RESUMO
Antibody drug conjugates (ADCs) can undergo in vivo biotransformation (e.g., payload metabolism, deconjugation) leading to reduced or complete loss of activity. The location/site of conjugation of payload-linker can have an effect on ADC stability and hence needs to be carefully optimized. Affinity capture LC-MS of intact ADCs or ADC subfragments has been extensively used to evaluate ADC biotransformation. However, the current methods have certain limitations such as the requirement of specific capture reagents, limited mass resolution of low mass change metabolites, low sensitivity, and use of capillary or nanoflow LC-MS. To address these challenges, we developed a generic affinity capture LC-MS assay that can be utilized to evaluate the biotransformation of any site-specific ADC independent of antibody type and site of conjugation (Fab and Fc) in preclinical studies. The method involves a combination of some or all of these steps: (1) "mono capture" or "dual capture" of ADCs from serum with streptavidin magnetic beads coated with a generic biotinylated antihuman capture reagent, (2) "on-bead" digestion with IdeS and/or PNGase F, and (3) reduction of interchain disulfide bonds to generate â¼25 kDa ADC subfragments, which are finally analyzed by LC-HRMS on a TOF mass spectrometer. The advantages of this method are that it can be performed using commercially available generic reagents and requires sample preparation time of less than 7 h. Furthermore, by reducing the size of intact ADC (â¼150 kDa) to subfragments (â¼25 kDa), the identification of conjugated payload and its metabolites can be achieved with excellent sensitivity and resolution (hydrolysis and other small mass change metabolites). This method was successfully applied to evaluate the in vitro and in vivo biotransformation of ADCs conjugated at different sites (LC, HC-Fab, and HC-Fc) with various classes of payload-linkers.
Assuntos
Biotransformação , Imunoconjugados/sangue , Imunoconjugados/metabolismo , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Cromatografia Líquida , Humanos , Espectrometria de MassasRESUMO
Antibody-drug conjugates (ADCs) use antibodies to deliver cytotoxic payloads directly into tumor cells via specifically binding to the target cell surface antigens. ADCs can enhance the anti-tumor effects of antibodies, and increase the delivery of cytotoxic payloads to cancer cells with a better therapeutic index. An ADC was prepared with a potent carbamate-containing tubulysin analogue attached to an anti-mesothelin antibody via a Cit-Val dipeptide linker. An aniline functionality in the tubulysin analogue was created to provide a site of linker attachment via an amide bond that would be stable in systemic circulation. Upon ADC internalization into antigen-positive cancer cells, the Cit-Val dipeptide linker was cleaved by lysosomal proteases, and the drug was released inside the tumor cells. The naturally occurring acetate of tubulysin was modified to a carbamate to reduce acetate hydrolysis of the ADC in circulation and to increase the hydrophilicity of the drug. The ADC bearing the monoclonal anti-mesothelin antibody and the carbamate-containing tubulysin was highly potent and immunologically specific to H226 human lung carcinoma cells in vitro, and efficacious at well-tolerated doses in a mesothelin-positive OVCAR3 ovarian cancer xenograft mouse model.
Assuntos
Antineoplásicos/química , Carbamatos/química , Proteínas Ligadas por GPI/antagonistas & inibidores , Imunoconjugados/química , Oligopeptídeos/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Carbamatos/síntese química , Carbamatos/farmacologia , Feminino , Humanos , Imunoconjugados/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Mesotelina , Camundongos , Camundongos SCID , Oligopeptídeos/síntese química , Oligopeptídeos/farmacologia , Neoplasias Ovarianas/tratamento farmacológicoRESUMO
The growing fraction (GF) of tumor has been reported as one of the predictive markers of the efficacy of chemotherapeutics. Therefore, a semi-mechanistic model has been developed that describes tumor growth on the basis of cell cycle, allowing the incorporation of the GF of a tumor in pharmacokinetic/pharmacodynamic (PK/PD) modeling. Efficacy data of anti-glypican 3 (GPC3) antibody drug conjugate (ADC) in a hepatocellular carcinoma (HCC) patient derived xenograft (PDX) model was used for evaluation of this proposed model. Our model was able to describe the kinetics of growth inhibition of HCC PDX models following treatment with anti-GPC3 ADC remarkably well. The estimated tumurostatic concentrations were used in tandem with human PKs translated from cynomolgus monkey for prediction of the efficacious dose. The projected efficacious human dose of anti-GPC3 ADC was in the range 0.20-0.63 mg/kg for the Q3W dosing regimen, with a median dose of 0.50 mg/kg. This publication is the first step in evaluating the applicability of GF in PK/PD modeling of ADCs. The authors are hopeful that incorporation of GF will result in an improved translation of the preclinical efficacy of ADCs to clinical settings and thereby better prediction of the efficacious human dose.
Assuntos
Antineoplásicos , Carcinoma Hepatocelular/tratamento farmacológico , Glipicanas/imunologia , Imunoconjugados , Neoplasias Hepáticas/tratamento farmacológico , Modelos Biológicos , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/imunologia , Antineoplásicos/sangue , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Ciclo Celular , Linhagem Celular Tumoral , Humanos , Imunoconjugados/sangue , Imunoconjugados/farmacocinética , Imunoconjugados/uso terapêutico , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Macaca fascicularis , Camundongos Nus , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Emerging evidence suggests that myeloid cells play a critical role in glioblastoma (GBM) immunosuppression. Disappointing results of recent checkpoint inhibitor trials suggest that combination immunotherapy with alternative agents could be fruitful in overcoming immunosuppression. Overexpression of chemokine receptor CXCR4 is associated with poor prognosis in GBM. We investigate the treatment effects of combination immunotherapy with anti-PD-1 and anti-CXCR4 in a murine glioma model. METHODS: C57BL/6 mice were implanted with GL261-Luc+ glioma cells and randomized into 4 arms: (1) control (2) anti-PD-1 (3) anti-CXCR4, and (4) anti-PD-1 and anti-CXCR4 therapy. Overall survival and median survival were assessed. Cell populations were assessed by flow cytometry. RESULTS: Combination therapy conferred a significant survival benefit compared to control and monotherapy arms. Mice that received combination therapy demonstrated immune memory and decreased populations of immunosuppressive tumor-infiltrating leukocytes, such as monocytic myeloid-derived suppressor cells and microglia within the brain. Furthermore, combination therapy improved CD4+/CD8+ ratios in the brain as well as contributed to increased levels of pro-inflammatory cytokines. CONCLUSIONS: Anti-CXCR4 and anti-PD-1 combination immunotherapy modulates tumor-infiltrating populations of the glioma microenvironment. Targeting myeloid cells with anti-CXCR4 facilitates anti-PD-1 to promote an antitumor immune response and improved survival rates.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Neoplasias Encefálicas/mortalidade , Glioblastoma/mortalidade , Imunoterapia , Células Mieloides/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptores CXCR4/antagonistas & inibidores , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Citocinas/metabolismo , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/imunologia , Glioblastoma/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/efeitos dos fármacos , Receptor de Morte Celular Programada 1/imunologia , Receptores CXCR4/imunologia , Taxa de Sobrevida , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Uncialamycin analogs were evaluated as potential cytotoxic agents in an antibody-drug conjugate (ADC) approach to treating human cancer. These analogs were synthesized using Hauser annulations of substituted phthalides as a key step. A highly potent uncialamycin analog 3c with a valine-citrulline dipeptide linker was conjugated to an anti-mesothelin monoclonal antibody (mAb) through lysines to generate a meso-13 conjugate. This conjugate demonstrated subnanomolar potency (IC50â¯=â¯0.88â¯nM, H226 cell line) in in vitro cytotoxicity experiments with good immunological specificity to mesothelin-positive lung cancer cell lines. The potency and mechanism of action of this uncialamycin class of enediyne antitumor antibiotics make them attractive payloads in ADC-based cancer therapy.
Assuntos
Antraquinonas/farmacologia , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Imunoconjugados/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Antraquinonas/química , Anticorpos Monoclonais/química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imunoconjugados/química , Neoplasias Pulmonares/patologia , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
The C-X-C chemokine receptor type 4 (CXCR4) plays a crucial role in modulating cell trafficking in hematopoietic stem cells and clonal B cells. We screened 418 patients with B-cell lymphoproliferative disorders and described the presence of the C1013G/CXCR4 warts, hypogammaglobulinemia, infections, and myelokathexis-associated mutation in 28.2% (37/131) of patients with lymphoplasmacytic lymphoma (Waldenström macroglobulinemia [WM]), being either absent or present in only 7% of other B-cell lymphomas. In vivo functional characterization demonstrates its activating role in WM cells, as demonstrated by significant tumor proliferation and dissemination to extramedullary organs, leading to disease progression and decreased survival. The use of a monoclonal antibody anti-CXCR4 led to significant tumor reduction in a C1013G/CXCR4 WM model, whereas drug resistance was observed in mutated WM cells exposed to Bruton's tyrosine kinase, mammalian target of rapamycin, and phosphatidylinositol 3-kinase inhibitors, but not proteasome inhibitors. These findings demonstrate that C1013G/CXCR4 is an activating mutation in WM and support its role as a critical regulator of WM molecular pathogenesis and as an important therapeutic target.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/farmacologia , Mutação de Sentido Incorreto , Receptores CXCR4/metabolismo , Macroglobulinemia de Waldenstrom/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Intervalo Livre de Doença , Feminino , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos SCID , Metástase Neoplásica , Transplante de Neoplasias , Receptores CXCR4/genética , Taxa de Sobrevida , Macroglobulinemia de Waldenstrom/tratamento farmacológico , Macroglobulinemia de Waldenstrom/genética , Macroglobulinemia de Waldenstrom/mortalidade , Macroglobulinemia de Waldenstrom/patologiaRESUMO
CD70 is a tumor necrosis factor (TNF)-like type II integral membrane protein that is transiently expressed on activated T- and B-lymphocytes. Aberrant expression of CD70 was identified in both solid tumors and haematologic malignancies. BMS-936561 (αCD70_MED-A) is an antibody-drug conjugate composed of a fully human anti-CD70 monoclonal antibody (αCD70) conjugated with a duocarmycin derivative, MED-A, through a maleimide-containing citrulline-valine dipeptide linker. MED-A is a carbamate prodrug that is activated by carboxylesterase to its active form, MED-B, to exert its DNA alkylation activity. In vitro serum stability studies suggested the efficiencies of hydrolyzing the carbamate-protecting group in αCD70_MED-A followed a rank order of mouse>rat > >monkey>dog~human. Pharmacokinetics of αCD70_MED-A was evaluated in mice, monkeys, and dogs after single intravenous doses. In mice, αCD70_MED-A was cleared rapidly, with no detectable exposures after 15 min following dosing. In contrast, αCD70_MED-A was much more stable in monkeys and dogs. The clearance of αCD70_MED-A in monkeys was 58 mL/d/kg, ~2-fold faster than that in dogs (31 mL/d/kg). The human PK profiles of the total αCD70 and αCD70_MED-A were predicted using allometrically scaled monkeys PK parameters of αCD70 and the carbamate hydrolysis rate constant estimated in dogs. Comparing the predicted and observed human PK from the phase I study, the dose-normalized concentration-time profiles of αCD70_MED-A and the total αCD70 were largely within the 5(th)-95(th) percentile of the predicted profiles.
Assuntos
Anticorpos Monoclonais/farmacocinética , Antineoplásicos Alquilantes/farmacocinética , Ligante CD27/antagonistas & inibidores , Imunoconjugados/farmacocinética , Indóis/farmacocinética , Pró-Fármacos/farmacocinética , Animais , Anticorpos Monoclonais/sangue , Antineoplásicos Alquilantes/sangue , Ligante CD27/imunologia , Cães , Humanos , Imunoconjugados/sangue , Indóis/sangue , Macaca fascicularis , Camundongos Endogâmicos BALB C , Modelos BiológicosRESUMO
OBJECTIVE: CXCL10 (also known as interferon-γ-inducible 10-kd protein [IP-10]) is a chemokine that potentially plays a role in the immunopathogenesis of rheumatoid arthritis (RA). We undertook this phase II study to evaluate the efficacy and safety of MDX-1100, a fully human, anti-CXCL10 (anti-IP-10) monoclonal antibody, in RA patients whose disease responded inadequately to methotrexate (MTX). METHODS: Patients with active RA receiving stable doses of MTX (10-25 mg weekly) were randomized to receive intravenous doses of 10 mg/kg MDX-1100 (n = 35) or placebo (n = 35) every other week. The primary end point was the proportion of patients meeting the American College of Rheumatology 20% improvement criteria (achieving an ACR20 response) on day 85, and patients were followed up for safety to day 141. RESULTS: The ACR20 response rate was significantly higher among MDX-1100-treated patients than among placebo-treated patients (54% versus 17%; P = 0.0024). Statistically significant differences in the ACR20 response rate between treatments were observed starting on day 43 (P < 0.05). The ACR50 and ACR70 response rates on day 85 did not differ between the groups. Overall, 51.4% of MDX-1100-treated patients and 30.3% of placebo-treated patients experienced at least 1 adverse event (AE). No study drug-related serious AEs were reported. CONCLUSION: MDX-1100 was well tolerated and demonstrated clinical efficacy in RA patients whose disease responded inadequately to MTX. This is the first study to demonstrate clinical efficacy of a chemokine inhibitor in RA and supports the notion of a potential role of IP-10 in the immunopathogenesis of RA.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Quimiocina CXCL10/antagonistas & inibidores , Metotrexato/uso terapêutico , Adulto , Idoso , Anticorpos Monoclonais Humanizados/efeitos adversos , Antirreumáticos/efeitos adversos , Método Duplo-Cego , Quimioterapia Combinada , Feminino , Humanos , Masculino , Metotrexato/efeitos adversos , Pessoa de Meia-Idade , Resultado do TratamentoRESUMO
Adequate mobilization of hematopoietic stem cells (HSCs), especially CD34+ cells, is necessary for stem cell transplantation in patients with hematological malignancies or autoimmune diseases. Burixafor is an inhibitor of the C-X-C Chemokine Receptor 4 that disrupts the C-X-C motif chemokine 12 (CXCL12)/CXCR4 axis in the bone marrow, releasing HSCs into circulation. In mice, a single intravenous dose of burixafor was rapidly absorbed (time to maximum concentration, 5 minutes) and increased peripheral white blood cell counts within 30 minutes. Additionally, burixafor was administered to 64 healthy subjects in a randomized, double-blind, placebo-controlled, single-ascending-dose study to evaluate safety, pharmacokinetics, and pharmacodynamics. Subjects received burixafor intravenous doses ranging from 0.10 to 4.40 mg/kg in 8 cohorts. Single doses were generally safe and well tolerated. Gastrointestinal events were reported at doses of 2.24 mg/kg or greater. Exposure (maximum concentration and area under the concentration-time curve) increased in an approximately dose-proportional manner. Time to maximum concentration occurred with a median of 0.26-0.30 hours that was not dose proportional. As expected, white blood cell, CD133+ cell, and CD34+ cell concentrations generally increased with the increases in burixafor dose from 0.10 to 3.14 mg/kg. At maximal levels, the CD34+ cell counts increased 3- to 14-fold from baseline levels. These results provide support for continued clinical development of burixafor.
Assuntos
Mobilização de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Hematopoéticas , Humanos , Animais , Camundongos , Voluntários Saudáveis , Células-Tronco Hematopoéticas/metabolismo , Receptores de Quimiocinas/metabolismoRESUMO
BACKGROUND: Emerging evidence suggests that the mechanism of chemotherapy-induced cell death may influence the antitumor immune response in patients with cancer. Unlike immunologically silent apoptosis, pyroptosis is a lytic and inflammatory form of programmed cell death characterized by pore formation in the cell membrane and release of proinflammatory factors. Gasdermin E (GSDME) has recently gained attention after cleavage of GSDME by certain chemotherapeutics has been shown to elicit pyroptosis. This study investigated the immunomodulatory effects of a mesothelin-targeting antibody drug conjugate (ADC) in mouse models of breast and colon cancer. METHODS: The antitumor effects of the ADC were studied in EMT6 breast cancer and CT26 colon cancer syngeneic mouse models. The immunomodulatory effects of the ADC were assessed by analysis of tumor-infiltrating immune cells using flow cytometry. ADC mechanism of action was evaluated by morphology, biological assays, ADC-mediated cleavage of key effector proteins, and CRISPR/Cas9-mediated knockout (KO). Finally, the antitumor effect of ADC and Fms-like tyrosine kinase-3 ligand (Flt3L) combination therapy was evaluated in tumors expressing GSDME as well as in GSDME-silenced tumors. RESULTS: The data demonstrated that the ADC controlled tumor growth and stimulated anticancer immune responses. Investigation of the mechanism of action revealed that tubulysin, the cytotoxic payload of the ADC, induced cleavage of GSDME and elicited pyroptotic cell death in GSDME-expressing cells. Using GSDME KO, we showed that GSDME expression is critical for the effectiveness of the ADC as a monotherapy. Combining the ADC with Flt3L, a cytokine that expands dendritic cells in both lymphoid and non-lymphoid tissues, restored control of GSDME KO tumors. CONCLUSIONS: Together, these results show for the first time that tubulysin and a tubulysin containing ADC can elicit pyroptosis, and that this fiery cell death is critical for antitumor immunity and therapeutic response.
Assuntos
Neoplasias do Colo , Imunoconjugados , Animais , Camundongos , Piroptose , Anticorpos , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Apoptose , Modelos Animais de DoençasRESUMO
Autologous Stem Cell Transplant (ASCT) is increasingly used to treat hematological malignancies. A key requisite for ASCT is mobilization of hematopoietic stem cells into peripheral blood, where they are collected by apheresis and stored for later transplantation. However, success is often hindered by poor mobilization due to factors including prior treatments. The combination of G-CSF and GPC-100, a small molecule antagonist of CXCR4, showed potential in a multiple myeloma clinical trial for sufficient and rapid collection of CD34+ stem cells, compared to the historical results from the standards of care, G-CSF alone or G-CSF with plerixafor, also a CXCR4 antagonist. In the present study, we show that GPC-100 has high affinity towards the chemokine receptor CXCR4, and it potently inhibits ß-arrestin recruitment, calcium flux and cell migration mediated by its ligand CXCL12. Proximity Ligation Assay revealed that in native cell systems with endogenous receptor expression, CXCR4 co-localizes with the beta-2 adrenergic receptor (ß2AR). Co-treatment with CXCL12 and the ß2AR agonist epinephrine synergistically increases ß-arrestin recruitment to CXCR4 and calcium flux. This increase is blocked by the co-treatment with GPC-100 and propranolol, a non-selective beta-adrenergic blocker, indicating a functional synergy. In mice, GPC-100 mobilized more white blood cells into peripheral blood compared to plerixafor. GPC-100 induced mobilization was further amplified by propranolol pretreatment and was comparable to mobilization by G-CSF. Addition of propranolol to the G-CSF and GPC-100 combination resulted in greater stem cell mobilization than the G-CSF and plerixafor combination. Together, our studies suggest that the combination of GPC-100 and propranolol is a novel strategy for stem cell mobilization and support the current clinical trial in multiple myeloma registered as NCT05561751 at www.clinicaltrials.gov.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Compostos Heterocíclicos , Mieloma Múltiplo , Animais , Camundongos , Mobilização de Células-Tronco Hematopoéticas/métodos , Mieloma Múltiplo/tratamento farmacológico , Propranolol/uso terapêutico , Cálcio/metabolismo , Compostos Heterocíclicos/uso terapêutico , Células-Tronco Hematopoéticas/metabolismo , Receptores CXCR4/metabolismo , Fator Estimulador de Colônias de Granulócitos/farmacologia , beta-Arrestinas/metabolismo , Benzilaminas/metabolismoRESUMO
A new series with the tetrahydroisoquinoline-fused benzodiazepine (TBD) ring system combined with the surrogates of (1-methyl-1H-pyrrol-3-yl)benzene ("MPB") payloads were designed and executed for conjugation with a monoclonal antibody for anticancer therapeutics. DNA models helped in rationally identifying modifications of the "MPB" binding component and guided structure-activity relationship generation. This hybrid series of payloads exhibited excellent in vitro activity when tested against a panel of various cancer cell lines. One of the payloads was appended with a lysosome-cleavable peptide linker and conjugated with an anti-mesothelin antibody via a site-specific conjugation method mediated by the enzyme bacterial transglutaminase (BTGase). Antibody-drug conjugate (ADC) 50 demonstrated good plasma stability and lysosomal cleavage. A single intravenous dose of ADC 50 (5 or 10 nmol/kg) showed robust efficacy in an N87 gastric cancer xenograft model.
RESUMO
A human anti-CD19 antibody was expressed in fucosyltransferase-deficient CHO cells to generate nonfucosylated MDX-1342. Binding of MDX-1342 to human CD19-expressing cells was similar to its fucosylated parental antibody. However, MDX-1342 exhibited increased affinity for FcγRIIIa-Phe158 and FcγRIIIa-Val158 receptors as well as enhanced effector cell function, as demonstrated by increased potency and efficacy in antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis assays. MDX-1342 showed dose-dependent improvement in survival using a murine B-cell lymphoma model in which Ramos cells were administered systemically. In addition, low nanomolar binding to cynomolgus monkey CD19 and increased affinity for cynomolgus monkey FcγRIIIa was observed. In vivo administration of MDX-1342 in cynomolgus monkeys revealed potent B-cell depletion, suggesting its potential utility as a B-lymphocyte depletive therapy for malignancies and autoimmune indications.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígenos CD19/imunologia , Linfócitos B/imunologia , Depleção Linfocítica , Linfoma de Células B/terapia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Células CHO , Cricetinae , Cricetulus , Humanos , Macaca fascicularis , Camundongos , Camundongos SCID , Camundongos Transgênicos , Fagocitose , Receptores de IgG/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: This study was undertaken to evaluate the effects of MDX-1401, a nonfucosylated fully human monoclonal antibody that binds to human CD30, and to determine whether it exhibits greater in vitro and in vivo activity than its parental antibody. EXPERIMENTAL DESIGN: Assays measuring antibody binding to CD30-expressing cells and FcgammaRIIIa (CD16) transfectants as well as antibody-dependent cellular cytotoxicity (ADCC) were conducted. Antitumor activity was determined using a Karpas-299 systemic model. RESULTS: The binding of MDX-1401 to CD30 antigen was identical to fucose-containing parental anti-CD30 antibody (MDX-060). In contrast, MDX-1401 showed increased binding affinity to FcgammaRIIIa-transfected cells resulting in increased effector function. MDX-1401 greatly improved ADCC activity as evidenced by a decrease in half-maximal effective concentration (EC(50)) and an increase in maximum cell lysis when compared with MDX-060. Increased ADCC activity was observed among a panel of cell lines, including one with very low CD30 antigen expression in which parental antibody failed to induce any detectable ADCC. MDX-1401 activity with all FcgammaRIIIa polymorphic variants, including less active Phe/Phe158 and Phe/Val158 effector cells, was shown. Furthermore, MDX-1401 was efficacious in inhibiting tumor growth in CD30(+) lymphoma xenografts. CONCLUSIONS: The low doses of antibody required for ADCC activity irrespective of donor genotype, the ability to mediate ADCC in target cells expressing low levels of CD30, and increased in vivo efficacy support the development of MDX-1401 for treatment of malignant lymphoma.
Assuntos
Anticorpos Monoclonais/farmacologia , Linfoma/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Afinidade de Anticorpos/efeitos dos fármacos , Afinidade de Anticorpos/imunologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Sítios de Ligação de Anticorpos/imunologia , Células CHO , Carboidratos/química , Carboidratos/imunologia , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Fucose/química , Fucose/imunologia , Humanos , Antígeno Ki-1/imunologia , Linfoma/imunologia , Linfoma/patologia , Masculino , Camundongos , Camundongos SCID , Receptores de IgG/química , Receptores de IgG/imunologiaRESUMO
A series of tetrahydroisoquinoline-based benzodiazepine dimers were synthesized and tested for in vitro cytotoxicity against a panel of cancer cell lines. Structure-activity relationship investigation of various spacers guided by molecular modeling studies helped to identify compounds with picomolar activity. Payload 17 was conjugated to anti-mesothelin and anti-fucosylated monosialotetrahexosylganglioside (FucGM1) antibodies using lysosome-cleavable valine-citrulline dipeptide linkers via heterogeneous lysine conjugation and bacterial transglutaminase-mediated site-specific conjugation. In vitro, these antibody drug conjugates (ADCs) exhibited significant cytotoxic and target-mediated selectivity on human cancer cell lines. The pharmacokinetics and efficacy of these ADCs were further evaluated in gastric and lung cancer xenograft models in mice. Consistent pharmacokinetic profiles, high target specificity, and robust antitumor activity were observed in these models after a single dose of the ADC-46 (0.02 µmol/kg).
Assuntos
Anticorpos Monoclonais/química , Antineoplásicos/farmacologia , Benzodiazepinas/química , Desenho de Fármacos , Imunoconjugados/farmacologia , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Neoplasias Gástricas/tratamento farmacológico , Tetra-Hidroisoquinolinas/química , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Antineoplásicos/química , Apoptose , Benzodiazepinas/metabolismo , Proliferação de Células , Feminino , Gangliosídeo G(M1)/análogos & derivados , Gangliosídeo G(M1)/imunologia , Proteínas Ligadas por GPI/imunologia , Humanos , Imunoconjugados/química , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Mesotelina , Camundongos , Camundongos SCID , Carcinoma de Pequenas Células do Pulmão/patologia , Neoplasias Gástricas/patologia , Relação Estrutura-Atividade , Tetra-Hidroisoquinolinas/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
N-glycosylation is critical to the function of monoclonal antibodies (mAbs) and distinguishes various systems used for their production. We expressed human mAbs in the small aquatic plant Lemna minor, which offers several advantages for manufacturing therapeutic proteins free of zoonotic pathogens. Glycosylation of a mAb against human CD30 was optimized by co-expressing the heavy and light chains of the mAb with an RNA interference construct targeting expression of the endogenous alpha-1,3-fucosyltransferase and beta-1,2-xylosyltransferase genes. The resultant mAbs contained a single major N-glycan species without detectable plant-specific N-glycans and had better antibody-dependent cell-mediated cytotoxicity and effector cell receptor binding activities than mAbs expressed in cultured Chinese hamster ovary (CHO) cells.
Assuntos
Anticorpos Monoclonais/biossíntese , Araceae/metabolismo , Polissacarídeos/imunologia , Biossíntese de Proteínas/imunologia , Interferência de RNA/fisiologia , Proteínas Recombinantes/biossíntese , Animais , Araceae/genética , Biotecnologia , Células CHO , Cricetinae , Cricetulus , Glicosilação , Humanos , Antígeno Ki-1/imunologia , Plantas Geneticamente Modificadas/metabolismo , Polissacarídeos/químicaRESUMO
The tubular gland of the chicken oviduct is an attractive system for protein expression as large quantities of proteins are deposited in the egg, the production of eggs is easily scalable and good manufacturing practices for therapeutics from eggs have been established. Here we examined the ability of upstream and downstream DNA sequences of ovalbumin, a protein produced exclusively in very high quantities in chicken egg white, to drive tissue-specific expression of human mAb in chicken eggs. To accommodate these large regulatory regions, we established and transfected lines of chicken embryonic stem (cES) cells and formed chimeras that express mAb from cES cell-derived tubular gland cells. Eggs from high-grade chimeras contained up to 3 mg of mAb that possesses enhanced antibody-dependent cellular cytotoxicity (ADCC), nonantigenic glycosylation, acceptable half-life, excellent antigen recognition and good rates of internalization.
Assuntos
Anticorpos Monoclonais/química , Animais , Southern Blotting , Western Blotting , Células CHO , Varredura Diferencial de Calorimetria , Carboidratos/química , Galinhas , Cricetinae , DNA/metabolismo , Clara de Ovo , Embrião de Mamíferos/citologia , Embrião não Mamífero , Ensaio de Imunoadsorção Enzimática , Feminino , Vetores Genéticos , Genoma , Glicosilação , Humanos , Imunoglobulina G , Imuno-Histoquímica , Focalização Isoelétrica , Camundongos , Camundongos Endogâmicos BALB C , Modelos Genéticos , Monossacarídeos/química , Oligossacarídeos/química , Ovalbumina/genética , Ovalbumina/metabolismo , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização por Electrospray , Células-Tronco/citologiaRESUMO
Purpose: The ganglioside fucosyl-GM1 (FucGM1) is a tumor-associated antigen expressed in a large percentage of human small cell lung cancer (SCLC) tumors, but absent in most normal adult tissues, making it a promising target in immuno-oncology. This study was undertaken to evaluate the preclinical efficacy of BMS-986012, a novel, nonfucosylated, fully human IgG1 antibody that binds specifically to FucGM1.Experimental Design: The antitumor activity of BMS-986012 was evaluated in in vitro assays using SCLC cells and in mouse xenograft and syngeneic tumor models, with and without chemotherapeutic agents and checkpoint inhibitors.Results: BMS-986012 showed a high binding affinity for FcγRIIIa (CD16), which resulted in enhanced antibody-dependent cellular cytotoxicity (ADCC) against FucGM1-expressing tumor cell lines. BMS-986012-mediated tumor cell killing was also observed in complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP) assays. In several mouse SCLC models, BMS-986012 demonstrated efficacy and was well tolerated. In the DMS79 xenograft model, tumor regression was achieved with BMS-986012 doses of 0.3 mg/kg and greater; antitumor activity was enhanced when BMS-986012 was combined with standard-of-care cisplatin or etoposide. In a syngeneic model, tumors derived from a genetically engineered model of SCLC were treated with BMS-986012 or anti-FucGM1 with a mouse IgG2a Fc and their responses evaluated; when BMS-986012 was combined with anti-PD-1 or anti-CD137 antibody, therapeutic responses significantly improved.Conclusions: Single-agent BMS-986012 demonstrated robust antitumor activity, with the addition of chemotherapeutic or immunomodulatory agents further inhibiting SCLC growth in the same models. These preclinical data supported evaluation of BMS-986012 in a phase I clinical trial of patients with relapsed, refractory SCLC. Clin Cancer Res; 24(20); 5178-89. ©2018 AACR.
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Antineoplásicos Imunológicos/farmacologia , Gangliosídeo G(M1)/análogos & derivados , Animais , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos de Neoplasias/imunologia , Carcinoma de Células Pequenas/tratamento farmacológico , Carcinoma de Células Pequenas/metabolismo , Carcinoma de Células Pequenas/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Gangliosídeo G(M1)/antagonistas & inibidores , Gangliosídeo G(M1)/imunologia , Gangliosídeo G(M1)/metabolismo , Humanos , Imuno-Histoquímica , Imunomodulação/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Ligação Proteica , Receptores de IgG/metabolismo , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Traditional chemotherapeutic drugs are often restricted by severe side effects and lack of tumor specificity. Peptide prodrugs cleavable by peptidases present in the tumor environment have been explored to improve the therapeutic index of cytotoxic drugs. One such prodrug of doxorubicin (Dox), CPI-0004Na [N-succinyl-beta-alanyl-L-leucyl-L-alanyl-L-leucyl-Dox (sALAL-Dox)] has been shown to have an improved antitumor efficacy profile with reduced toxicity compared with Dox in tumor xenograft models (V. Dubois et al., Cancer Res., 62: 2327-2331, 2002). In this study, we demonstrate that CD10, a cell surface metalloprotease expressed on a variety of tumor cell types, is capable of cleaving CPI-0004Na and related peptide prodrugs such as N-succinyl-beta-alanyl-L-isoleucyl-L-alanyl-L-leucyl-Dox (sAIAL-Dox). This proteolytic cleavage generates leucyl-Dox, which is capable of entering cells and generating intracellular Dox. In a [(3)H]thymidine proliferation assay, analogues of CPI-0004Na showed a 100-300-fold increase in potency on CD10(+) cells compared with CD10(-) cells. Cytotoxicity of CPI-0004Na was inhibited by phosphoramidon, a known inhibitor of CD10 enzymatic activity. Furthermore, Chinese hamster ovary CHO-S cells, which are resistant to CPI-0004Na, could be sensitized to the cytotoxic effect of the prodrug by transfection of a CD10 cDNA. Tumor xenograft studies using LNCaP prostate tumor cells support the important role of CD10 in the antitumor efficacy of these prodrugs against tumors expressing CD10. CPI-0004Na and sAIAL-Dox achieved statistically significant 70% tumor growth inhibition at day 22. CD10 is expressed on many types of human tumors including B-cell lymphoma, leukemia, and prostate, breast, colorectal, and lung carcinomas; therefore, CD10-cleavable prodrugs may be effective in a range of different tumor types.
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Doxorrubicina/análogos & derivados , Doxorrubicina/farmacocinética , Neprilisina/metabolismo , Oligopeptídeos/farmacocinética , Pró-Fármacos/farmacocinética , Animais , Células CHO , Cricetinae , Relação Dose-Resposta a Droga , Doxorrubicina/efeitos adversos , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Nus , Neprilisina/antagonistas & inibidores , Neprilisina/biossíntese , Neprilisina/genética , Oligopeptídeos/efeitos adversos , Pró-Fármacos/efeitos adversos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/enzimologia , Transfecção , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0161779.].