RESUMO
What happens to macromolecules in vivo? What drives the structure-activity relationship and in vivo stability for antibody-drug conjugates (ADCs)? These interrelated questions are increasingly relevant due to the re-emerging importance of ADCs as an impactful therapeutic modality and the gaps that exist in our understanding of ADC structural determinants that underlie ADC in vivo stability. Complex macromolecules, such as ADCs, may undergo changes in vivo due to their intricate structure as biotransformations may occur on the linker, the payload, and/or at the modified conjugation site. Furthermore, the dissection of ADC metabolism presents a substantial analytical challenge due to the difficulty in the identification or quantification of minor changes on a large macromolecule. We employed immunocapture-LCMS methods to evaluate in vivo changes in the drug-antibody ratio (DAR) profile in four different lead ADCs. This comprehensive characterization revealed that a critical structural determinant contributing to the ADC design was the linker, and competition of the thio-succinimide hydrolysis reaction over retro-Michael deconjugation can result in superb conjugation stability in vivo. These data, in conjunction with additional factors, informed the selection of AZD8205, puxitatug samrotecan, a B7-H4-directed cysteine-conjugated ADC bearing a novel topoisomerase I inhibitor payload, with durable DAR, currently being studied in the clinic for the potential treatment of solid malignancies (NCT05123482). These results highlight the relevance of studying macromolecule biotransformation and elucidating the ADC structure-in vivo stability relationship. The comprehensive nature of this work increases our confidence in the understanding of these processes. We hope this analytical approach can inform future development of bioconjugate drug candidates.
Assuntos
Biotransformação , Imunoconjugados , Imunoconjugados/metabolismo , Imunoconjugados/química , Animais , Camundongos , Humanos , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/metabolismo , Estabilidade de Medicamentos , Feminino , Camptotecina/análogos & derivadosRESUMO
MEDI2228 is an antibody drug conjugate (ADC) comprised of a fully human B-cell maturation antigen (BCMA) antibody conjugated to a pyrrolobenzodiazepine (PBD) dimer. This phase 1 trial evaluated MEDI2228 in patients with relapsed/refractory (R/R) multiple myeloma (MM), who received prior treatment with approved agents from 3 classes of antimyeloma drugs (proteasome inhibitors, immunomodulatory drugs, and monoclonal antibodies). Primary endpoint was safety and tolerability; secondary endpoints included efficacy, pharmacokinetics, and immunogenicity. A total of 107 patients were treated and the maximum tolerated dose (MTD) was 0.14 mg/kg Q3W. Two patients had dose-limiting toxicities (DLTs; thrombocytopenia; 0.20 mg/kg Q3W). The most frequent treatment-related adverse events were photophobia (43.9%), rash (29.0%), and thrombocytopenia (19.6%). In MTD cohort A (n = 41), the objective response rate (ORR) was 56.1%, with 1 stringent complete response, 9 very good partial responses, and 13 partial responses. ORR was 53.3% in triple refractory patients. In cohort B (n=25), ORR was 32%. Although MEDI2228 demonstrated efficacy in R/R MM, ocular toxicity precluded further development of this drug.
MEDI2228 is an ADC comprised of a fully human anti-BCMA antibody conjugated to the cytotoxic PBD payload, tesirine.MEDI2228 monotherapy demonstrated efficacy across all dose levels; and responses were observed in patients with triple refractory MM.This study further validates BCMA as a target for ADC-based therapy in MM; ocular toxicity precluded further clinical development.
RESUMO
PURPOSE: Platinum and PARP inhibitors (PARPi) demonstrate activity in breast and ovarian cancers, but drug resistance ultimately emerges. Here, we examine B7-H4 expression in primary and recurrent high-grade serous ovarian carcinoma (HGSOC) and the activity of a B7-H4-directed antibody-drug conjugate (B7-H4-ADC), using a pyrrolobenzodiazepine-dimer payload, in PARPi- and platinum-resistant HGSOC patient-derived xenograft (PDX) models. EXPERIMENTAL DESIGN: B7-H4 expression was quantified by flow cytometry and IHC. B7-H4-ADC efficacy was tested against multiple cell lines in vitro and PDX in vivo. The effect of B7-H4-ADC on cell cycle, DNA damage, and apoptosis was measured using flow cytometry. RESULTS: B7-H4 is overexpressed in 92% of HGSOC tumors at diagnosis (n = 12), persisted in recurrent matched samples after platinum treatment, and was expressed at similar levels across metastatic sites after acquired multi-drug resistance (n = 4). Treatment with B7-H4-ADC resulted in target-specific growth inhibition of multiple ovarian and breast cancer cell lines. In platinum- or PARPi-resistant ovarian cancer cells, B7-H4-ADC significantly decreased viability and colony formation while increasing cell-cycle arrest and DNA damage, ultimately leading to apoptosis. Single-dose B7-H4-ADC led to tumor regression in 65.5% of breast and ovarian PDX models (n = 29), with reduced activity in B7-H4 low or negative models. In PARPi and platinum-resistant HGSOC PDX models, scheduled B7-H4-ADC dosing led to sustained tumor regression and increased survival. CONCLUSIONS: These data support B7-H4 as an attractive ADC target for treatment of drug-resistant HGSOC and provide evidence for activity of an ADC with a DNA-damaging payload in this population. See related commentary by Veneziani et al., p. 1434.
Assuntos
Imunoconjugados , Neoplasias Ovarianas , Feminino , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Platina/farmacologia , Platina/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Apoptose , Carcinoma Epitelial do Ovário/tratamento farmacológico , Linhagem Celular TumoralRESUMO
PURPOSE: Adenoid cystic carcinoma (ACC) is a heterogeneous malignancy, and no effective systemic therapy exists for metastatic disease. We previously described two prognostic ACC molecular subtypes with distinct therapeutic vulnerabilities, ACC-I and ACC-II. In this study, we explored the ACC tumor microenvironment (TME) using RNA-sequencing and spatial biology to identify potential therapeutic targets. EXPERIMENTAL DESIGN: Tumor samples from 62 ACC patients with available RNA-sequencing data that had been collected as part of previous studies were stained with a panel of 28 validated metal-tagged antibodies. Imaging mass cytometry (IMC) was performed using the Fluidigm Helios CyTOF instrument and analyzed with Visiopharm software. The B7-H4 antibody-drug conjugate AZD8205 was tested in ACC patient-derived xenografts (PDX). RESULTS: RNA deconvolution revealed that most ACCs are immunologically "cold," with approximately 30% being "hot." ACC-I tumors with a poor prognosis harbored a higher density of immune cells; however, spatial analysis by IMC revealed that ACC-I immune cells were significantly restricted to the stroma, characterizing an immune-excluded TME. ACC-I tumors overexpressed the immune checkpoint B7-H4, and the degree of immune exclusion was directly correlated with B7-H4 expression levels, an independent predictor of poor survival. Two ACC-I/B7-H4-high PDXs obtained 90% complete responses to a single dose of AZD8205, but none were observed with isotype-conjugated payload or in an ACC-II/B7-H4 low PDX. CONCLUSIONS: Spatial analysis revealed that ACC subtypes have distinct TMEs, with enrichment of ACC-I immune cells that are restricted to the stroma. B7-H4 is highly expressed in poor-prognosis ACC-I subtype and is a potential therapeutic target.
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Carcinoma Adenoide Cístico , Humanos , Carcinoma Adenoide Cístico/tratamento farmacológico , Carcinoma Adenoide Cístico/genética , Inibidor 1 da Ativação de Células T com Domínio V-Set , Prognóstico , Biomarcadores Tumorais , Microambiente TumoralRESUMO
PURPOSE: We evaluated the activity of AZD8205, a B7-H4-directed antibody-drug conjugate (ADC) bearing a novel topoisomerase I inhibitor (TOP1i) payload, alone and in combination with the PARP1-selective inhibitor AZD5305, in preclinical models. EXPERIMENTAL DESIGN: IHC and deep-learning-based image analysis algorithms were used to assess prevalence and intratumoral heterogeneity of B7-H4 expression in human tumors. Several TOP1i-ADCs, prepared with Val-Ala or Gly-Gly-Phe-Gly peptide linkers, with or without a PEG8 spacer, were compared in biophysical, in vivo efficacy, and rat toxicology studies. AZD8205 mechanism of action and efficacy studies were conducted in human cancer cell line and patient-derived xenograft (PDX) models. RESULTS: Evaluation of IHC-staining density on a per-cell basis revealed a range of heterogeneous B7-H4 expression across patient tumors. This informed selection of bystander-capable Val-Ala-PEG8-TOP1i payload AZ14170133 and development of AZD8205, which demonstrated improved stability, efficacy, and safety compared with other linker-payload ADCs. In a study of 26 PDX tumors, single administration of 3.5 mg/kg AZD8205 provided a 69% overall response rate, according to modified RECIST criteria, which correlated with homologous recombination repair (HRR) deficiency (HRD) and elevated levels of B7-H4 in HRR-proficient models. Addition of AZD5305 sensitized very low B7-H4-expressing tumors to AZD8205 treatment, independent of HRD status and in models representing clinically relevant mechanisms of PARPi resistance. CONCLUSIONS: These data provide evidence for the potential utility of AZD8205 for treatment of B7-H4-expressing tumors and support the rationale for an ongoing phase 1 clinical study (NCT05123482). See related commentary by Pommier and Thomas, p. 991.
Assuntos
Imunoconjugados , Neoplasias , Ratos , Humanos , Animais , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Inibidores da Topoisomerase I , Neoplasias/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Poli(ADP-Ribose) Polimerase-1/genéticaRESUMO
PURPOSE: Efforts are required to improve the potency and durability of CD38- and BCMA-based immunotherapies in human multiple myeloma. We here delineated the molecular and cellular mechanisms underlying novel immunomodulatory effects triggered by BCMA pyrrolobenzodiazepine (PBD) antibody drug conjugate (ADC) MEDI2228 which can augment efficacy of these immunotherapies. EXPERIMENTAL DESIGN: MEDI2228-induced transcriptional and protein changes were investigated to define significantly impacted genes and signaling cascades in multiple myeloma cells. Mechanisms whereby MEDI2228 combination therapies can enhance cytotoxicity or overcome drug resistance in multiple myeloma cell lines and patient multiple myeloma cells were defined using in vitro models of tumor in the bone marrow (BM) microenvironment, as well as in human natural killer (NK)-reconstituted NOD/SCID gamma (NSG) mice bearing MM1S tumors. RESULTS: MEDI2228 enriched IFN I signaling and enhanced expression of IFN-stimulated genes in multiple myeloma cell lines following the induction of DNA damage-ATM/ATR-CHK1/2 pathways. It activated cGAS-STING-TBK1-IRF3 and STAT1-IRF1-signaling cascades and increased CD38 expression in multiple myeloma cells but did not increase CD38 expression in BCMA-negative NK effector cells. It overcame CD38 downregulation on multiple myeloma cells triggered by IL6 and patient BM stromal cell-culture supernatant via activation of STAT1-IRF1, even in immunomodulatory drug (IMiD)- and bortezomib-resistant multiple myeloma cells. In vitro and in vivo upregulation of NKG2D ligands and CD38 in MEDI2228-treated multiple myeloma cells was further associated with synergistic daratumumab (Dara) CD38 MoAb-triggered NK-mediated cytotoxicity of both cell lines and autologous drug-resistant patient multiple myeloma cells. CONCLUSIONS: These results provide the basis for clinical evaluation of combination MEDI2228 with Dara to further improve patient outcome in multiple myeloma.
Assuntos
Imunoconjugados , Mieloma Múltiplo , ADP-Ribosil Ciclase 1 , Animais , Anticorpos Monoclonais , Antígeno de Maturação de Linfócitos B , Linhagem Celular Tumoral , Humanos , Imunidade , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Glicoproteínas de Membrana , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Microambiente TumoralRESUMO
To target mechanisms critical for multiple myeloma (MM) plasma cell adaptations to genomic instabilities and further sustain MM cell killing, we here specifically trigger DNA damage response (DDR) in MM cells by a novel BCMA antibody-drug conjugate (ADC) delivering the DNA cross-linking PBD dimer tesirine, MEDI2228. MEDI2228, more effectively than its anti-tubulin MMAF-ADC homolog, induces cytotoxicity against MM cells regardless of drug resistance, BCMA levels, p53 status, and the protection conferred by bone marrow stromal cells and IL-6. Distinctly, prior to apoptosis, MEDI2228 activates DDRs in MM cells via phosphorylation of ATM/ATR kinases, CHK1/2, CDK1/2, and H2AX, associated with expression of DDR-related genes. Significantly, MEDI2228 synergizes with DDR inhibitors (DDRi s) targeting ATM/ATR/WEE1 checkpoints to induce MM cell lethality. Moreover, suboptimal doses of MEDI2228 and bortezomib (btz) synergistically trigger apoptosis of even drug-resistant MM cells partly via modulation of RAD51 and accumulation of impaired DNA. Such combination further induces superior in vivo efficacy than monotherapy via increased nuclear γH2AX-expressing foci, irreversible DNA damages, and tumor cell death, leading to significantly prolonged host survival. These results indicate leveraging MEDI2228 with DDRi s or btz as novel combination strategies, further supporting ongoing clinical development of MEDI2228 in patients with relapsed and refractory MM.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Antígeno de Maturação de Linfócitos B/imunologia , Bortezomib/uso terapêutico , Proteínas de Ciclo Celular/antagonistas & inibidores , Imunoconjugados/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Proteínas Tirosina Quinases/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Benzodiazepinas/administração & dosagem , Linhagem Celular Tumoral , Dano ao DNA , Sinergismo Farmacológico , Humanos , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Pirróis/administração & dosagemRESUMO
Experimental procedures and 1H and 13C NMR of the heterotrifunctional linker used for preparation of dual drug conjugates and PBD payload are included. Procedure for carrying preparation of antibody linker conjugate via thiol maleimide conjugation and antibody drug conjugates (ADCs) using copper assisted click reaction and oxime ligation, their cell viability assay and western blotting procedures of the resultant conjugates are detailed. Also, reduced mass spectroscopy results and in vitro cytotoxicity of antibody drug conjugates used in this article are shown.
RESUMO
Codelivery of multiple therapeutic agents with different anticancer mechanisms can overcome drug resistance as well as generate additive or synergistic anticancer effects that may enhance the antitumor efficacy. Antibody-drug conjugates (ADCs) can be used for highly specific delivery of multiple therapeutic agents with different anticancer mechanisms, though more research is required towards designing flexible platforms on which dual drug ADCs could be prepared. Herein, we describe the synthesis of a heterotrifunctional linker that could be used to construct flexible platforms for preparing dual-cytotoxic drug conjugates in a site-specific manner. As a proof of concept, we synthesized dual drug ADCs carrying monomethyl auristain E (MMAE, tubulin polymerization inhibitor) and pyrrolobenzodiazepine dimer (PBD, DNA minor groove alkylator). We then evaluated the dual drug ADCs for in vitro efficacy and confirmed the dual mechanism of action.
Assuntos
Imunoconjugados/química , Moduladores de Tubulina/química , Aminobenzoatos/química , Anticorpos Monoclonais/química , Antineoplásicos Alquilantes/química , Benzodiazepinas/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Química Click , Humanos , Imunoconjugados/farmacologia , Oligopeptídeos/química , Pirróis/químicaRESUMO
PURPOSE: Antibody-drug conjugates (ADC) utilizing noncleavable linker drugs have been approved for clinical use, and several are in development targeting solid and hematologic malignancies including multiple myeloma. Currently, there are no reliable biomarkers of activity for these ADCs other than presence of the targeted antigen. We observed that certain cell lines are innately resistant to such ADCs, and sought to uncover the underlying mechanism of resistance. EXPERIMENTAL DESIGN: The expression of 43 lysosomal membrane target genes was evaluated in cell lines resistant to ADCs bearing the noncleavable linker, pyrrolobenzodiazepine payload SG3376, in vitro. The functional relevance of SLC46A3, a lysosomal transporter of noncleavable ADC catabolites whose expression uniquely correlated with SG3376 resistance, was assessed using EPHA2-, HER2-, and BCMA-targeted ADCs and isogenic cells overexpressing or genetically inactivated for SLC46A3. SLC46A3 expression was also examined in patient-derived xenograft and in vitro models of acquired T-DM1 resistance and multiple myeloma bone marrow samples by RT-PCR. RESULTS: Loss of SLC46A3 expression was found to be a mechanism of innate and acquired resistance to ADCs bearing DM1 and SG3376. Sensitivity was restored in refractory lines upon introduction of SLC46A3, suggesting that expression of SLC46A3 may be more predictive of activity than target antigen levels alone. Interrogation of primary multiple myeloma samples indicated a range of SLC46A3 expression, including samples with undetectable levels like multiple myeloma cell lines resistant to BCMA-targeting DM1 and SG3376 ADCs. CONCLUSIONS: Our findings support SLC46A3 as a potential patient selection biomarker with immediate relevance to clinical trials involving these ADCs.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Benzodiazepinas/farmacologia , Biomarcadores , Imunoconjugados/farmacologia , Maitansina/farmacologia , Pirróis/farmacologia , Animais , Antineoplásicos Imunológicos/química , Benzodiazepinas/química , Linhagem Celular Tumoral , Modelos Animais de Doenças , Expressão Gênica , Inativação Gênica , Humanos , Imunoconjugados/química , Maitansina/química , Melanoma Experimental , Camundongos , Pirróis/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Antibody-drug conjugates (ADCs) are a class of biopharmaceuticals that combine the specificity of antibodies with the high-potency of cytotoxic drugs. Engineering cysteine residues in the antibodies using mutagenesis is a common method to prepare site-specific ADCs. With this approach, solvent accessible amino acids in the antibody have been selected for substitution with cysteine for conjugating maleimide-bearing cytotoxic drugs, resulting in homogeneous and stable site-specific ADCs. Here we describe a cysteine engineering approach based on the insertion of cysteines before and after selected sites in the antibody, which can be used for site-specific preparation of ADCs. Cysteine-inserted antibodies have expression level and monomeric content similar to the native antibodies. Conjugation to a pyrrolobenzodiazepine dimer (SG3249) resulted in comparable efficiency of site-specific conjugation between cysteine-inserted and cysteine-substituted antibodies. Cysteine-inserted ADCs were shown to have biophysical properties, FcRn, and antigen binding affinity similar to the cysteine-substituted ADCs. These ADCs were comparable for serum stability to the ADCs prepared using cysteine-mutagenesis and had selective and potent cytotoxicity against human prostate cancer cells. Two of the cysteine-inserted variants abolish binding of the resulting ADCs to FcγRs in vitro, thereby potentially preventing non-target mediated uptake of the ADCs by cells of the innate immune system that express FcγRs, which may result in mitigating off-target toxicities. A selected cysteine-inserted ADC demonstrated potent dose-dependent anti-tumor activity in a xenograph tumor mouse model of human breast adenocarcinoma expressing the oncofetal antigen 5T4.
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Anticorpos Monoclonais/química , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Cisteína/química , Animais , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/uso terapêutico , Linhagem Celular Tumoral , Feminino , Humanos , Imunoconjugados/química , Imunoconjugados/uso terapêutico , Neoplasias Mamárias Experimentais/tratamento farmacológico , Camundongos , Camundongos Nus , Trastuzumab/química , Trastuzumab/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
HER3/ERBB3 is a kinase-deficient member of the EGFR family receptor tyrosine kinases (RTK) that is broadly expressed and activated in human cancers. HER3 is a compelling cancer target due to its important role in activation of the oncogenic PI3K/AKT pathway. It has also been demonstrated to confer tumor resistance to a variety of cancer therapies, especially targeted drugs against EGFR and HER2. HER3 can be activated by its ligand (heregulin/HRG), which induces HER3 heterodimerization with EGFR, HER2, or other RTKs. Alternatively, HER3 can be activated in a ligand-independent manner through heterodimerization with HER2 in HER2-amplified cells. We developed a fully human mAb against HER3 (KTN3379) that efficiently suppressed HER3 activity in both ligand-dependent and independent settings. Correspondingly, KTN3379 inhibited tumor growth in divergent tumor models driven by either ligand-dependent or independent mechanisms in vitro and in vivo Most intriguingly, while investigating the mechanistic underpinnings of tumor response to KTN3379, we discovered an interesting dichotomy in that PTEN loss, a frequently occurring oncogenic lesion in a broad range of cancer types, substantially blunted the tumor response in HER2-amplified cancer, but not in the ligand-driven cancer. To our knowledge, this represents the first study ascertaining the impact of PTEN loss on the antitumor efficacy of a HER3 mAb. KTN3379 is currently undergoing a phase Ib clinical trial in patients with advanced solid tumors. Our current study may help us optimize patient selection schemes for KTN3379 to maximize its clinical benefits. Mol Cancer Ther; 15(4); 689-701. ©2016 AACR.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Neoplasias/metabolismo , Receptor ErbB-3/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Expressão Gênica , Humanos , Ligantes , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosforilação , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Receptor ErbB-3/química , Receptor ErbB-3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
EphB4 is a member of the Eph receptor tyrosine kinase family shown to act in neuronal guidance and mediate venal/arterial separation. In contrast to these more established roles, EphB4's function in cancer is much less clear. Here we illustrate both tumor promoting as well as suppressing roles of EphB4, by showing that its activation resulted in inhibition of the Ras/ERK pathway in endothelial cells but activation of the same pathway in MCF-7 breast cancer cells. This was true if EphB4 was stimulated with EphrinB2, its natural ligand, or an agonistic monoclonal antibody for EphB4. Correspondingly, EphB4 activation stimulated MCF7 growth while inhibiting HUVEC cell proliferation. The reason for these dramatic differences is due to functional coupling of EphB4 to different downstream effectors. Reduction of p120 RasGAP in HUVEC cells attenuated the inhibitory effect of EphB4 activation on the ERK pathway, whereas knockdown of PP2A in MCF7 cells attenuated EphB4 activation of the ERK pathway. This represents the first time a functional coupling between Eph receptor and PP2A has been demonstrated leading to activation of an oncogenic pathway. Our study illustrates the caveats and potential challenges of targeting EphB4 for cancer therapy due to the conflicting effects on cancer cell and endothelial cell compartments.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Receptor EphB4/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Efrina-B2/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias/enzimologia , Receptor EphB4/agonistas , Receptor EphB4/imunologiaRESUMO
The EphA2 receptor tyrosine kinase is selectively expressed on the surface of many different human tumors. We have previously shown that tumor cells can be targeted by EphA2 monoclonal antibodies and that these antibodies function, in part, by inducing EphA2 internalization and degradation. In this report, we describe the isolation and characterization of a fully human monoclonal antibody (1C1) that selectively binds both the human and rodent EphA2 receptor. After cell binding, the antibody induces rapid tyrosine phosphorylation, internalization, and degradation of the EphA2 receptor. Because monoclonal antibodies that selectively bind tumor cells and internalize provide a vehicle for targeted delivery of cytotoxics, 1C1 was conjugated to the microtubule inhibitor monomethylauristatin phenylalanine using a stable maleimidocaproyl linker. The anti-EphA2 antibody-drug conjugate [1C1-maleimidocaproyl-MMAF (mcMMAF)] stimulated the activation of caspase-3/caspase-7 and the death of EphA2-expressing cells with IC(50) values as low as 3 ng/mL. Similarly, the conjugate induced degradation of the EphA2 receptor and inhibited tumor growth in vivo. Administration of 1C1-mcMMAF at doses as low as 1 mg/kg once weekly resulted in significant growth inhibition of EphA2-expressing tumors without any observable adverse effects in mouse xenograft and rat syngeneic tumor models. Our data support the use of an antibody-drug conjugate approach to selectively target and inhibit the growth of EphA2-expressing tumors.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Imunoconjugados/uso terapêutico , Neoplasias Experimentais/tratamento farmacológico , Receptor EphA2/antagonistas & inibidores , Animais , Feminino , Humanos , Camundongos , Oligopeptídeos/uso terapêutico , Ratos , Ratos Endogâmicos F344 , Receptor EphA2/análise , Receptor EphA2/fisiologiaRESUMO
The humanized monoclonal antibody Abegrin, currently in phase II trials for treatment of solid tumors, specifically recognizes the integrin alphavbeta3. Due to its high expression on mature osteoclasts, angiogenic endothelial cells, and tumor cells, integrin alphavbeta3 functions in several pathologic processes important to tumor growth and metastasis. Targeting of this integrin with Abegrin results in antitumor, antiangiogenic, and antiosteolytic activities. Here, we exploit the species specificity of Abegrin to evaluate the effects of direct targeting of tumor cells (independent of targeting of endothelia or osteoclasts). Flow cytometry analysis of human tumor cell lines shows high levels of alphavbeta3 on many solid tumors, including cancers of the prostate, skin, ovary, kidney, lung, and breast. We also show that tumor growth of alphavbeta3-expressing tumor cells is inhibited by Abegrin in a dose-dependent manner. We present a novel finding that high-dose administration can actively impair the antitumor activity of Abegrin. We also provide evidence that antibody-dependent cellular cytotoxicity contributes to in vitro and in vivo antitumor activity. Finally, it was observed that peak biological activity of Abegrin arises at serum levels that are consistent with those achieved in clinical trials. These results support a concept that Abegrin can be used to achieve selective targeting of the many tumor cells that express alphavbeta3 integrin. In combination with the well-established concept that alphavbeta3 plays a key role in cancer-associated angiogenesis and osteolytic activities, this triad of activity could provide new opportunities for therapeutic targeting of cancer.
Assuntos
Anticorpos Monoclonais/farmacologia , Integrina alfaVbeta3/imunologia , Neoplasias/terapia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Citotoxicidade Celular Dependente de Anticorpos , Linhagem Celular Tumoral , Relação Dose-Resposta Imunológica , Feminino , Humanos , Integrina alfaVbeta3/biossíntese , Camundongos , Camundongos Nus , Camundongos SCID , Neoplasias/imunologia , Especificidade da Espécie , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
TCDD (2,3,7,8-tetrachlorodibenzo- p -dixoin) induces phase II drug-metabolizing enzyme NQO1 [NAD(P)H:quinone oxidoreductase; EC 1.6.99.2; DT-diaphorase] in a wide range of mammalian tissues and cells. Here, we analysed the molecular pathway mediating NQO1 induction by TCDD in mouse hepatoma cells. Inhibition of protein synthesis with CHX (cycloheximide) completely blocks induction of NQO1 by TCDD as well as the basal expression and induction by phenolic antioxidant tBHQ (2-t-butylbenzene-1,4-diol), implicating a labile factor in NQO1 mRNA expression. The inhibition is both time- and concentration-dependent, requires inhibition of protein synthesis, and occurs at a transcriptional level. Inhibition of NQO1 transcription by CHX correlates with a rapid reduction of the CNC bZip (cap 'n' collar basic leucine zipper) transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) through the 26 S proteasome pathway. Moreover, blocking Nrf2 degradation with proteasome inhibitor MG132 increases the amount of Nrf2 and superinduces NQO1 in the presence of TCDD or tBHQ. Finally, genetic experiments using AhR (aryl hydrocarbon receptor)-, Arnt (aryl hydrocarbon receptor nuclear translocator)- or Nrf2-deficient cells reveal that, while induction of NQO1 by TCDD depends on the presence of AhR and Arnt, the basal and inducible expression of NQO1 by either TCDD or tBHQ requires functional Nrf2. The findings demonstrate a novel role of Nrf2 in the induction of NQO1 by TCDD and provide new insights into the mechanism by which Nrf2 regulates the induction of phase II enzymes by both phenolic antioxidants and AhR ligands.
Assuntos
Proteínas de Ligação a DNA/fisiologia , NAD(P)H Desidrogenase (Quinona)/biossíntese , Dibenzodioxinas Policloradas/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Transativadores/fisiologia , Animais , Antioxidantes/farmacologia , Linhagem Celular Tumoral , Células Cultivadas , Cicloeximida/farmacologia , Cisteína Endopeptidases/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Indução Enzimática , Hidroquinonas/farmacologia , Zíper de Leucina , Ligantes , Camundongos , Complexos Multienzimáticos/metabolismo , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2 , Complexo de Endopeptidases do Proteassoma , Inibidores da Síntese de Proteínas/farmacologia , Transdução de Sinais , Transativadores/química , Transativadores/metabolismo , Transcrição Gênica , Ubiquitinas/metabolismoRESUMO
Phenolic antioxidants inhibit the induction of inflammatory cytokines by inflammatory stimuli. Here, we analyzed the mechanism by which the antioxidants inhibit LPS-induced expression of tumor necrosis factor alpha (TNFalpha) in macrophages. Hydroquinone and tert-butyl hydroquinone, prototypes of phenolic antioxidants, block lipopolysaccharide (LPS)-induced transcription of TNFalpha and a nuclear factor (NF)-kappaB-mediated reporter gene expression, suggesting NF-kappaB as a target in the inhibition. Analyses of the NF-kappaB activation pathway revealed that the antioxidants do not inhibit LPS-induced activation of the IkappaB kinase activity, degradation of IkappaBalpha, or translocation of activated NF-kappaB into the nucleus, but they do block the formation of NF-kappaB/DNA binding complexes. In vitro experiments showed that the antioxidants do not directly interfere with DNA binding of NF-kappaB. Structure-activity analyses suggest that inhibition of NF-kappaB function involves the redox cycling property of the antioxidants. These findings implicate a redox-sensitive factor important for the binding of NF-kappaB to its DNA recognition sequence as a target molecule in the inhibition of NF-kappaB function and inflammatory cytokine expression by phenolic antioxidants.
Assuntos
Antioxidantes/farmacologia , Citocinas/metabolismo , Macrófagos/efeitos dos fármacos , NF-kappa B/antagonistas & inibidores , Fenóis/farmacologia , Animais , Células Cultivadas , DNA/efeitos dos fármacos , DNA/metabolismo , Macrófagos/metabolismo , Camundongos , Oxirredução , Transdução de Sinais , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Phenolic antioxidants exhibit anti-inflammatory activity in protection against chemical toxicity and cancer. To investigate the molecular mechanism of anti-inflammation, we analyzed the regulation of tumor necrosis factor alpha (TNF-alpha) expression in macrophages, a key step in inflammation, by the antioxidants. Whereas lipopolysaccharide (LPS), an inflammatory inducer, stimulates rapid synthesis of TNF-alpha protein, phenolic antioxidants, exemplified by tert-butyl hydroquinone and 1,4-dihydroquinone, block LPS-induced production of TNF-alpha protein in a time- and dose-dependent manner. Inhibition of TNF-alpha induction correlates with the capacity of the antioxidants to undergo oxidation-reduction cycling, implicating oxidative signaling in the inhibition. The antioxidants blocked LPS-induced increase of the steady-state mRNA of TNF-alpha but did not affect the half-life of the mRNA. Electrophoretic mobility shift assay reveals a total inhibition of LPS-induced formation of nuclear factor kappaB.DNA binding complexes by phenolic antioxidants. Finally, 1,4-dihydroquinone blocks the induction of TNF-alpha target genes interleukin 1beta and interleukin 6 at both mRNA and protein levels. Our findings demonstrate that phenolic antioxidants potently inhibit signal-induced TNF-alpha transcription and suggest a mechanism of anti-inflammation by the antioxidants through control of cytokine induction during inflammation.