RESUMO
Antibody-drug conjugates (ADCs) are a promising cancer treatment modality that enables the selective delivery of highly cytotoxic payloads to tumours. However, realizing the full potential of this platform necessitates innovative molecular designs to tackle several clinical challenges such as drug resistance, tumour heterogeneity and treatment-related adverse effects. Several emerging ADC formats exist, including bispecific ADCs, conditionally active ADCs (also known as probody-drug conjugates), immune-stimulating ADCs, protein-degrader ADCs and dual-drug ADCs, and each offers unique capabilities for tackling these various challenges. For example, probody-drug conjugates can enhance tumour specificity, whereas bispecific ADCs and dual-drug ADCs can address resistance and heterogeneity with enhanced activity. The incorporation of immune-stimulating and protein-degrader ADCs, which have distinct mechanisms of action, into existing treatment strategies could enable multimodal cancer treatment. Despite the promising outlook, the importance of patient stratification and biomarker identification cannot be overstated for these emerging ADCs, as these factors are crucial to identify patients who are most likely to derive benefit. As we continue to deepen our understanding of tumour biology and refine ADC design, we will edge closer to developing truly effective and safe ADCs for patients with treatment-refractory cancers. In this Review, we highlight advances in each ADC component (the monoclonal antibody, payload, linker and conjugation chemistry) and provide more-detailed discussions on selected examples of emerging novel ADCs of each format, enabled by engineering of one or more of these components.
Assuntos
Antineoplásicos , Imunoconjugados , Neoplasias , Humanos , Imunoconjugados/química , Anticorpos Monoclonais/efeitos adversos , Antineoplásicos/uso terapêutico , Preparações FarmacêuticasRESUMO
Valine-citrulline is a protease-cleavable linker commonly used in many drug delivery systems, including antibody-drug conjugates (ADC) for cancer therapy. However, its suboptimal in vivo stability can cause various adverse effects such as neutropenia and hepatotoxicity, leading to dose delays or treatment discontinuation. Here, we report that glutamic acid-glycine-citrulline (EGCit) linkers have the potential to solve this clinical issue without compromising the ability of traceless drug release and ADC therapeutic efficacy. We demonstrate that our EGCit ADC resists neutrophil protease-mediated degradation and spares differentiating human neutrophils. Notably, our anti-HER2 ADC shows almost no sign of blood and liver toxicity in healthy mice at 80 mg kg-1. In contrast, at the same dose level, the FDA-approved anti-HER2 ADCs Kadcyla and Enhertu show increased levels of serum alanine aminotransferase and aspartate aminotransferase and morphologic changes in liver tissues. Our EGCit conjugates also exert greater antitumor efficacy in multiple xenograft tumor models compared with Kadcyla and Enhertu. This linker technology could substantially broaden the therapeutic windows of ADCs and other drug delivery agents, providing clinical options with improved efficacy and safety.
Assuntos
Antineoplásicos , Imunoconjugados , Ado-Trastuzumab Emtansina , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Citrulina , Humanos , Imunoconjugados/metabolismo , Imunoconjugados/farmacologia , Camundongos , Peptídeo Hidrolases , Índice TerapêuticoRESUMO
Glioblastoma multiforme (GBM) is the most aggressive and fatal disease of all brain tumor types. Most therapies rarely provide clinically meaningful outcomes in the treatment of GBM. Although antibody-drug conjugates (ADCs) are promising anticancer drugs, no ADCs have been clinically successful for GBM, primarily because of poor blood-brain barrier (BBB) penetration. Here, we report that ADC homogeneity and payload loading rate are critical parameters contributing to this discrepancy. Although both homogeneous and heterogeneous conjugates exhibit comparable in vitro potency and pharmacokinetic profiles, the former shows enhanced payload delivery to brain tumors. Our homogeneous ADCs provide improved antitumor effects and survival benefits in orthotopic brain tumor models. We also demonstrate that overly drug-loaded species in heterogeneous conjugates are particularly poor at crossing the BBB, leading to deteriorated overall brain tumor targeting. Our findings indicate the importance of homogeneous conjugation with optimal payload loading in generating effective ADCs for intractable brain tumors.
Assuntos
Antineoplásicos , Neoplasias Encefálicas , Glioblastoma , Imunoconjugados , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Linhagem Celular Tumoral , Glioblastoma/tratamento farmacológico , Humanos , Imunoconjugados/farmacocinética , Imunoconjugados/uso terapêutico , Preparações FarmacêuticasRESUMO
Antibody-based therapy has shown great success in the treatment of many diseases, including cancers. While antibodies and antibody-drug conjugates (ADCs) have also been evaluated for central nervous system (CNS) disorders as well as brain tumors, their therapeutic efficacy can be substantially limited due to low permeability across the blood-brain barrier (BBB). Thus, improving BBB permeability of therapeutic antibodies is critical in establishing this drug class as a reliable clinical option for CNS diseases. Here, we report that, compared with a conventional heterogeneous conjugation, homogeneous conjugation of the synthetic BBB shuttle peptide angiopep-2 (Ang2) to a monoclonal antibody (mAb) provides improved binding affinity for brain microvascular endothelial cells in vitro and accumulation into normal brain tissues in vivo. In a mouse model, we also demonstrate that the homogeneous anti-EGFR mAb-Ang2 conjugate administered intravenously efficiently accumulates in intracranial tumors. These findings suggest that homogeneous conjugation of BBB shuttle peptides such as Ang2 is a promising approach to enhancing the therapeutic efficacy of antibody agents for CNS diseases.
RESUMO
Glioblastoma (GBM) is a common and aggressive brain cancer that accounts for 60% of adult brain tumors. Anti-angiogenesis therapy is an attractive option due to the high vasculature density of GBM. However, the best-known anti-angiogenic therapeutics, bevacizumab, and aflibercept, have failed to show significant benefits in GBM patients. One of the reasons is the limited brain penetration of antibody-based therapies due to existence of the blood-brain barrier (BBB), which is further strengthened by the blood vessel normalization effects induced by anti-angiogenic therapies. To investigate if increased drug concentration in the brain by transferrin receptor (TfR)-mediated delivery across the BBB can enhance efficacy of anti-angiogenic antibody therapies, we first identified an antibody that binds to the apical domain of the mouse TfR and does not compete with the natural ligand transferrin (Tf) binding to TfR. Then, we engineered two bispecific antibodies fusing a vascular endothelial growth factor (VEGF)-Trap with the TfR-targeting antibody. Characterization of the two bispecific formats using multiple in vitro assays, which include endocytosis, cell surface and whole-cell TfR levels, human umbilical vein endothelial cell growth inhibition, and binding affinity, demonstrated that the VEGF-Trap fused with a monovalent αTfR (VEGF-Trap/moAb4) has desirable endocytosis without the induction of TfR degradation. Peripherally administered VEGF-Trap/moAb4 improved the brain concentration of VEGF-Trap by more than 10-fold in mice. The distribution of VEGF-Trap/moAb4 was validated to be in the brain parenchyma, indicating the molecule was not trapped inside the vasculature. Moreover, improved VEGF-Trap brain distribution significantly inhibited the angiogenesis of U-87 MG GBM tumors in a mouse model.
Assuntos
Anticorpos Biespecíficos , Glioblastoma , Inibidores da Angiogênese/farmacologia , Animais , Anticorpos Biespecíficos/metabolismo , Glioblastoma/metabolismo , Humanos , Camundongos , Receptores da Transferrina , Receptores de Fatores de Crescimento do Endotélio Vascular , Proteínas Recombinantes de Fusão , Transferrina/metabolismo , Fator A de Crescimento do Endotélio VascularRESUMO
Breast tumors generally consist of a diverse population of cells with varying gene expression profiles. Breast tumor heterogeneity is a major factor contributing to drug resistance, recurrence, and metastasis after chemotherapy. Antibody-drug conjugates (ADCs) are emerging chemotherapeutic agents with striking clinical success, including T-DM1 for HER2-positive breast cancer. However, these ADCs often suffer from issues associated with intratumor heterogeneity. Here, we show that homogeneous ADCs containing two distinct payloads are a promising drug class for addressing this clinical challenge. Our conjugates show HER2-specific cell killing potency, desirable pharmacokinetic profiles, minimal inflammatory response, and marginal toxicity at therapeutic doses. Notably, a dual-drug ADC exerts greater treatment effect and survival benefit than does co-administration of two single-drug variants in xenograft mouse models representing intratumor HER2 heterogeneity and elevated drug resistance. Our findings highlight the therapeutic potential of the dual-drug ADC format for treating refractory breast cancer and perhaps other cancers.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/tratamento farmacológico , Imunoconjugados/farmacologia , Receptor ErbB-2/imunologia , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/complicações , Linhagem Celular Tumoral , Feminino , Humanos , Imunoconjugados/farmacocinética , Imunoconjugados/uso terapêutico , Imunoconjugados/toxicidade , Imuno-Histoquímica , Inflamação/complicações , Camundongos , Oligopeptídeos/farmacologia , Oligopeptídeos/uso terapêutico , Receptor ErbB-2/metabolismo , Trastuzumab/farmacologia , Trastuzumab/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Antibody-drug conjugates (ADCs) hold great therapeutic promise for cancer indications; however, treating tumors with intratumor heterogeneity remains challenging. We hypothesized that ADCs that can simultaneously target two different cancer antigens could address this issue. Here, we report controlled production and evaluation of bispecific ADCs chemically functionalized with tumor-targeting small molecules. Enzyme-mediated conjugation of bi-functional branched linkers and following sequential orthogonal click reactions with payload and tumor targeting modules (folic acid or RGD peptide) afforded homogeneous bispecific ADCs with defined ligand/drug-to-antibody ratios ranging from 4 + 4 to 16 + 4 (ligand/payload). Most bispecific ADCs were stable under physiological conditions for 14 days. Functionalization with the cancer-specific ligands did not impair cathepsin B-mediated payload release from ADCs. Bispecific ADCs targeting the folate receptor (FR)/human epidermal growth factor receptor 2 (HER2) demonstrated specific binding and high cell killing potency only in cells expressing either antigen (FR or HER2). Integrin/HER2 bispecific ADCs equipped with RGD peptides also showed target-specific binding and cytotoxicity in integrin- or HER2-positive cells. These findings suggest that our small-molecule based bispecific ADCs have the potential to effectively treat tumors with heterogeneous antigen expression.
Assuntos
Antineoplásicos/farmacologia , Receptor 1 de Folato/antagonistas & inibidores , Imunoconjugados/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Receptor 1 de Folato/metabolismo , Humanos , Imunoconjugados/química , Estrutura Molecular , Receptor ErbB-2/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
The architecturally symmetrical and synthetically challenging marine natural products lomaiviticins A and B present alluring synthetic targets due to their molecular complexity, potent antitumor properties, and natural scarcity. Herein, we report the total synthesis of the fully glycosylated monomeric unit of lomaiviticin A, monolomaiviticin A. The retrosynthetically derived synthetic strategy relied on an intramolecular palladium-catalyzed coupling reaction to complete the tetracyclic aglycon scaffold and gold-promoted glycosylations to install the synthetically challenging α- and ß-glycoside moieties of the target molecule. This accomplishment paves a path for the eventual total synthesis of lomaiviticins A and B and opens opportunities for biological investigations within this family of compounds.
RESUMO
We established a methodology for initiating cross-linking of antibodies selectively on the cell surface through intermolecular copper-free click reactions facilitated by increased effective concentrations of antibodies binding to target antigens. Upon cross-linking of tetrazine- and bicyclononyne-modified trastuzumab on the surface of HER2-overexpressing cells, increased antibody uptake and activation of intracellular signaling were observed. Our findings demonstrate that the cross-linking reaction can significantly alter the biophysical properties of proteins, activating their unique functionalities on targeted cells to realize an increased cargo delivery and synthetic manipulation of cellular signaling.
Assuntos
Compostos Aza/imunologia , Compostos Bicíclicos com Pontes/imunologia , Reagentes de Ligações Cruzadas/química , Trastuzumab/imunologia , Células 3T3 , Animais , Compostos Aza/química , Compostos Bicíclicos com Pontes/química , Linhagem Celular Tumoral , Humanos , Camundongos , Estrutura Molecular , Receptor ErbB-2/química , Receptor ErbB-2/imunologia , Propriedades de Superfície , Trastuzumab/químicaRESUMO
Acute myeloid leukemia (AML) is the most common and aggressive blood cancer in adults. In particular, significant unmet medical needs exist for effective treatment strategies for acute myelomonocytic leukemia (M4) and acute monocytic leukemia (M5) AML subtypes. Antibody-drug conjugates (ADC) are a promising drug class for AML therapy, as demonstrated by the FDA-approved anti-CD33 ADC, gemtuzumab ozogamicin (Mylotarg). However, CD33 is expressed in normal hematopoietic stem cells, highlighting the critical need to identify AML-specific targets to minimize the risk of potential adverse effects. We have demonstrated that the leukocyte immunoglobulin-like receptor subfamily B4 (LILRB4) is expressed at significantly higher levels on monocytic M4 and M5 AML cells than on normal counterparts. Here, we test whether LILRB4 is a promising ADC target to kill monocytic AML cells while sparing healthy counterparts. To this end, we generated ADCs from a humanized anti-LILRB4 mAb and the antimitotic payload, monomethyl auristatin F. The conjugates constructed were characterized and evaluated for LILRB4-specific cell killing potency, toxicity to progenitor cells, pharmacokinetics, and therapeutic efficacy. Our ADC linker technology platform efficiently generated homogeneous anti-LILRB4 ADCs with defined drug-to-antibody ratios. The homogeneous anti-LILRB4 ADCs demonstrated the capacity for LILRB4-mediated internalization, suitable physicochemical properties, and high cell killing potency against LILRB4-positive AML cells. Importantly, our data indicate that these ADCs spare normal progenitor cells. One of our homogeneous conjugates exerted a remarkable therapeutic effect and no significant toxicity in a xenograft mouse model of disseminated human AML. Our findings highlight the clinical potential of anti-LILRB4 ADCs in monocytic AML therapy.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Imunoconjugados/farmacologia , Glicoproteínas de Membrana/antagonistas & inibidores , Receptores Imunológicos/antagonistas & inibidores , Animais , Antineoplásicos Imunológicos/química , Linhagem Celular Tumoral , Modelos Animais de Doenças , Estabilidade de Medicamentos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunoconjugados/química , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/etiologia , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Camundongos , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Microbial transglutaminase (MTGase) catalyzes site-specific transpeptidation between a primary amine within linkers and the side chain of glutamine 295 within deglycosylated chimeric, humanized, and human IgG1s, affording homogeneous antibody-drug conjugates (ADCs). This method can be empowered by mutation of asparagine 297, insertion of a glutamine-containing peptide tag, and the use of branched linkers. Such modifications facilitate the conjugation process and provide flexibility in adjusting the conjugation site and drug-to-antibody ratio (DAR). Here, we present a protocol optimized in our group for MTGase-mediated linker incorporation and subsequent click chemistry-based payload installation. Both small linear linkers and bulky branched linkers can be incorporated into the Fc moiety within various antibodies, affording homogeneous ADCs with defined DARs. Thanks to the high homogeneity, ADCs constructed using this method can be analyzed using a single-quadrupole electrospray ionization (ESI) mass spectrometer, which many laboratories own for regular analysis of small molecules and peptides. The approach presented here allows for facile and cost-effective production of various homogeneous ADCs and other antibody conjugates for research and clinical purposes.
Assuntos
Anticorpos Monoclonais/química , Imunoconjugados/química , Transglutaminases/química , Transglutaminases/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Cromatografia Líquida , Humanos , Espectrometria de Massas , Relação Estrutura-AtividadeRESUMO
Therapeutic strategies are urgently needed for patients with acute myeloid leukemia (AML). Leukocyte immunoglobulin-like receptor B4 (LILRB4), which suppresses T-cell activation and supports tissue infiltration of AML cells, represents an attractive drug target for anti-AML therapeutics. Here, we report the identification and development of an LILRB4-specific humanized mAb that blocks LILRB4 activation. This mAb, h128-3, showed potent activity in blocking the development of monocytic AML in various models including patient-derived xenograft mice and syngeneic immunocompetent AML mice. MAb h128-3 enhanced the anti-AML efficacy of chemotherapy treatment by stimulating mobilization of leukemia cells. Mechanistic studies revealed four concordant modes of action for the anti-AML activity of h128-3: (i) reversal of T-cell suppression, (ii) inhibition of monocytic AML cell tissue infiltration, (iii) antibody-dependent cellular cytotoxicity, and (iv) antibody-dependent cellular phagocytosis. Therefore, targeting LILRB4 with antibody represents an effective therapeutic strategy for treating monocytic AML.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Apolipoproteínas E/metabolismo , Transformação Celular Neoplásica/imunologia , Transformação Celular Neoplásica/metabolismo , Leucemia Mieloide Aguda/etiologia , Leucemia Mieloide Aguda/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Imunológicos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Anticorpos Bloqueadores , Anticorpos Monoclonais , Citotoxicidade Celular Dependente de Anticorpos , Antineoplásicos Imunológicos/uso terapêutico , Apolipoproteínas E/química , Apoptose , Linhagem Celular , Transformação Celular Neoplásica/efeitos dos fármacos , Técnicas de Cocultura , Modelos Animais de Doenças , Humanos , Leucemia Mieloide Aguda/patologia , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/química , Camundongos , Camundongos Knockout , Modelos Biológicos , Modelos Moleculares , Ligação Proteica/efeitos dos fármacos , Coelhos , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/química , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Antibody-drug conjugates (ADCs), monoclonal antibodies conjugated with highly potent drugs (payloads) through chemical linkers, are an emerging class of therapeutic agents for cancer chemotherapy. Their clinical success has been demonstrated by the 4 ADCs already approved by the U.S. Food and Drug Administration (FDA), and more than 60 promising ADCs now in clinical trials. Further advancement of this novel molecular platform could potentially revolutionize current strategies and regimens for treating cancers. The linker structure and antibody-linker conjugation modality critically contribute to ADC homogeneity, circulation stability, pharmacokinetic profiles, tolerability, and overall treatment efficacy. Despite extensive efforts to improve these parameters, most ADC linkers used to date possess linear structures, and therefore accommodate only single payloads. The clinical potential of branched ADC linkers, enabling the installation of two payload molecules, remains unexplored because of the lack of efficient conjugation methods. In addition, according to a recent report, the stability of enzymatically cleavable linkers in mouse circulation is another crucial factor for the successful evaluation of ADCs in preclinical studies. In this review, I present my research group's effort to develop both branched linkers and efficient conjugation methods for constructing dual-loading ADCs with high homogeneity and enhanced potency. I also present a novel tripeptide ADC linker with enhanced stability in mouse circulation. Multidisciplinary experience, approaches, and collaboration are key to successfully advancing our ADC research programs. I herein describe how my experience in the U.S. has helped to develop and manage complex biomedical research projects in a small academic laboratory setting.
Assuntos
Descoberta de Drogas/métodos , Imunoconjugados/química , Animais , Anticorpos Monoclonais/química , Antineoplásicos , Pesquisa Biomédica , Sistemas de Liberação de Medicamentos , Descoberta de Drogas/tendências , Humanos , Pesquisa Interdisciplinar , Japão , Camundongos , Estados UnidosRESUMO
Valine-citrulline linkers are commonly used as enzymatically cleavable linkers for antibody-drug conjugates. While stable in human plasma, these linkers are unstable in mouse plasma due to susceptibility to an extracellular carboxylesterase. This instability often triggers premature release of drugs in mouse circulation, presenting a molecular design challenge. Here, we report that an antibody-drug conjugate with glutamic acid-valine-citrulline linkers is responsive to enzymatic drug release but undergoes almost no premature cleavage in mice. We demonstrate that this construct exhibits greater treatment efficacy in mouse tumor models than does a valine-citrulline-based variant. Notably, our antibody-drug conjugate contains long spacers facilitating the protease access to the linker moiety, indicating that our linker assures high in vivo stability despite a high degree of exposure. This technology could add flexibility to antibody-drug conjugate design and help minimize failure rates in pre-clinical studies caused by linker instability.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Ácido Glutâmico/química , Imunoconjugados/farmacologia , Oligopeptídeos/química , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacocinética , Antineoplásicos/química , Antineoplásicos/farmacocinética , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Hidrolases de Éster Carboxílico/sangue , Linhagem Celular Tumoral , Citrulina/química , Desenho de Fármacos , Estabilidade de Medicamentos , Feminino , Expressão Gênica , Humanos , Hidrólise , Imunoconjugados/química , Imunoconjugados/farmacocinética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Análise de Sobrevida , Valina/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The antibody-drug conjugate (ADC), a humanized or human monoclonal antibody conjugated with highly cytotoxic small molecules (payloads) through chemical linkers, is a novel therapeutic format and has great potential to make a paradigm shift in cancer chemotherapy. This new antibody-based molecular platform enables selective delivery of a potent cytotoxic payload to target cancer cells, resulting in improved efficacy, reduced systemic toxicity, and preferable pharmacokinetics (PK)/pharmacodynamics (PD) and biodistribution compared to traditional chemotherapy. Boosted by the successes of FDA-approved Adcetris® and Kadcyla®, this drug class has been rapidly growing along with about 60 ADCs currently in clinical trials. In this article, we briefly review molecular aspects of each component (the antibody, payload, and linker) of ADCs, and then mainly discuss traditional and new technologies of the conjugation and linker chemistries for successful construction of clinically effective ADCs. Current efforts in the conjugation and linker chemistries will provide greater insights into molecular design and strategies for clinically effective ADCs from medicinal chemistry and pharmacology standpoints. The development of site-specific conjugation methodologies for constructing homogeneous ADCs is an especially promising path to improving ADC design, which will open the way for novel cancer therapeutics.
Assuntos
Anticorpos Monoclonais/química , Imunoconjugados/química , Aminoácidos/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Antígenos/metabolismo , Engenharia Genética , Humanos , Imunoconjugados/metabolismoRESUMO
Antibody-drug conjugates (ADCs) are emerging therapeutic agents in the treatment of cancer, and various conjugation strategies and chemical linkers have been developed to efficiently construct ADCs. Despite previous extensive efforts for improving conjugation efficiency and ADC homogeneity, most ADC linkers developed to date load only single payloads. Branched linkers that can load multiple payload molecules have yet to be fully explored. It is logical to envisage that a multi-loading strategy allows for increase in drug-to-antibody ratio (DAR) with less chemical or enzymatic modification to the antibody structure compared to traditional linear linkers, leading to efficient ADC construction, minimal destabilization of the antibody structure, and enhanced ADC efficacy. Herein, we report that the branched linkers we designed can be quantitatively installed on an anti-HER2 monoclonal antibody by microbial transglutaminase (MTGase)-mediated conjugation without impairing its antigen binding affinity, enabling modular installation of payload molecules and construction of homogeneous ADCs with increased DARs (up to 8). An anti-HER2 antibody-monomethyl auristatin F conjugate constructed using our branched linkers showed greater in vitro cytotoxicity against HER2-expressing breast cancer cell lines than that consisting of linear linkers, demonstrating the effectiveness of the branched linker-based payload delivery. Our finding demonstrates that enzymatic ADC construction using branched linkers is a promising strategy, which may lead to innovative cancer therapeutics.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Oligopeptídeos/farmacologia , Transglutaminases/química , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Química Click , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/biossíntese , Relação Estrutura-Atividade , Transglutaminases/metabolismoRESUMO
The accessory gene regulator (agr) of Staphylococcus aureus coordinates various pathogenic events and is recognized as a promising therapeutic target for virulence control. S. aureus utilizes autoinducing peptides (AIPs), cyclic-peptide signaling molecules, to mediate the agr system. Despite the high potency of synthetic AIP analogues in agr inhibition, the potential of AIP molecules as a delivery vehicle for antibacterial agents remains unexplored. Herein, we report that truncated AIP scaffolds can be fused with fluorophore and cytotoxic photosensitizer molecules without compromising their high agr inhibitory activity, binding affinity to the receptor AgrC, or cell specificity. Strikingly, a photosensitizer-AIP conjugate exhibited 16-fold greater efficacy in a S. aureus cell-killing assay than a nontargeting analogue. These findings highlight the potential of truncated AIP conjugates as useful chemical tools for in-depth biological studies and as effective anti-S. aureus agents.