavage by nuclease at 37 °C, although more free drug was released from the monoimine compared to the diimine adduct, which presumably was partly cross-linked. The strong binding of the monoimine IGN drug to duplex DNA results from both the noncovalent IGN-DNA interaction and the covalent bond formation between the 2-amino group of a guanine residue and the imine moiety in IGN.
Assuntos
Antineoplásicos/química , Benzodiazepinas/química , Adutos de DNA/química , DNA/química , Imunoconjugados/farmacologia , Indóis/química , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Adutos de DNA/metabolismo , Dimerização , ELISPOT , Humanos , Iminas/química , Imunoconjugados/administração & dosagem , Oligonucleotídeos/química , Pirróis/químicaRESUMO
Although peptide linkers are used in multiple clinical-stage ADCs, there are only few reports on optimizing peptide linkers for efficient lysosomal proteolysis and for stability in circulation. We screened multiple dipeptide linkers for efficiency of proteolysis and compared them to the dipeptide linkers currently being evaluated in the clinic: Val-Cit, Val-Ala, and Ala-Ala. Lead dipeptide linkers selected from the initial screen were incorporated into ADCs with indolinobenzodiazepine dimer (IGN) payloads to evaluate cellular processing, in vitro cytotoxic activity, plasma stability, and in vivo efficacy. ADCs with several dipeptide linkers bearing l-amino acids showed faster lysosomal processing in target cancer cells compared to the l-Ala-l-Ala linked ADC. These variances in linker processing rates did not result in different in vitro and in vivo activities among peptide linker ADCs, presumably due to accumulation of threshold cytotoxic catabolite levels for ADCs of several peptide linkers in the cell lines and xenografts tested. ADCs with l-amino acid dipeptide linkers exhibited superior in vitro cytotoxic potencies in multiple cell lines compared to an ADC with a d-Ala-d-Ala dipeptide linker and an ADC with a noncleavable linker. This work adds to the toolbox of stable, lysosomally cleavable peptide linkers for ADCs.
Assuntos
Anticorpos/química , Biopolímeros/química , Dipeptídeos/química , Imunoconjugados/química , Lisossomos/metabolismo , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Humanos , Camundongos , Camundongos SCID , Estrutura Molecular , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
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
Antibody-drug conjugates (ADCs) that incorporate potent indolinobenzodiazepine DNA alkylators as the payload component are currently undergoing clinical evaluation. In one ADC design, the payload molecules are linked to the antibody through a peptidase-labile l-Ala-l-Ala linker. In order to determine the role of amino acid stereochemistry on antitumor activity and tolerability, we incorporated l- and d-alanyl groups in the dipeptide, synthesized all four diastereomers, and prepared and tested the corresponding ADCs. Results of our preclinical evaluation showed that the l-Ala-l-Ala configuration provided the ADC with the highest therapeutic index (antitumor activity vs toxicity).
RESUMO
Indolinobenzodiazepine DNA alkylators (IGNs) are the cytotoxic payloads in antibody-drug conjugates (ADCs) currently undergoing Phase I clinical evaluation (IMGN779, IMGN632, and TAK164). These ADCs possess linkers that have been incorporated into a central substituted phenyl spacer. Here, we present an alternative strategy for the IGNs, linking through a carbamate at the readily available N-10 amine present in the monoimine containing dimer. As a result, we have designed a series of N-10 linked IGN ADCs with a wide range of in vitro potency and tolerability, which may allow us to better match an IGN with a particular target based on the potential dosing needs.
RESUMO
Antibody-drug conjugates (ADCs) incorporating potent indolinobenzodiazepine (IGN) DNA alkylators as the cytotoxic payload are currently undergoing clinical evaluation. The optimized design of these payloads consists of an unsymmetrical dimer possessing both an imine and an amine effectively eliminating DNA crosslinking and demonstrating improved tolerability in mice. Here we present an alternate approach to generating DNA alkylating ADCs by linking the IGN monomer with a biaryl system which has a high DNA binding affinity to potentially enhance tolerability. These BIA ADCs were found to be highly cytotoxic in vitro and demonstrated potent antitumor activity in vivo.
Assuntos
Alquilantes/química , Desenho de Fármacos , Imunoconjugados/química , Animais , Anticorpos Monoclonais/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , DNA/metabolismo , Humanos , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Camundongos , Camundongos SCID , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Relação Estrutura-Atividade , Transplante HeterólogoRESUMO
The outlook for patients with refractory/relapsed acute myeloid leukemia (AML) remains poor, with conventional chemotherapeutic treatments often associated with unacceptable toxicities, including severe infections due to profound myelosuppression. Thus there exists an urgent need for more effective agents to treat AML that confer high therapeutic indices and favorable tolerability profiles. Because of its high expression on leukemic blast and stem cells compared with normal hematopoietic stem cells and progenitors, CD123 has emerged as a rational candidate for molecularly targeted therapeutic approaches in this disease. Here we describe the development and preclinical characterization of a CD123-targeting antibody-drug conjugate (ADC), IMGN632, that comprises a novel humanized anti-CD123 antibody G4723A linked to a recently reported DNA mono-alkylating payload of the indolinobenzodiazepine pseudodimer (IGN) class of cytotoxic compounds. The activity of IMGN632 was compared with X-ADC, the ADC utilizing the G4723A antibody linked to a DNA crosslinking IGN payload. With low picomolar potency, both ADCs reduced viability in AML cell lines and patient-derived samples in culture, irrespective of their multidrug resistance or disease status. However, X-ADC exposure was >40-fold more cytotoxic to the normal myeloid progenitors than IMGN632. Of particular note, IMGN632 demonstrated potent activity in all AML samples at concentrations well below levels that impacted normal bone marrow progenitors, suggesting the potential for efficacy in AML patients in the absence of or with limited myelosuppression. Furthermore, IMGN632 demonstrated robust antitumor efficacy in multiple AML xenograft models. Overall, these findings identify IMGN632 as a promising candidate for evaluation as a novel therapy in AML.
Assuntos
Imunoconjugados/uso terapêutico , Subunidade alfa de Receptor de Interleucina-3/imunologia , Leucemia Mieloide Aguda/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Xenoenxertos , Humanos , Imunoconjugados/imunologia , Camundongos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Tumor-selective delivery of cytotoxic agents in the form of antibody-drug conjugates (ADCs) is now a clinically validated approach for cancer treatment. In an attempt to improve the clinical success rate of ADCs, emphasis has been recently placed on the use of DNA-cross-linking pyrrolobenzodiazepine compounds as the payload. Despite promising early clinical results with this class of ADCs, doses achievable have been low due to systemic toxicity. Here, we describe the development of a new class of potent DNA-interacting agents wherein changing the mechanism of action from a cross-linker to a DNA alkylator improves the tolerability of the ADC. ADCs containing the DNA alkylator displayed similar in vitro potency, but improved bystander killing and in vivo efficacy, compared with those of the cross-linker. Thus, the improved in vivo tolerability and antitumor activity achieved in rodent models with ADCs of the novel DNA alkylator could provide an efficacious, yet safer option for cancer treatment. Mol Cancer Ther; 17(3); 650-60. ©2018 AACR.
Assuntos
Imunoconjugados/farmacologia , Substâncias Intercalantes/farmacologia , Neoplasias/tratamento farmacológico , Índice Terapêutico do Medicamento , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/metabolismo , Antineoplásicos Alquilantes/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Reagentes de Ligações Cruzadas/química , DNA/genética , DNA/metabolismo , Desenho de Fármacos , Humanos , Imunoconjugados/química , Imunoconjugados/metabolismo , Substâncias Intercalantes/química , Substâncias Intercalantes/metabolismo , Camundongos , Neoplasias/patologia , Carga Tumoral/efeitos dos fármacosRESUMO
Antibody-drug conjugates (ADCs) represent an emerging new paradigm in cancer therapy. The approval of two ADCs has spurred considerable interest in this area of research, and over 55 ADCs are currently in clinical testing. In order to improve the clinical success rate of ADC therapy, all three components of the ADC: the antibody, linker, and payload have to be optimized. While considerable improvements have been made in antibody properties and target selection, medicinal chemistry efforts have lagged behind, and there is a significant need for innovation in linker design and payloads.
RESUMO
The promise of tumor-selective delivery of cytotoxic agents in the form of antibody-drug conjugates (ADC) has now been realized, evidenced by the approval of two ADCs, both of which incorporate highly cytotoxic tubulin-interacting agents, for cancer therapy. An ongoing challenge remains in identifying potent agents with alternative mechanisms of cell killing that can provide ADCs with high therapeutic indices and favorable tolerability. Here, we describe the development of a new class of potent DNA alkylating agents that meets these objectives. Through chemical design, we changed the mechanism of action of our novel DNA cross-linking agent to a monofunctional DNA alkylator. This modification, coupled with linker optimization, generated ADCs that were well tolerated in mice and demonstrated robust antitumor activity in multiple tumor models at doses 1.5% to 3.5% of maximally tolerated levels. These properties underscore the considerable potential of these purpose-created, unique DNA-interacting conjugates for broadening the clinical application of ADC technology. Mol Cancer Ther; 15(8); 1870-8. ©2016 AACR.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Imunoconjugados/farmacologia , Animais , Antineoplásicos Alquilantes/química , Efeito Espectador , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , DNA/química , DNA/metabolismo , Adutos de DNA , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Imunoconjugados/química , Camundongos , Estrutura Molecular , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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
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.
Assuntos
Compostos de Anilina/química , Antineoplásicos Fitogênicos/química , Imunoconjugados/química , Maitansina/química , Compostos de Anilina/farmacocinética , Compostos de Anilina/uso terapêutico , Animais , Antineoplásicos Fitogênicos/farmacocinética , Antineoplásicos Fitogênicos/uso terapêutico , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Imunoconjugados/farmacocinética , Imunoconjugados/uso terapêutico , Maitansina/farmacocinética , Maitansina/uso terapêutico , Camundongos , Neoplasias/tratamento farmacológicoRESUMO
Ado-trastuzumab emtansine (T-DM1) is an antibody-drug conjugate that combines the antitumor properties of the humanized anti-human epidermal growth factor receptor 2 (HER2) antibody, trastuzumab, with the maytansinoid, DM1, a potent microtubule-disrupting agent, joined by a stable linker. Upon binding to HER2, the conjugate is internalized via receptor-mediated endocytosis, and an active derivative of DM1 is subsequently released by proteolytic degradation of the antibody moiety within the lysosome. Initial clinical evaluation led to a phase III trial in advanced HER2-positive breast cancer patients who had relapsed after prior treatment with trastuzumab and a taxane, which showed that T-DM1 significantly prolonged progression-free and overall survival with less toxicity than lapatinib plus capecitabine. In 2013, T-DM1 received FDA approval for the treatment of patients with HER2-positive metastatic breast cancer who had previously received trastuzumab and a taxane, separately or in combination, the first ADC to receive full approval based on a randomized study.
Assuntos
Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Maitansina/análogos & derivados , Receptor ErbB-2/metabolismo , Ado-Trastuzumab Emtansina , Animais , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/farmacocinética , Anticorpos Monoclonais Humanizados/toxicidade , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Neoplasias da Mama/metabolismo , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Ensaios Clínicos Fase III como Assunto , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Maitansina/efeitos adversos , Maitansina/química , Maitansina/farmacocinética , Maitansina/farmacologia , Maitansina/toxicidade , Camundongos , Ratos , TrastuzumabRESUMO
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.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , HumanosRESUMO
The synthesis and biological evaluation of phosphate prodrugs of analogues of 1 (CC-1065) and their conjugates with antibodies are described. The phosphate group on the 1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI) portion of the compounds confers enhanced solubility and stability in aqueous solutions. In the presence of phosphatases, these compounds convert into active DNA-alkylating agents. The synthesis of the prodrugs was achieved sequentially through coupling of CBI with a bis-indolyl moiety, followed by attachment of a thiol-containing linker, and conversion of the hydroxyl group of CBI into a phosphate prodrug. The linkers incorporated into the prodrugs enable conjugation to an antibody via either a stable disulfide or thioether bond, in aqueous buffer solutions containing as little as 5% organic cosolvent, resulting in exclusively monomeric and stable antibody-cytotoxic prodrug conjugates. Two disulfide-containing linkers differing in the degree of steric hindrance were used in antibody conjugates to test the effect of different rates of intracellular disulfide cleavage and effector release on biological activity. The prodrugs can be converted to the active cytotoxic compounds through the action of endogenous phosphatases. Antibody-prodrug conjugates displayed potent antigen-selective cytotoxic activity in vitro and antitumor activity in vivo.
Assuntos
Anticorpos Monoclonais Humanizados/química , Antineoplásicos Alquilantes/síntese química , Indóis/síntese química , Compostos Organofosforados/síntese química , Pró-Fármacos/síntese química , Animais , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/farmacologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Estabilidade de Medicamentos , Duocarmicinas , Feminino , Humanos , Indóis/química , Indóis/farmacologia , Camundongos , Camundongos SCID , Transplante de Neoplasias , Compostos Organofosforados/química , Compostos Organofosforados/farmacologia , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Solubilidade , Estereoisomerismo , Relação Estrutura-Atividade , Transplante HeterólogoRESUMO
Antibody conjugates are a diverse class of therapeutics consisting of a cytotoxic agent linked covalently to an antibody or antibody fragment directed toward a specific cell surface target expressed by tumor cells. The notion that antibodies directed toward targets on the surface of malignant cells could be used for drug delivery is not new. The history of antibody conjugates is marked by hurdles that have been identified and overcome. Early conjugates used mouse antibodies; cytotoxic agents that were immunogenic (proteins), too toxic, or not sufficiently potent; and linkers that were not sufficiently stable in circulation. Investigators have explored 4 main avenues using antibodies to target cytotoxic agents to malignant cells: antibody-protein toxin (or antibody fragment-protein toxin fusion) conjugates, antibody-chelated radionuclide conjugates, antibody-small-molecule drug conjugates, and antibody-enzyme conjugates administered along with small-molecule prodrugs that require metabolism by the conjugated enzyme to release the activated species. Only antibody-radionuclide conjugates and antibody-drug conjugates have reached the regulatory approval stage, and nearly 20 antibody conjugates are currently in clinical trials. The time may have come for this technology to become a major contributor to improving treatment for cancer patients.
Assuntos
Imunoconjugados/uso terapêutico , Neoplasias/terapia , Animais , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/administração & dosagem , Sistemas de Liberação de Medicamentos , Humanos , Imunotoxinas/uso terapêutico , Camundongos , Terapia de Alvo Molecular , RadioimunoterapiaRESUMO
A novel pathway for ex vivo maytansinoid release from thioether linked antibody maytansinoid conjugates (AMCs) upon incubation in human plasma has been identified. A thioether succinimide-linked AMC can undergo chemical oxidation followed by sulfoxide elimination under mild aqueous conditions (pH 5.5-7.5, 37 °C). Oxidized thioether-linked AMCs exhibit high, target-specific cytotoxicity toward cancer cells.
Assuntos
Anticorpos/química , Imunoconjugados/química , Maitansina/química , Linhagem Celular Tumoral , Humanos , Concentração de Íons de Hidrogênio , Imunoconjugados/sangue , Imunoconjugados/toxicidade , Maleimidas/química , Oxirredução , Polietilenoglicóis/química , Ácidos Sulfênicos/químicaRESUMO
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.
Assuntos
Anticorpos/química , Imunoconjugados/química , Maitansina/química , Animais , Química Farmacêutica/métodos , Desenho de Fármacos , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos , Modelos Químicos , Transplante de Neoplasias , Polietilenoglicóis/química , Succinimidas/química , Sulfonas/químicaRESUMO
In this report, we describe the synthesis of a panel of disulfide-linked huC242 (anti-CanAg) antibody maytansinoid conjugates (AMCs), which have varying levels of steric hindrance around the disulfide bond, in order to investigate the relationship between stability to reduction of the disulfide linker and antitumor activity of the conjugate in vivo. The conjugates were first tested for stability to reduction by dithiothreitol in vitro and for plasma stability in CD1 mice. It was found that the conjugates having the more sterically hindered disulfide linkages were more stable to reductive cleavage of the maytansinoid in both settings. When the panel of conjugates was tested for in vivo efficacy in two human colon cancer xenograft models in SCID mice, it was found that the conjugate with intermediate disulfide bond stability having two methyl groups on the maytansinoid side of the disulfide bond and no methyl groups on the linker side of the disulfide bond (huC242-SPDB-DM4) displayed the best efficacy. The ranking of in vivo efficacies of the conjugates was not predicted by their in vitro potencies, since all conjugates were highly active in vitro, including a huC242-SMCC-DM1 conjugate with a noncleavable linkage which showed only marginal activity in vivo. These data suggest that factors in addition to intrinsic conjugate potency and conjugate half-life in plasma influence the magnitude of antitumor activity observed for an AMC in vivo. We provide evidence that bystander killing of neighboring nontargeted tumor cells by diffusible cytotoxic metabolites produced from target cell processing of disulfide-linked antibody-maytansinoid conjugates may be one additional factor contributing to the activity of these conjugates in vivo.