RESUMO
The normal electron-demand Diels-Alder (DA) cycloaddition is a classic transformation routinely used in synthesis; however, applications in biological systems are limited. Here, we report a spiro[2.4]hepta-4,6-diene-containing noncanonical amino acid (SCpHK) capable of efficient incorporation into antibodies and subsequent coupling with maleimide via a DA reaction. SCpHK was stable throughout protein expression in mammalian cells and enabled covalent attachment of maleimide drug-linkers yielding DA antibody-drug conjugates (DA-ADCs) with nearly quantitative conversion in a one-step process. The uncatalyzed DA reaction between SCpHK and maleimide in aqueous buffer was rapid (1.8-5.4 M-1 s-1), and the antibody-drug adduct was stable in rat serum for at least 1 week at 37 °C. Anti-EphA2 DA-ADCs containing AZ1508 or SG3249 maleimide drug-linkers were potent inhibitors of tumor growth in PC3 tumor models in vivo. The DA bioconjugation strategy described here represents a simple method to produce site-specific and stable ADCs with maleimide drug-linkers.
Assuntos
Imunoconjugados/química , Maleimidas/química , Animais , Células CHO , Sobrevivência Celular/efeitos dos fármacos , Cricetulus , Reação de Cicloadição , Humanos , Imunoconjugados/farmacologia , Modelos Moleculares , Células PC-3 , Conformação Proteica , Compostos de Espiro/químicaRESUMO
Soluble ligands have commonly been targeted by antibody therapeutics for cancers and other diseases. Although monoclonal antibodies targeting such ligands can block their interactions with their cognate receptors, they can also significantly increase the half-life of their ligands by FcRn-mediated antibody recycling, thereby evading ligand renal clearance and requiring increasingly high antibody doses to neutralize the increasing pool of target. To overcome this issue, we generated a bispecific/biparatopic antibody (BiSAb) that targets two different epitopes on IL-6 to block IL-6-mediated signaling. The BiSAb formed large immune complexes with IL-6 that can bind Fcγ receptors on phagocytic cells and are rapidly internalized. In addition, rapid clearance of the BiSAb·IL-6 complex was observed in mice while the parental antibodies prolonged the serum half-life of IL-6. Intravital imaging of the liver in mice confirmed that the rapid clearance of these large immune complexes was associated with Fcγ receptor-dependent binding to Kupffer cells in the liver. The approach described here provides a general strategy for therapeutic antibodies with the ability to not only neutralize but also actively drive clearance of their soluble antigens.
Assuntos
Anticorpos Biespecíficos/metabolismo , Anticorpos Monoclonais/metabolismo , Complexo Antígeno-Anticorpo/imunologia , Interleucina-6/antagonistas & inibidores , Receptores de IgG/metabolismo , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais/imunologia , Células HEK293 , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Interleucina-6/imunologia , Células de Kupffer/citologia , Células de Kupffer/metabolismo , Fígado/citologia , Fígado/metabolismo , Camundongos , Ligação Proteica , Receptores de IgG/imunologiaRESUMO
The thiol-maleimide linkage is widely used for antibody-drug conjugate (ADC) production; however, conjugation of maleimide-drugs could be improved by simplified procedures and reliable conjugate stability. Here, we report the evaluation of electron-rich and cyclic dienes that can be appended to antibodies and reacted with maleimide-containing drugs through the Diels-Alder (DA) reaction. Drug conjugation is fast and quantitative due to reaction acceleration in water, and the linkage is more stable in serum than in the corresponding thiol-maleimide adduct with the same drug. ADCs produced using the DA reaction (DAADCs) are effective in vitro and in vivo, demonstrating the utility of this reaction in producing effective biotherapeutics. Given the large number of commercially available maleimide compounds, this conjugation approach could be readily applied to the production of a wide range of antibody (or protein) conjugates.
Assuntos
Reação de Cicloadição/métodos , Imunoconjugados/química , Maleimidas/química , Alcenos , Anticorpos/química , Reagentes de Ligações Cruzadas/química , Estabilidade de Medicamentos , Maleimidas/uso terapêutico , Preparações Farmacêuticas/químicaRESUMO
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.
Assuntos
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
Prostate cancer is generally considered an immunologically "cold" tumor type that is insensitive to immunotherapy. Targeting surface antigens on tumors through cellular therapy can induce a potent antitumor immune response to "heat up" the tumor microenvironment. However, many antigens expressed on prostate tumor cells are also found on normal tissues, potentially causing on-target, off-tumor toxicities and a suboptimal therapeutic index. Our studies revealed that six-transmembrane epithelial antigen of prostate-2 (STEAP2) was a prevalent prostate cancer antigen that displayed high, homogeneous cell surface expression across all stages of disease with limited distal normal tissue expression, making it ideal for therapeutic targeting. A multifaceted lead generation approach enabled development of an armored STEAP2 chimeric antigen receptor T cell (CAR-T) therapeutic candidate, AZD0754. This CAR-T product was armored with a dominant-negative TGF-ß type II receptor, bolstering its activity in the TGF-ß-rich immunosuppressive environment of prostate cancer. AZD0754 demonstrated potent and specific cytotoxicity against antigen-expressing cells in vitro despite TGF-ß-rich conditions. Further, AZD0754 enforced robust, dose-dependent in vivo efficacy in STEAP2-expressing cancer cell line-derived and patient-derived xenograft mouse models, and exhibited encouraging preclinical safety. Together, these data underscore the therapeutic tractability of STEAP2 in prostate cancer as well as build confidence in the specificity, potency, and tolerability of this potentially first-in-class CAR-T therapy.
Assuntos
Neoplasias da Próstata , Receptores de Antígenos Quiméricos , Masculino , Humanos , Camundongos , Animais , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Imunoterapia Adotiva , Neoplasias da Próstata/patologia , Linfócitos T , Fator de Crescimento Transformador beta/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Linhagem Celular Tumoral , Microambiente Tumoral , Oxirredutases/metabolismoRESUMO
Most strategies used to prepare homogeneous site-specific antibody-drug conjugates (ADCs) result in ADCs with a drug-to-antibody ratio (DAR) of two. Here, we report a disulfide re-bridging strategy to prepare homogeneous ADCs with DAR of one using a dual-maleimide pyrrolobenzodiazepine (PBD) dimer (SG3710) and an engineered antibody (Flexmab), which has only one intrachain disulfide bridge at the hinge. We demonstrate that SG3710 efficiently re-bridge a Flexmab targeting human epidermal growth factor receptor 2 (HER2), and the resulting ADC was highly resistant to payload loss in serum and exhibited potent anti-tumor activity in a HER2-positive gastric carcinoma xenograft model. Moreover, this ADC was tolerated in rats at twice the dose compared to a site-specific ADC with DAR of two prepared using a single-maleimide PBD dimer (SG3249). Flexmab technologies, in combination with SG3710, provide a platform for generating site-specific homogenous PBD-based ADCs with DAR of one, which have improved biophysical properties and tolerability compared to conventional site-specific PBD-based ADCs with DAR of two.
Assuntos
Antineoplásicos , Benzodiazepinas/química , Imunoconjugados , Pirróis/química , Receptor ErbB-2/antagonistas & inibidores , Neoplasias Gástricas/tratamento farmacológico , Trastuzumab , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Feminino , Humanos , Imunoconjugados/química , Imunoconjugados/farmacologia , Células MCF-7 , Camundongos Nus , Ratos , Receptor ErbB-2/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Trastuzumab/química , Trastuzumab/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Pyrrolobenzodiazepine dimers (PBD) form cross-links within the minor groove of DNA causing double-strand breaks (DSB). DNA repair genes such as BRCA1 and BRCA2 play important roles in homologous recombination repair of DSB. We hypothesized that PBD-based antibody-drug conjugates (ADC) will have enhanced killing of cells in which homologous recombination processes are defective by inactivation of BRCA1 or BRCA2 genes. To support this hypothesis, we found 5T4-PBD, a PBD-dimer conjugated to anti-5T4 antibody, elicited more potent antitumor activity in tumor xenografts that carry defects in DNA repair due to BRCA mutations compared with BRCA wild-type xenografts. To delineate the role of BRCA1/2 mutations in determining sensitivity to PBD, we used siRNA knockdown and isogenic BRCA1/2 knockout models to demonstrate that BRCA deficiency markedly increased cell sensitivity to PBD-based ADCs. To understand the translational potential of treating patients with BRCA deficiency using PBD-based ADCs, we conducted a "mouse clinical trial" on 23 patient-derived xenograft (PDX) models bearing mutations in BRCA1 or BRCA2 Of these PDX models, 61% to 74% had tumor stasis or regression when treated with a single dose of 0.3 mg/kg or three fractionated doses of 0.1 mg/kg of a PBD-based ADC. Furthermore, a suboptimal dose of PBD-based ADC in combination with olaparib resulted in significantly improved antitumor effects, was not associated with myelotoxicity, and was well tolerated. In conclusion, PBD-based ADC alone or in combination with a PARP inhibitor may have improved therapeutic window in patients with cancer carrying BRCA mutations.
Assuntos
Antineoplásicos Imunológicos/administração & dosagem , Benzodiazepinas/química , Imunoconjugados/administração & dosagem , Neoplasias Experimentais/tratamento farmacológico , Ftalazinas/administração & dosagem , Piperazinas/administração & dosagem , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagem , Pirróis/química , Administração Intravenosa , Animais , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/farmacologia , Proteína BRCA1/genética , Proteína BRCA2/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Células HeLa , Humanos , Imunoconjugados/química , Imunoconjugados/farmacologia , Glicoproteínas de Membrana/antagonistas & inibidores , Camundongos , Mutação , Neoplasias Experimentais/genética , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Sequenciamento do Exoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
By simultaneous binding two disease mediators, bispecific antibodies offer the opportunity to broaden the utility of antibody-based therapies. Herein, we describe the design and characterization of Bs4Ab, an innovative and generic bispecific tetravalent antibody platform. The Bs4Ab format comprises a full-length IgG1 monoclonal antibody with a scFv inserted into the hinge domain. The Bs4Ab design demonstrates robust manufacturability as evidenced by MEDI3902, which is currently in clinical development. To further demonstrate the applicability of the Bs4Ab technology, we describe the molecular engineering, biochemical, biophysical, and in vivo characterization of a bispecific tetravalent Bs4Ab that, by simultaneously binding vascular endothelial growth factor and angiopoietin-2, inhibits their function. We also demonstrate that the Bs4Ab platform allows Fc-engineering similar to that achieved with IgG1 antibodies, such as mutations to extend half-life or modulate effector functions.
Assuntos
Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais/biossíntese , Imunoglobulina G/farmacologia , Engenharia de Proteínas/métodos , Anticorpos de Cadeia Única/farmacologia , Angiopoietina-2/antagonistas & inibidores , Animais , Anticorpos Biespecíficos/biossíntese , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Humanos , Imunoglobulina G/biossíntese , Camundongos , Anticorpos de Cadeia Única/biossíntese , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: The cytoprotective drug amifostine (Ethyol) protects rats from oral mucositis resulting from a single dose of gamma-irradiation. We expanded earlier studies to determine whether multiple doses of amifostine protect against fractionated or hyperfractionated radiation and whether the active metabolite of amifostine (WR-1065) accumulates in tissues upon repeated administration. METHODS AND MATERIALS: Rats received amifostine daily for 5 days in conjunction with a 1-week fractionated radiation schedule and were evaluated for oral mucositis. Rats also received amifostine before the am or pm exposure or b.i.d. in conjunction with hyperfractionated radiation. To determine the pharmacokinetics of WR-1065 after repeated dosing, amifostine was given 5 days a week for 1 or 3 weeks, and rat tissue and plasma were collected at intervals during and after treatment and analyzed for WR-1065. RESULTS: Amifostine protected rats from mucositis resulting from fractionated or hyperfractionated radiation. When the number of days of amifostine administration was reduced, protection was diminished. A dose of 100 mg/kg given in the morning or 2 doses at 50 mg/kg provided the best protection against hyperfractionated radiation. WR-1065 did not accumulate in tissues or tumor upon repeated administration. CONCLUSIONS: Amifostine prevented radiation-induced mucositis in a rat model; protection was dose and schedule dependent.
Assuntos
Amifostina/uso terapêutico , Lesões Experimentais por Radiação/prevenção & controle , Protetores contra Radiação/uso terapêutico , Estomatite/prevenção & controle , Amifostina/farmacocinética , Animais , Fracionamento da Dose de Radiação , Avaliação Pré-Clínica de Medicamentos , Feminino , Injeções Intravenosas , Injeções Subcutâneas , Neoplasias Mamárias Experimentais/metabolismo , Mercaptoetilaminas/administração & dosagem , Mercaptoetilaminas/farmacocinética , Mucosa Bucal/efeitos da radiação , Protetores contra Radiação/farmacocinética , Ratos , Ratos Endogâmicos F344 , Ratos Sprague-Dawley , Distribuição TecidualRESUMO
Amifostine (Ethyol) has been evaluated clinically as a radioprotective agent for the prevention of xerostomia and mucositis for patients receiving radiotherapy (RT). Currently, amifostine is approved for the prevention of xerostomia in head and neck cancer patients receiving RT when administered intravenously (IV) before RT. For the clinician, there would be several advantages to administering the drug subcutaneously and to being able to show its protective effects on mucositis. The authors have developed a rat RT model to examine the protective effects of amifostine after IV and subcutaneous (SC) administration in a mucositis model. Rats (5 per group) were given 200 mg/kg (human dose equivalent of approximately 1,300 mg/m(2)) of amifostine either IV or SC, and their head and neck regions were exposed to 15.3 Gy of gamma radiation 0.5, 2, 4, and 8 hours after amifostine administration. For 10 days after treatment, the oral cavities of the rats were examined for signs of mucositis. Mucosal erythema and mucosal edema were scored according to 0 through 5 and 0 through 2 scales, respectively, with the scores added to indicate overall mucositis. The average mucositis score for the untreated animals was 3.5. Rats were protected from mucositis up to 4 hours when given amifostine either IV or SC. Rats that received amifostine SC, but not IV, were protected from mucositis 8 hours after administration. Preliminary pharmacokinetic data have revealed slightly higher active metabolite (WR-1065) levels in the parotid gland and small intestine in the rats given amifostine SC compared with IV and equivalent levels in the plasma and kidney. The data showed that SC administration of amifostine gave radioprotection comparable to IV administration up to 4 hours before RT and may be more effective than IV administration at longer pretreatment intervals.
Assuntos
Amifostina/administração & dosagem , Cabeça/efeitos da radiação , Lesões por Radiação/prevenção & controle , Protetores contra Radiação/administração & dosagem , Amifostina/farmacocinética , Animais , Feminino , Injeções Intravenosas , Injeções Subcutâneas , Mucosa Bucal , Doses de Radiação , Protetores contra Radiação/farmacocinética , Ratos , Ratos Sprague-Dawley , Estomatite/patologia , Estomatite/prevenção & controleRESUMO
The radioprotective effects and pharmacokinetics of subcutaneously (SC) administered amifostine have been investigated in animal studies. Studies in rats using a single dose of amifostine showed that SC administration gave protection from radiation-induced mucositis that is at least equivalent to that achieved by intravenous administration of the drug. These studies also indicate that tissue levels of the active metabolite WR-1065 correlated better with the radioprotective effects of amifostine than do plasma WR-1065 levels. Multiple-dose studies in rats show radioprotective effects equal to or greater than those obtained with intravenous dosing in the setting of fractionated irradiation. In addition, there is no evidence of drug accumulation in either normal or tumor tissue, with tumor WR-1065 levels peaking just above the limits of quantitation during treatment. Preliminary data from studies of SC amifostine in monkeys indicate a plasma pharmacokinetic profile similar to that reported earlier in humans. Tissue WR-1065 levels were higher at 30 minutes after SC dosing than they were after intravenous dosing and were comparable for the two routes at 60 minutes.
Assuntos
Amifostina/farmacocinética , Citoproteção , Protetores contra Radiação/farmacocinética , Amifostina/administração & dosagem , Amifostina/metabolismo , Animais , Disponibilidade Biológica , Avaliação Pré-Clínica de Medicamentos , Injeções Intravenosas , Injeções Subcutâneas , Macaca fascicularis , Mercaptoetilaminas/metabolismo , Lesões por Radiação/prevenção & controle , Protetores contra Radiação/administração & dosagem , Protetores contra Radiação/metabolismo , RatosRESUMO
The chemo- and radioprotectant drug amifostine (Ethyol; MedImmune, Inc, Gaithersburg, MD) is approved for intravenous (IV) administration; however, the subcutaneous (SC) route is being explored as a practical alternative. We have previously reported equivalence between IV and SC administration using a rat model of radioprotection and active metabolite (WR-1065) tissue pharmacokinetics. To examine the more clinically relevant fractionated and hyperfractionated radiation schedules and the effects of variations in the time of amifostine administration, we expanded these studies to include radioprotection and pharmacokinetic studies of WR-1065 using multiple dosing. To measure radioprotection using a fractionated radioprotection model, rats were given amifostine over a 1-week period at various doses (25 mg/kg, 50 mg/kg, 100 mg/kg; or 162.5 mg/m(2), 325 mg/m(2), 650 mg/m(2), respectively) IV or SC daily 30 minutes before exposure to 7.5 Gy/dose. Rats were fully protected from mucositis at the highest amifostine dose, with protection diminishing as the amifostine was decreased. Equivalent protection was observed whether the drug was given IV or SC. When the number of days of amifostine administration was reduced, protection was diminished. Amifostine also protected against radiation delivered using a 1-week hyperfractionated schedule (4.5 Gy/exposure twice daily), with optimal protection occurring when the drug was administered bid 30 minutes before each exposure (50 mg/kg) or every day before the morning exposure (100 mg/kg). The need for daily dosing to achieve optimal radioprotection was consistent with the tissue pharmacokinetics of the active metabolite. We found that WR-1065 did not accumulate in tissues or in SC-implanted tumors when amifostine was administered daily for 3 weeks. In addition, tissue and tumor levels of WR-1065 declined to baseline 24 hours after each amifostine dose. In a monkey pharmacokinetic model, plasma levels of WR-1065 (characterized by a pronounced spike of WR-1065 immediately after IV administration that was absent when the drug was given SC) were similar to those of humans; however, levels of WR-1065 in the tissues were higher 30 minutes following SC administration and were equivalent 60 minutes following IV or SC administration. These results suggest that maximum tissue levels and protection occur when amifostine is given 30 to 60 minutes before radiation exposure, that treatment breaks reduce the radioprotection by amifostine, and that protection from hyperfractionated radiation is dependent on amifostine dose and schedule.
Assuntos
Amifostina/administração & dosagem , Lesões por Radiação/prevenção & controle , Protetores contra Radiação/administração & dosagem , Amifostina/farmacocinética , Animais , Injeções Intravenosas , Injeções Subcutâneas , Macaca fascicularis , Mercaptoetilaminas/farmacocinética , Modelos Animais , Mucosa/efeitos dos fármacos , Mucosa/efeitos da radiação , Protetores contra Radiação/farmacocinética , RatosRESUMO
PURPOSE: Amifostine (Ethyol) is currently approved for intravenous (IV) administration to prevent xerostomia in patients receiving radiotherapy for head-and-neck cancer. Recently, subcutaneous (SC) administration has been explored as an alternative route. To determine whether SC administration was equivalent to IV administration, we used models to follow pharmacokinetics and oral mucosal protection in rats. METHODS: Amifostine was administered to rats at doses of 200, 100, or 50 mg/kg (1300, 650, or 325 mg/m(2)) IV or SC at various times before radiation at 15.3 Gy (protection studies) or harvest of blood and tissues for analysis by HPLC (pharmacokinetic studies). RESULTS: Amifostine administered IV or SC 1 h before radiation protected rats from mucositis, but the protective effect was more prolonged when amifostine was administered SC. Tissue levels of the active metabolite (WR-1065) were equivalent after SC administration. The correlation between tissue levels of WR-1065 and protection was strong, but that between blood levels of WR-1065 and protection was only weak. CONCLUSION: These data demonstrate that, in a rat model, SC administration of amifostine was at least as effective as that by IV.
Assuntos
Amifostina/administração & dosagem , Lesões por Radiação/prevenção & controle , Protetores contra Radiação/administração & dosagem , Estomatite/prevenção & controle , Amifostina/metabolismo , Animais , Feminino , Injeções Intravenosas , Injeções Subcutâneas , Mercaptoetilaminas/metabolismo , Modelos Animais , Mucosa Bucal/efeitos dos fármacos , Mucosa Bucal/efeitos da radiação , Lesões por Radiação/metabolismo , Protetores contra Radiação/metabolismo , Ratos , Ratos Sprague-Dawley , Estomatite/etiologia , Estomatite/metabolismo , XerostomiaRESUMO
MEDI-573 is a human antibody that neutralizes insulin-like growth factor (IGF) I and IGFII. IGFs are overexpressed in multiple types of cancer; their overexpression is a potential mechanism for resistance to IGFI receptor (IGFIR)-targeting therapy. Effects of IGF on cell proliferation, differentiation, and survival are mediated through its binding to and activation of IGFIR or insulin receptor A (IR-A). In this study, we measured the mRNA levels of IGFI, IGFII, and IGFIR in human pediatric sarcoma xenografts, and protein levels in sarcoma cell lines. MEDI-573 potently inhibited in vitro proliferation of sarcoma cell lines, with Ewing sarcoma cell lines being the most sensitive. In addition, MEDI-573 inhibited IGFI- and IGFII-induced sarcoma cell proliferation in vitro. The effect of MEDI-573 on IGF signaling was also examined. Treatment with MEDI-573 markedly reduced levels of pIGFIR, pIR-A, and pAKT and significantly blocked IGFI- and IGFII-induced activation of the IGFIR and AKT pathways. MEDI-573 inhibited the growth of sarcoma xenografts in vivo and inhibition correlated with neutralization of IGFI and IGFII. Combination of MEDI-573 with either rapamycin or AZD2014, another mTOR inhibitor (mTORi), significantly enhanced the antitumor activity of MEDI-573, and this response correlated with modulation of AKT and mTOR signaling. In summary, sarcoma cells respond to autocrine or paracrine growth stimulation by IGFI and IGFII, and inhibition of IGFI and IGFII by MEDI-573 results in significant slowing of tumor growth rate in sarcoma models, particularly in Ewing sarcoma. These data provide evidence for the potential benefits of MEDI-573 and mTORi combinations in patients with Ewing sarcoma.
Assuntos
Anticorpos Neutralizantes/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Sarcoma/tratamento farmacológico , Somatomedinas/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados , Benzamidas , Anticorpos Amplamente Neutralizantes , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Ligantes , Camundongos , Camundongos Nus , Morfolinas/administração & dosagem , Morfolinas/farmacologia , Fosforilação , Pirimidinas , Distribuição Aleatória , Sarcoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/administração & dosagem , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/administração & dosagemRESUMO
Angiopoietin 2 (Ang2) is an important regulator of angiogenesis, blood vessel maturation and integrity of the vascular endothelium. The correlation between the dynamic expression of Ang2 in tumors with regions of high angiogenic activity and a poor prognosis in many tumor types makes Ang2 an ideal drug target. We have generated MEDI3617, a human anti-Ang2 monoclonal antibody that neutralizes Ang2 by preventing its binding to the Tie2 receptor in vitro, and inhibits angiogenesis and tumor growth in vivo. Treatment of mice with MEDI3617 resulted in inhibition of angiogenesis in several mouse models including: FGF2-induced angiogenesis in a basement extract plug model, tumor and retinal angiogenesis. In xenograft tumor models, treatment with MEDI3617 resulted in a reduction in tumor angiogenesis and an increase in tumor hypoxia. The administration of MEDI3617 as a single agent to mice bearing human tumor xenografts resulted in tumor growth inhibition against a broad spectrum of tumor types. Combining MEDI3617 with chemotherapy or bevacizumab resulted in a delay in tumor growth and no body weight loss was observed in the combination groups. These results, combined with pharmacodynamic studies, demonstrate that treatment of tumor-bearing mice with MEDI3617 significantly inhibited tumor growth as a single agent by blocking tumor angiogenesis. Together, these data show that MEDI3617 is a robust antiangiogenic agent and support the clinical evaluation and biomarker development of MEDI3617 in cancer patients.
Assuntos
Inibidores da Angiogênese/farmacologia , Angiopoietina-2/antagonistas & inibidores , Anticorpos Monoclonais/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias/tratamento farmacológico , Neovascularização Patológica/prevenção & controle , Inibidores da Angiogênese/administração & dosagem , Angiopoietina-2/imunologia , Angiopoietina-2/metabolismo , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados/administração & dosagem , Bevacizumab , Linhagem Celular Tumoral , Molde por Corrosão , Relação Dose-Resposta a Droga , Feminino , Fluorescência , Células HEK293 , Humanos , Camundongos , Camundongos Nus , Neoplasias/irrigação sanguínea , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , Neovascularização Patológica/imunologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Paclitaxel/administração & dosagem , Fosforilação , Receptor TIE-2/genética , Receptor TIE-2/metabolismo , Neovascularização Retiniana/imunologia , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/prevenção & controle , Fatores de Tempo , Transfecção , Carga Tumoral/efeitos dos fármacos , Microtomografia por Raio-X , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
IPI-504 is a novel, highly soluble small-molecule inhibitor of heat shock protein 90 (Hsp90), a protein chaperone essential for regulating homeostasis of oncoproteins and cell signaling proteins. Human epidermal growth factor receptor 2 (HER2; ErbB2) oncoprotein, expressed in a subset of metastatic breast cancers, is a Hsp90 client protein. In this study, we investigated the antitumor activity and the mechanism of action of IPI-504 in HER2(+), trastuzumab-sensitive and trastuzumab-refractory cell lines in vitro and in vivo. IPI-504 exhibited potent antiproliferative activities (range of IC(50), 10-40 nmol/L) against several tumor cell lines examined, whereby mechanism of action was mediated through HER2 and Akt degradation. Both intravenous and oral administration of IPI-504 assessed in multiple schedules showed potent tumor growth inhibition in vivo with corresponding degradation of HER2. The tolerability and efficacy of IPI-504 combined with either trastuzumab or lapatinib were also investigated in HER2(+) tumor xenograft models. Combination of IPI-504 with trastuzumab significantly enhanced tumor growth delay and induced greater responses when compared with either agent alone. Although, as expected, trastuzumab alone did not exhibit any significant antitumor activity in the trastuzumab-resistant JIMT-1 model, IPI-504 administered in combination with trastuzumab yielded greater antitumor efficacy than either agent alone. Finally, combination of IPI-504 and lapatinib was well tolerated up to 50 mg/kg IPI-504 and 100 mg/kg lapatinib and resulted in significant delay in tumor growth, including partial and complete tumor responses. These lines of evidence support the development of IPI-504 in HER2-positive breast cancers as a single agent and in combination with either trastuzumab or lapatinib
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzoquinonas/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Lactamas Macrocíclicas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Lapatinib , Quinazolinas/farmacologia , Receptor ErbB-2/metabolismo , Trastuzumab , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: EphA2 is overexpressed in many types of human cancer but is absent or expressed at low levels in normal epithelial tissues. We investigated whether a novel immunoconjugate containing an anti-EphA2 monoclonal antibody (1C1) linked to a chemotherapeutic agent (monomethyl auristatin phenylalanine [MMAF]) through a noncleavable linker maleimidocaproyl (mc) had antitumor activity against ovarian cancer cell lines and tumor models. METHODS: Specificity of 1C1-mcMMAF was examined in EphA2-positive HeyA8 and EphA2-negative SKMel28 ovarian cancer cells by antibody binding and internalization assays. Controls were phosphate-buffered saline (PBS), 1C1, or control IgG-mcMMAF. Viability and apoptosis were investigated in ovarian cancer cell lines and tumor models (10 mice per group). Antitumor activities were tested in the HeyA8-luc and SKOV3ip1 orthotopic mouse models of ovarian cancer. Endothelial cells were identified by use of immunohistochemistry and anti-CD31 antibodies. All statistical tests were two-sided. RESULTS: The 1C1-mcMMAF immunoconjugate specifically bound to EphA2-positive HeyA8 cells but not to EphA2-negative cells and was internalized by HeyA8 cells. Treatment with 1C1-mcMMAF decreased the viability of HeyA8-luc cells in an EphA2-specific manner. In orthotopic mouse models, treatment with 1C1-mcMMAF inhibited tumor growth by 85%-98% compared with that in control mice (eg, for weight of HeyA8 tumors, 1C1-mcMMAF = 0.05 g and control = 1.03 g; difference = 0.98 g, 95% confidence interval [CI] = 0.40 to 1.58 g; P = .001). Even in bulkier disease models with HeyA8-luc cells, 1C1-mcMMAF treatment, compared with control treatment, caused regression of established tumors and increased survival of the mice (eg, 1C1-mcMMAF vs control, mean = 60.6 days vs 29.4 days; difference = 31.2 days, 95% CI = 27.6 to 31.2 days; P = .001). The antitumor effects of 1C1-mcMMAF therapy, in SKOV3ip1 tumors, for example, were statistically significantly related to decreased proliferation (eg, 1C1-mcMMAF vs control, mean = 44.1% vs 55.8% proliferating cells; difference = 11.7%, 95% CI = 2.45% to 20.9%; P = .01) and increased apoptosis of tumor cells (eg, 1C1-mcMMAF vs control, mean = 8.6% vs 0.9% apoptotic cells; difference = 7.7%, 95% CI = 3.8% to 11.7%; P < .001) and of mouse endothelial cells (eg, 1C1-mcMMAF vs control, mean 2.8% vs 0.4% apoptotic endothelial cells; difference = 2.4%, 95% CI = 1.4% to 4.6%; P = .034). CONCLUSION: The 1C1-mcMMAF immunoconjugate had antitumor activity in preclinical models of ovarian carcinoma.
Assuntos
Antineoplásicos/farmacologia , Imunoconjugados/farmacologia , Oligopeptídeos/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/imunologia , Receptor EphA2/antagonistas & inibidores , Animais , Anticorpos Monoclonais/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Imunofluorescência , Humanos , Imunoconjugados/uso terapêutico , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Oligopeptídeos/uso terapêutico , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Antígeno Nuclear de Célula em Proliferação/análise , Receptor EphA2/análise , Receptor EphA2/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
EphA2 is a receptor tyrosine kinase that has been shown to be overexpressed in a variety of human tumor types. Previous studies demonstrated that agonist monoclonal antibodies targeting EphA2 induced the internalization and degradation of the receptor, thereby abolishing its oncogenic effects. In this study, the in vitro and in vivo antibody-dependent cell-mediated cytotoxicity (ADCC) activity of EphA2 effector-enhanced agonist monoclonal antibodies was evaluated. With tumor cell lines and healthy human peripheral blood monocytes, the EphA2 antibodies demonstrated approximately 80% tumor cell killing. In a dose-dependent manner, natural killer (NK) cells were required for the in vitro ADCC activity and became activated as demonstrated by the induction of cell surface expression of CD107a. To assess the role of NK cells on antitumor efficacy in vivo, the EphA2 antibodies were evaluated in xenograft models in severe compromised immunodeficient (SCID) mice (which have functional NK cells and monocytes) and SCID nonobese diabetic (NOD) mice (which largely lack functional NK cells and monocytes). Dosing of EphA2 antibody in the SCID murine tumor model resulted in a 6.2-fold reduction in tumor volume, whereas the SCID/nonobese diabetic model showed a 1.6-fold reduction over the isotype controls. Together, these results demonstrate that the anti-EphA2 monoclonal antibodies may function through at least two mechanisms of action: EphA2 receptor activation and ADCC-mediated activity. These novel EphA2 monoclonal antibodies provide additional means by which host effector mechanisms can be activated for selective destruction of EphA2-expressing tumor cells.
Assuntos
Anticorpos Monoclonais/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Neoplasias/imunologia , Receptor EphA2/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Genótipo , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Proteína 1 de Membrana Associada ao Lisossomo/imunologia , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Fosforilação/efeitos dos fármacos , Polimorfismo Genético , Receptor EphA2/agonistas , Receptor EphA2/metabolismo , Receptores de IgG/genética , Ressonância de Plasmônio de Superfície , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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
Amifostine (Ethyol) is a cytoprotective drug approved for the reduction of xerostomia in head and neck cancer when administered to patients receiving postoperative radiation therapy. Although amifostine is approved for intravenous infusion, the off-label subcutaneous route of administration has become more prevalent. Although human patient data indicate higher plasma bioavailability of the active metabolite (WR-1065) following intravenous compared to subcutaneous administration, there are no corresponding data showing human tissue levels of WR-1065 following either route of administration due to the difficulty in obtaining human specimens. In our study we compared plasma and tissue pharmacokinetics of WR-1065 in primates following both routes of administration. Monkeys received amifostine at a dose of 260 mg/m2 either intravenously or subcutaneously. Plasma samples were analyzed for total WR-1065 by reverse-phase high-pressure liquid chromatography (HPLC) and fluorescence detection up to 4 h after amifostine administration. Tissues were analyzed for free WR-1065 by reverse-phase HPLC and electrochemical detection 30 and 60 min after administration. Following intravenous administration, plasma WR-1065 levels peaked rapidly and showed a bi-exponential decline, while following subcutaneous administration WR-1065 levels rose slowly and declined exponentially. The relative plasma bioavailability of WR-1065 given subcutaneously was lower at 30 and 60 min. Interestingly, after 30 min, tissues showed equal or slightly greater concentrations of WR-1065 following subcutaneous administration. Levels following 60 min were comparable following both routes. The plasma bioavailability studies performed in primates confirm human plasma data. Expanding the study to evaluate primate tissue levels of WR-1065 revealed that despite lower plasma bioavailability following subcutaneous administration, tissue levels of the active metabolite were surprisingly greater than or equal to those measured in animals that received the drug intravenously. These studies strengthen the argument for subcutaneous administration of amifostine in radiation oncology.