RESUMO
Dysregulation of MET signaling has been implicated in tumorigenesis and metastasis. ARGX-111 combines complete blockade of this pathway with enhanced tumor cell killing and was investigated in 24 patients with MET-positive advanced cancers in a phase 1b study at four dose levels (0.3-10 mg/kg). ARGX-111 was well tolerated up to 3 mg/kg (MTD). Anti-tumor activity was observed in nearly half of the patients (46%) with a mean duration of treatment of 12 weeks. NHance® mutations in the Fc of ARGX-111 increased affinity for the neonatal Fc receptor (FcRn) at acidic pH, stimulating transcytosis across FcRn-expressing cells and radiolabeled ARGX-111 accumulated in lymphoid tissues, bone and liver, organs expressing FcRn at high levels in a biodistribution study using human FcRn transgenic mice. In line with this, we observed, in a patient with MET-amplified (>10 copies) gastric cancer, diminished metabolic activity in multiple metastatic lesions in lymphoid and bone tissues by 18F-FDG-PET/CT after two infusions with 0.3 mg/kg ARGX-111. When escalated to 1 mg/kg, a partial response was reached. Furthermore, decreased numbers of CTC (75%) possibly by the enhanced tumor cell killing witnessed the modes of action of the drug, warranting further clinical investigation of ARGX-111.
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Primary immune thrombocytopenia (ITP) is an acquired autoimmune bleeding disorder, characterized by a low platelet count (<100 × 109 /L) in the absence of other causes associated with thrombocytopenia. In most patients, IgG autoantibodies directed against platelet receptors can be detected. They accelerate platelet clearance and destruction, inhibit platelet production, and impair platelet function, resulting in increased risk of bleeding and impaired quality of life. Efgartigimod is a human IgG1 antibody Fc-fragment, a natural ligand of the neonatal Fc receptor (FcRn), engineered for increased affinity to FcRn, while preserving its characteristic pH-dependent binding. Efgartigimod blocks FcRn, preventing IgG recycling, and causing targeted IgG degradation. In this Phase 2 study, 38 patients were randomized 1:1:1 to receive four weekly intravenous infusions of either placebo (N = 12) or efgartigimod at a dose of 5 mg/kg (N = 13) or 10 mg/kg (N = 13). This short treatment cycle of efgartigimod in patients with ITP, predominantly refractory to previous lines of therapy, was shown to be well tolerated, and demonstrated a favorable safety profile consistent with Phase 1 data. Efgartigimod induced a rapid reduction of total IgG levels (up to 63.7% mean change from baseline), which was associated with clinically relevant increases in platelet counts (46% patients on efgartigimod vs 25% on placebo achieved a platelet count of ≥50 × 109 /L on at least two occasions, and 38% vs 0% achieved ≥50 × 109 /L for at least 10 cumulative days), and a reduced proportion of patients with bleeding. Taken together, these data warrant further evaluation of FcRn antagonism as a novel therapeutic approach in ITP.
Assuntos
Fragmentos Fc das Imunoglobulinas/uso terapêutico , Imunoglobulina G/uso terapêutico , Púrpura Trombocitopênica Idiopática , Receptores Fc/antagonistas & inibidores , Adulto , Idoso , Método Duplo-Cego , Feminino , Seguimentos , Antígenos de Histocompatibilidade Classe I/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Contagem de Plaquetas , Púrpura Trombocitopênica Idiopática/sangue , Púrpura Trombocitopênica Idiopática/tratamento farmacológico , Receptores Fc/sangueRESUMO
OBJECTIVE: To investigate safety and explore efficacy of efgartigimod (ARGX-113), an anti-neonatal Fc receptor immunoglobulin G1 Fc fragment, in patients with generalized myasthenia gravis (gMG) with a history of anti-acetylcholine receptor (AChR) autoantibodies, who were on stable standard-of-care myasthenia gravis (MG) treatment. METHODS: A phase 2, exploratory, randomized, double-blind, placebo-controlled, 15-center study is described. Eligible patients were randomly assigned (1:1) to receive 4 doses over a 3-week period of either 10 mg/kg IV efgartigimod or matched placebo combined with their standard-of-care therapy. Primary endpoints were safety and tolerability. Secondary endpoints included efficacy (change from baseline to week 11 of Myasthenia Gravis Activities of Daily Living, Quantitative Myasthenia Gravis, and Myasthenia Gravis Composite disease severity scores, and of the revised 15-item Myasthenia Gravis Quality of Life scale), pharmacokinetics, pharmacodynamics, and immunogenicity. RESULTS: Of the 35 screened patients, 24 were enrolled and randomized: 12 received efgartigimod and 12 placebo. Efgartigimod was well-tolerated in all patients, with no serious or severe adverse events reported, no relevant changes in vital signs or ECG findings observed, and no difference in adverse events between efgartigimod and placebo treatment. All patients treated with efgartigimod showed a rapid decrease in total immunoglobulin G (IgG) and anti-AChR autoantibody levels, and assessment using all 4 efficacy scales consistently demonstrated that 75% showed a rapid and long-lasting disease improvement. CONCLUSIONS: Efgartigimod was safe and well-tolerated. The correlation between reduction of levels of pathogenic IgG autoantibodies and disease improvement suggests that reducing pathogenic autoantibodies with efgartigimod may offer an innovative approach to treat MG. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that efgartigimod is safe and well-tolerated in patients with gMG.
Assuntos
Fatores Imunológicos/uso terapêutico , Miastenia Gravis/tratamento farmacológico , Receptores Fc/antagonistas & inibidores , Atividades Cotidianas , Corticosteroides/uso terapêutico , Adulto , Idoso , Autoanticorpos/imunologia , Inibidores da Colinesterase/uso terapêutico , Método Duplo-Cego , Feminino , Antígenos de Histocompatibilidade Classe I , Humanos , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Imunossupressores/uso terapêutico , Masculino , Pessoa de Meia-Idade , Miastenia Gravis/imunologia , Receptores Colinérgicos/imunologia , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: Intravenous Ig (IVIg), plasma exchange, and immunoadsorption are frequently used in the management of severe autoimmune diseases mediated by pathogenic IgG autoantibodies. These approaches modulating IgG levels can, however, be associated with some severe adverse reactions and a substantial burden to patients. Targeting the neonatal Fc receptor (FcRn) presents an innovative and potentially more effective, safer, and more convenient alternative for clearing pathogenic IgGs. METHODS: A randomized, double-blind, placebo-controlled first-in-human study was conducted in 62 healthy volunteers to explore single and multiple ascending intravenous doses of the FcRn antagonist efgartigimod. The study objectives were to assess safety, tolerability, pharmacokinetics, pharmacodynamics, and immunogenicity. The findings of this study were compared with the pharmacodynamics profile elicited by efgartigimod in cynomolgus monkeys. RESULTS: Efgartigimod treatment resulted in a rapid and specific clearance of serum IgG levels in both cynomolgus monkeys and healthy volunteers. In humans, single administration of efgartigimod reduced IgG levels up to 50%, while multiple dosing further lowered IgGs on average by 75% of baseline levels. Approximately 8 weeks following the last administration, IgG levels returned to baseline. Efgartigimod did not alter the homeostasis of albumin or Igs other than IgG, and no serious adverse events related to efgartigimod infusion were observed. CONCLUSION: Antagonizing FcRn using efgartigimod is safe and results in a specific, profound, and sustained reduction of serum IgG levels. These results warrant further evaluation of this therapeutic approach in IgG-driven autoimmune diseases. TRIAL REGISTRATION: Clinicaltrials.gov NCT03457649. FUNDING: argenx BVBA.
Assuntos
Doenças Autoimunes , Fragmentos Fc das Imunoglobulinas/administração & dosagem , Imunoglobulina G/sangue , Receptores Fc/antagonistas & inibidores , Adulto , Animais , Autoanticorpos/sangue , Doenças Autoimunes/sangue , Doenças Autoimunes/tratamento farmacológico , Células CHO , Cricetulus , Método Duplo-Cego , Feminino , Antígenos de Histocompatibilidade Classe I , Humanos , Fragmentos Fc das Imunoglobulinas/efeitos adversos , Macaca fascicularis , MasculinoRESUMO
Purpose: The purpose of this study was to evaluate safety, pharmacokinetics, pharmacodynamics, and preliminary antitumor efficacy of ARGX-110, a glyco-engineered monoclonal antibody, targeting CD70, in patients with CD70 expressing advanced malignancies.Experimental Design: Dose escalation with a sequential 3+3 design was performed in five steps at the 0.1, 1, 2, 5, and 10 mg/kg dose levels (N = 26). ARGX-110 was administered intravenously every 3 weeks until progression or intolerable toxicity. Dose-limiting toxicity was evaluated in the 21 days following the first ARGX-110 administration (Cycle 1). Samples for pharmacokinetics and pharmacodynamics were collected.Results: Dose-limiting toxicity was not observed and the maximum tolerated dose was not reached. ARGX-110 was generally well tolerated, with no dose-related increase in treatment-emergent adverse events (TEAE). The most common TEAE were fatigue and drug related infusion-related reactions (IRR). Of the 20 SAEs reported, five events, all IRRs, were considered related to ARGX-110. ARGX-110 demonstrates dose proportionality over the dose range 1 to 10 mg/kg, but not at 0.1 mg/kg and a terminal half-life of 10 to 13 days. The best overall response was stable disease (14/26) in all 26 evaluable patients with various malignancies and the mean duration of treatment was 15 weeks. No dose-response related antitumor activity was observed, but biomarker readouts provided signs of biological activity, particularly in patients with hematologic malignancies.Conclusions: This dose-escalation phase I trial provides evidence of good tolerability of ARGX-110, pharmacokinetics, and preliminary antitumor activity at all dose levels in generally heavily pretreated patients with advanced CD70-positive malignancies. Clin Cancer Res; 23(21); 6411-20. ©2017 AACR.
Assuntos
Anticorpos Anti-Idiotípicos/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Ligante CD27/imunologia , Neoplasias/tratamento farmacológico , Adulto , Idoso , Anticorpos Anti-Idiotípicos/efeitos adversos , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/farmacocinética , Ligante CD27/antagonistas & inibidores , Relação Dose-Resposta a Droga , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/classificação , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Feminino , Humanos , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Neoplasias/imunologia , Neoplasias/patologiaRESUMO
The activity of tumor necrosis factor (TNF), a cytokine involved in inflammatory pathologies, can be inhibited by antibodies or trap molecules. Herein, llama-derived variable heavy-chain domains of heavy-chain antibody (VHH, also called Nanobodies™) were generated for the engineering of bivalent constructs, which antagonize the binding of TNF to its receptors with picomolar potencies. Three monomeric VHHs (VHH#1, VHH#2, and VHH#3) were characterized in detail and found to bind TNF with sub-nanomolar affinities. The crystal structures of the TNF-VHH complexes demonstrate that VHH#1 and VHH#2 share the same epitope, at the center of the interaction area of TNF with its TNFRs, while VHH#3 binds to a different, but partially overlapping epitope. These structures rationalize our results obtained with bivalent constructs in which two VHHs were coupled via linkers of different lengths. Contrary to conventional antibodies, these bivalent Nanobody™ constructs can bind to a single trimeric TNF, thus binding with avidity and blocking two of the three receptor binding sites in the cytokine. The different mode of binding to antigen and the engineering into bivalent constructs supports the design of highly potent VHH-based therapeutic entities.
RESUMO
Highly potent human antibodies are required to therapeutically neutralize cytokines such as interleukin-6 (IL-6) that is involved in many inflammatory diseases and malignancies. Although a number of mutagenesis approaches exist to perform antibody affinity maturation, these may cause antibody instability and production issues. Thus, a robust and easy antibody affinity maturation strategy to increase antibody potency remains highly desirable. By immunizing llama, cloning the 'immune' antibody repertoire and using phage display, we selected a diverse set of IL-6 antagonistic Fabs. Heavy chain shuffling was performed on the Fab with lowest off-rate, resulting in a panel of variants with even lower off-rate. Structural analysis of the Fab:IL-6 complex suggests that the increased affinity was partly due to a serine to tyrosine switch in HCDR2. This translated into neutralizing capacity in an in vivo model of IL-6 induced SAA production. Finally, a novel Fab library was designed, encoding all variations found in the natural repertoire of VH genes identified after heavy chain shuffling. High stringency selections resulted in identification of a Fab with 250-fold increased potency when re-formatted into IgG1. Compared with a heavily engineered anti-IL-6 monoclonal antibody currently in clinical development, this IgG was at least equally potent, showing the engineering process to have had led to a highly potent anti-IL-6 antibody.
Assuntos
Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Mutação/genética , Biblioteca de Peptídeos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos , Camelídeos Americanos/genética , Humanos , Fragmentos Fab das Imunoglobulinas/química , Interleucina-6/imunologia , Modelos Imunológicos , Modelos Moleculares , Proteínas Recombinantes/química , Alinhamento de SequênciaRESUMO
Hepatocyte growth factor (HGF) and its receptor MET represent validated targets for cancer therapy. However, HGF/MET inhibitors being explored as cancer therapeutics exhibit cytostatic activity rather than cytotoxic activity, which would be more desired. In this study, we engineered an antagonistic anti-MET antibody that, in addition to blocking HGF/MET signaling, also kills MET-overexpressing cancer cells by antibody-dependent cellular cytotoxicity (ADCC). As a control reagent, we engineered the same antibody in an ADCC-inactive form that is similarly capable of blocking HGF/MET activity, but in the absence of any effector function. In comparing these two antibodies in multiple mouse models of cancer, including HGF-dependent and -independent tumor xenografts, we determined that the ADCC-enhanced antibody was more efficacious than the ADCC-inactive antibody. In orthotopic mammary carcinoma models, ADCC enhancement was crucial to deplete circulating tumor cells and to suppress metastases. Prompted by these results, we optimized the ADCC-enhanced molecule for clinical development, generating an antibody (ARGX-111) with improved pharmacologic properties. ARGX-111 competed with HGF for MET binding, inhibiting ligand-dependent MET activity, downregulated cell surface expression of MET, curbing HGF-independent MET activity, and engaged natural killer cells to kill MET-expressing cancer cells, displaying MET-specific cytotoxic activity. ADCC assays confirmed the cytotoxic effects of ARGX-111 in multiple human cancer cell lines and patient-derived primary tumor specimens, including MET-expressing cancer stem-like cells. Together, our results show how ADCC provides a therapeutic advantage over conventional HGF/MET signaling blockade and generates proof-of-concept for ARGX-111 clinical testing in MET-positive oncologic malignancies.
Assuntos
Anticorpos Monoclonais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Fator de Crescimento de Hepatócito/metabolismo , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Ligação Competitiva , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Humanos , Camundongos Nus , Neoplasias/metabolismo , Neoplasias/patologia , Ligação Proteica , Proteínas Proto-Oncogênicas c-met/imunologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Activation of MET by HGF plays a key role in tumor progression. Using a recently developed llama platform that generates human-like immunoglobulins, we selected 68 different antibodies that compete with HGF for binding to MET. HGF-competing antibodies recognized 4 distinct hotspots localized in different MET domains. We identified 1 hotspot that coincides with the known HGF ß chain binding site on blades 2-3 of the SEMA domain ß-propeller. We determined that a second and a third hotspot lie within blade 5 of the SEMA domain and IPT domains 2-3, both of which are thought to bind to HGF α chain. Characterization of the fourth hotspot revealed a region across the PSI-IPT 1 domains not previously associated with HGF binding. Individual or combined targeting of these hotspots effectively interrupted HGF/MET signaling in multiple cell-based biochemical and biological assays. Selected antibodies directed against SEMA blades 2-3 and the PSI-IPT 1 region inhibited brain invasion and prolonged survival in a glioblastoma multiforme model, prevented metastatic disease following neoadjuvant therapy in a triple-negative mammary carcinoma model, and suppressed cancer cell dissemination to the liver in a KRAS-mutant metastatic colorectal cancer model. These results identify multiple regions of MET responsible for HGF-mediated tumor progression, unraveling the complexity of HGF-MET interaction, and provide selective molecular tools for targeting MET activity in cancer.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Anticorpos Monoclonais , Afinidade de Anticorpos , Sítios de Ligação , Ligação Competitiva , Neoplasias Encefálicas/patologia , Camelídeos Americanos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Progressão da Doença , Glioblastoma/patologia , Fator de Crescimento de Hepatócito/química , Fator de Crescimento de Hepatócito/imunologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas c-met/químicaRESUMO
Overexpression of CD70 has been documented in a variety of solid and hematological tumors, where it is thought to play a role in tumor proliferation and evasion of immune surveillance. Here, we describe ARGX-110, a defucosylated IgG1 monoclonal antibody (mAb) that selectively targets and neutralizes CD70, the ligand of CD27. ARGX-110 was generated by immunization of outbred llamas. The antibody was germlined to 95% human identity, and its anti-tumor efficacy was tested in several in vitro assays. ARGX-110 binds CD70 with picomolar affinity. In depletion studies, ARGX-110 lyses tumor cells with greater efficacy than its fucosylated version. In addition, ARGX-110 demonstrates strong complement-dependent cytotoxicity and antibody-dependent cellular phagocytosis activity. ARGX-110 inhibits signaling of CD27, which results in blocking of the activation and proliferation of Tregs. In a Raji xenograft model, administration of the fucosylated version of ARGX-110 resulted in a prolonged survival at doses of 0.1 mg/kg and above. The pharmacokinetics of ARGX-110 was tested in cynomolgus monkeys; the calculated half-life is 12 days. In conclusion, ARGX-110 is a potent blocking mAb with a dual mode of action against both CD70-bearing tumor cells and CD70-dependent Tregs. This antibody is now in a Phase 1 study in patients with advanced malignancies expressing CD70 (NCT01813539).
Assuntos
Anticorpos Monoclonais/metabolismo , Antineoplásicos/metabolismo , Ligante CD27/imunologia , Imunoglobulina G/metabolismo , Imunoterapia/métodos , Neoplasias/terapia , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Anticorpos Monoclonais/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Antineoplásicos/imunologia , Camelídeos Americanos , Pontos de Checagem do Ciclo Celular/imunologia , Células Cultivadas , Humanos , Imunoglobulina G/imunologia , Ativação Linfocitária/efeitos dos fármacos , Neoplasias/imunologia , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/antagonistas & inibidoresRESUMO
The approximately 15-kDa variable domains of camelid heavy-chain-only antibodies (called Nanobodies) can easily be formatted as multivalent or multispecific single-chain proteins. Because of fast excretion, however, they are less suitable for therapy of cancer. In this study, we aimed for improved tumor targeting of a bivalent anti-epidermal growth factor receptor (EGFR) Nanobody (alphaEGFR-alphaEGFR) by fusion to a Nanobody unit binding to albumin (alphaAlb). Biodistributions of alphaEGFR-alphaEGFR, alphaEGFR-alphaEGFR-alphaAlb ( approximately 50 kDa), alphaTNF-alphaTNF-alphaAlb (control, binding tumor necrosis factor-alpha), and the approximately 150-kDa anti-EGFR antibody cetuximab were compared in A431 xenograft-bearing mice. The proteins were radiolabeled with (177)Lu to facilitate quantification. Tumor uptake of (177)Lu-alphaEGFR-alphaEGFR decreased from 5.0 +/- 1.4 to 1.1 +/- 0.1 %ID/g between 6 and 72 h after injection. Due to its rapid blood clearance, tumor-to-blood ratios >80 were obtained within 6 h after injection. Blood clearance became dramatically slower and tumor uptake became significantly higher by introduction of alphaAlb. Blood levels of alphaEGFR-alphaEGFR-alphaAlb were 21.2 +/- 2.5, 11.9 +/- 0.6, and 4.0 +/- 1.4 and tumor levels were 19.4 +/- 5.5, 35.2 +/- 7.5, and 28.0 +/- 6.8 %ID/g at 6, 24, and 72 h after injection, respectively. Tumor uptake was at least as high as for cetuximab (15.5 +/- 3.9, 27.1 +/- 7.9, and 25.6 +/- 6.1 %ID/g) and significantly higher than for alphaTNF-alphaTNF-alphaAlb. alphaEGFR-alphaEGFR-alphaAlb showed faster and deeper tumor penetration than cetuximab. These data show that simple fusion of alphaEGFR and alphaAlb building blocks results in a bifunctional Nanobody format, which seems more favorable for therapy as far as pharmacokinetics and tumor deposition are concerned.
Assuntos
Albuminas/metabolismo , Anticorpos/imunologia , Receptores ErbB/imunologia , Nanotecnologia , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais Humanizados , Cetuximab , Receptores ErbB/metabolismo , Ligação Proteica , Distribuição TecidualRESUMO
OBJECTIVE: The advent of tumor necrosis factor (TNF)-blocking drugs has provided rheumatologists with an effective, but highly expensive, treatment for the management of established rheumatoid arthritis (RA). Our aim was to explore preclinically the application of camelid anti-TNF VHH proteins, which are single-domain antigen binding (VHH) proteins homologous to human immunoglobulin V(H) domains, as TNF antagonists in a mouse model of RA. METHODS: Llamas were immunized with human and mouse TNF, and antagonistic anti-TNF VHH proteins were isolated and cloned for bacterial production. The resulting anti-TNF VHH proteins were recombinantly linked to yield bivalent mouse and human TNF-specific molecules. To increase the serum half-life and targeting properties, an anti-serum albumin anti-TNF VHH domain was incorporated into the bivalent molecules. The TNF-neutralizing potential was analyzed in vitro. Mouse TNF-specific molecules were tested in a therapeutic protocol in murine collagen-induced arthritis (CIA). Disease progression was evaluated by clinical scoring and histologic evaluation. Targeting properties were evaluated by 99mTc labeling and gamma camera imaging. RESULTS: The bivalent molecules were up to 500 times more potent than the monovalent molecules. The antagonistic potency of the anti-human TNF VHH proteins exceeded even that of the anti-TNF antibodies infliximab and adalimumab that are used clinically in RA. Incorporation of binding affinity for albumin into the anti-TNF VHH protein significantly prolonged its serum half-life and promoted its targeting to inflamed joints in the murine CIA model of RA. This might explain the excellent therapeutic efficacy observed in vivo. CONCLUSION: These data suggest that because of the flexibility of their format, camelid anti-TNF VHH proteins can be converted into potent therapeutic agents that can be produced and purified cost-effectively.
Assuntos
Antirreumáticos/uso terapêutico , Artrite Experimental/terapia , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/uso terapêutico , Região Variável de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/uso terapêutico , Fator de Necrose Tumoral alfa/imunologia , Adalimumab , Animais , Anticorpos/imunologia , Anticorpos/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Camelídeos Americanos/imunologia , Meia-Vida , Cadeias Pesadas de Imunoglobulinas/sangue , Região Variável de Imunoglobulina/sangue , Infliximab , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Pro-inflammatory cytokines regulate the growth, differentiation, and activation of immune cells and can play a role in antitumor responses. GM-CSF and IL-2 induce tumor rejection in animal models when expressed by tumor cells, and IL-2 is used for the treatment of melanoma and renal cell cancer. However, high doses of GM-CSF and IL-2 are associated with severe side effects in cancer patients. We generated a dual cytokine fusion protein for simultaneous targeted delivery of human GM-CSF and IL-2 to human tumors. The fusion protein is based on a heterodimeric core structure formed by human CH1 and C kappa domains (heterominibody) with C-terminally fused human cytokines and N-terminally fused human single-chain Ab fragments (scFv) specific for the tumor-associated surface antigen epithelial cell adhesion molecule (Ep-CAM). The dual cytokine heterominibody (DCH) was well expressed and secreted by CHO cells, preserved the specific proliferative activities of the two cytokines, and showed Ep-CAM-specific binding to tumor cells. DCH induced potent tumor cell lysis in vitro by two distinct mechanisms. One was activating PBMCs to lyse tumor cells, which was superior to cytotoxicity induced by equimolar ratios of free recombinant human IL-2 and GM-CSF. The other mechanism was redirected lysis, as seen with isolated human T cells, which was solely dependent on the IL-2 fusion part. The therapeutic principle of dual cytokine targeting may warrant in vivo testing of murine-specific analogues in appropriate mouse models and further preclinical development of the less immunogenic, human cytokine- and human Ep-CAM-specific DCH molecule described here.
Assuntos
Antineoplásicos/administração & dosagem , Moléculas de Adesão Celular/genética , Sistemas de Liberação de Medicamentos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Interleucina-2/administração & dosagem , Animais , Anticorpos/administração & dosagem , Antígenos de Neoplasias/administração & dosagem , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Antineoplásicos/imunologia , Células CHO , Moléculas de Adesão Celular/administração & dosagem , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/imunologia , Linhagem Celular Tumoral , Cricetinae , Citotoxicidade Imunológica , Modelos Animais de Doenças , Combinação de Medicamentos , Eletroforese em Gel de Poliacrilamida , Molécula de Adesão da Célula Epitelial , Células Epiteliais/química , Células Epiteliais/citologia , Variação Genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Interleucina-2/análogos & derivados , Interleucina-2/genética , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes , Linfócitos T/imunologiaRESUMO
BscCD19xCD3 is a bispecific single-chain antibody construct with exceptional cytotoxic potency in vitro and in vivo. Here, we have investigated the biological activity of bscCD19xCD3 in chimpanzee, the only animal species identified in which bscCD19xCD3 showed bispecific binding, redirected B-cell lysis and cytokine production comparable to human cells. Pharmacokinetic analysis following 2-h intravenous infusion of 0.06, 0.1 or 0.12 mug/kg of bscCD19xCD3 as part of a dose escalation study in a single female chimpanzee revealed a half-life of approximately 2 h and elimination of the bispecific antibody from circulation within approximately 8 h after the end of infusion. This short exposure to bscCD19xCD3 elicited a transient increase in serum levels of IFNgamma, IL-6, IL-2, soluble CD25, and transiently upregulated expression of CD69 and MHC class II on CD8-positive cells. Cytokine release and upregulation of T-cell activation markers were not observed with vehicle controls. A multiple-dose study using 5 weekly doses of 0.1 mug/kg in two animals also showed transient cytokine release and an activation of peripheral T cells with a first-dose effect, accompanied by a transient lymphopenia. While oscillations of T-cell counts were relatively even during repeated treatments, the amplitudes of peripheral B cells declined with every infusion, which was not observed in a vehicle control animal. Our data show that bscCD19xCD3 can be safely administered to chimpanzees at dose levels that cause fully reversible T-cell activation and, despite a very short exposure time, cumulative loss of peripheral B lymphocytes. A clinical trial testing prolonged administration of bscCD19xCD3 (MT103) for improving efficacy is currently ongoing.
Assuntos
Anticorpos Biespecíficos/farmacocinética , Linfócitos B/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Depleção Linfocítica , Linfócitos T/efeitos dos fármacos , Animais , Antígenos CD19/imunologia , Linfócitos B/imunologia , Complexo CD3/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Testes Imunológicos de Citotoxicidade , Feminino , Citometria de Fluxo , Humanos , Imunoterapia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Ativação Linfocitária/imunologia , Masculino , Pan troglodytes , Linfócitos T/imunologiaRESUMO
PURPOSE: The epithelial cell adhesion molecule (Ep-CAM) is a clinically validated target for antibody-based therapy of cancer. The aim of this work was to evaluate the specific cytotoxic activity of a novel fully human Ep-CAM-specific IgG1 antibody, called MT201, against primary ovarian tumor cells and an ovarian tumor cell line. METHODS: The anti-tumor efficacy of MT201 was examined both in coculture of the ovarian cancer cell line OvCAR-3 and peripheral blood mononuclear cells (PBMCs) from healthy donors, and in primary metastatic tumor specimens freshly dissected from 21 patients with ovarian cancer using only the tumor-resident autologous effector cells. The extent of tumor cell depletion was determined by flow cytometry using Ep-CAM/CA-125 double-labeling or Ep-CAM labeling, both combined with propidium iodide uptake as cell lysis marker. RESULTS: MT201 at sub- micro g/ml concentrations effectively eliminated OvCar-3 cells in the presence of PBMC. In freshly dissected tumor specimen, endogenous autologous immune cells could lyse, in a MT201-dependent fashion, Ep-CAM-positive tumor cells in 17 out of 21 patients showing an ex vivo response rate of 81%. In certain samples, up to 80% lysis of Ep-CAM-positive tumor cells by MT201 were observed after 16-30 h of incubation. CONCLUSIONS: These data indicate that MT201 can effectively redirect tumor-resident effector cells against Ep-CAM-positive ovarian cancer cells and may therefore offer an effective therapy for ovarian cancer.
Assuntos
Anticorpos Monoclonais/farmacologia , Moléculas de Adesão Celular/farmacologia , Neoplasias Ovarianas/patologia , Anticorpos Monoclonais Humanizados , Antígenos de Neoplasias/imunologia , Biomarcadores Tumorais/imunologia , Complexo CD3/imunologia , Moléculas de Adesão Celular/imunologia , Morte Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Molécula de Adesão da Célula Epitelial , Feminino , Humanos , Leucócitos Mononucleares , Células Tumorais CultivadasRESUMO
Antigen-specific B cells are key players in many autoimmune diseases through the production of autoreactive antibodies that can cause severe tissue damage and malfunction. We have designed and expressed a fusion protein, referred to as MOG-Fc, composed of the extracellular Ig-like domain of human myelin oligodendrocyte glycoprotein (MOG) and the C(H)2 and C(H)3 domains of the human IgG1 heavy chain. The dimerized fusion protein was capable of mediating cytotoxicity against a MOG-reactive hybridoma line in vitro. Likewise, MOG-Fc significantly reduced the number of circulating MOG-reactive B cells in an anti-MOG Ig heavy chain knock-in mouse model. Our study shows that autoantigen-reactive B lymphocytes can be efficiently and selectively eliminated by an autoantigen Fcgamma1 fusion protein in vitro as well as in vivo. Such fusion proteins may provide a platform for the development of highly selective therapeutic approaches.
Assuntos
Autoantígenos/genética , Autoimunidade , Linfócitos B/imunologia , Depleção Linfocítica , Glicoproteína Associada a Mielina/genética , Animais , Anticorpos Antinucleares/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Células CHO , Cricetinae , Humanos , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Camundongos , Proteínas da Mielina , Glicoproteína Mielina-Oligodendrócito , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/toxicidadeRESUMO
We have recently demonstrated that a recombinant single-chain bispecific Ab construct, bscCD19xCD3, in vitro induces rapid B lymphoma-directed cytotoxicity at picomolar concentrations with unstimulated peripheral T cells. In this study, we show that treatment of nonobese diabetic SCID mice with submicrogram doses of bscCD19xCD3 could prevent growth of s.c. human B lymphoma xenografts and essentially cured animals when given at an early tumor stage. The effect was dose dependent, dependent on E:T ratio and the time between tumor inoculation and administration of bscCD19xCD3. No therapeutic effect was seen in the presence of human lymphocytes alone, a vehicle control, or with a bispecific single-chain construct of identical T cell-binding activity but different target specificity. In a leukemic nonobese diabetic SCID mouse model, treatment with bscCD19xCD3 prolonged survival of mice in a dose-dependent fashion. The human lymphocytes used as effector cells in both animal models did not express detectable T cell activation markers at the time of coinoculation with tumor cells. The bispecific Ab therefore showed an in vivo activity comparable to that observed in cell culture with respect to high potency and T cell costimulus independence. These properties make bscCD19xCD3 superior to previously investigated CD19 bispecific Ab-based therapies.
Assuntos
Anticorpos Biespecíficos/farmacologia , Antígenos CD19/imunologia , Antineoplásicos/farmacologia , Complexo CD3/imunologia , Inibidores do Crescimento/farmacologia , Leucemia de Células B/prevenção & controle , Linfoma de Células B/prevenção & controle , Subpopulações de Linfócitos T/imunologia , Transplante Heterólogo , Animais , Anticorpos Biespecíficos/genética , Antineoplásicos/síntese química , Células Cultivadas , Sobrevivência de Enxerto/genética , Sobrevivência de Enxerto/imunologia , Inibidores do Crescimento/síntese química , Humanos , Injeções Intravenosas , Injeções Subcutâneas , Leucemia de Células B/genética , Leucemia de Células B/imunologia , Leucemia de Células B/patologia , Ativação Linfocitária/genética , Linfoma de Células B/genética , Linfoma de Células B/imunologia , Linfoma de Células B/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/prevenção & controle , Fatores de Tempo , Transplante Heterólogo/métodos , Células Tumorais CultivadasRESUMO
The epithelial cell adhesion molecule (Ep-CAM) is expressed on the surface of most human carcinomas, including ovarian, breast, lung, prostate and colorectal carcinoma. Ep-CAM was shown to be a valid target for monoclonal antibody-based therapies. We have investigated whether an Ep-CAM-/CD3-bispecific single-chain antibody called bscEp-CAM x CD3 is effective in tumor cell elimination within the cellular microenvironment of primary ovarian cancer tissue. The ex vivo elimination of ovarian cancer cells in tumor preparations from 21 patients was monitored by flow cytometry using Ep-CAM/CA-125 double-labeling or Ep-CAM single-labeling combined with propidium iodide uptake of cells. Methodology was established by the ovarian cancer cell line OvCAR. A total of 17 (81%) patient samples showed a dose-dependent tumor cell elimination by bscEp-CAM x CD3. High and specific tumor cell lysis was seen at bscEp-CAM x CD3 concentrations as low as 1 ng/ml, at very low effector:target ratios and in the absence of T cell costimulation. The high efficacy of the bispecific antibody may be due to the non-restricted activation of tumor-resident cytotoxic T lymphocytes. In clinical trials, the ex vivo data with the T cell-recruiting bispecific antibody bscEp-CAM x CD3 may translate into a high response rate and efficacy of tumor cell elimination.
Assuntos
Anticorpos Biespecíficos/farmacologia , Antígenos de Neoplasias/imunologia , Complexo CD3/imunologia , Moléculas de Adesão Celular/imunologia , Citotoxicidade Imunológica/imunologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias Ovarianas/patologia , Neoplasias Peritoneais/secundário , Linfócitos T/imunologia , Sobrevivência Celular , Molécula de Adesão da Célula Epitelial , Feminino , Citometria de Fluxo , Humanos , Ativação Linfocitária/efeitos dos fármacos , Neoplasias Ovarianas/imunologia , Neoplasias Peritoneais/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Células Tumorais CultivadasRESUMO
A recent study reported on an anti-CD19/anti-CD3 single-chain bispecific antibody (bscCD19xCD3) exhibiting high activity against human B lymphoma cell lines (Löffler et al., Blood 2000;95:2098-103). In the present study, we have explored in detail the in vitro efficacy, T-cell donor variability, binding characteristics, specificity, kinetics and interleukin-2 (IL-2) dependence of bscCD19xCD3. We found that a majority of human donor T cells tested (n = 86) gave half-maximal B-lymphoma cell lysis (ED(50)) within a range of 10-50 pg/ml bscCD19xCD3, corresponding to sub-picomolar concentrations of the bispecific antibody. Under identical experimental conditions, the anti-CD20 monoclonal antibody rituximab had an at least 100,000-fold lower in vitro efficacy. The extreme potency of bscCD19xCD3 was in sharp contrast to the relatively low affinity of the anti-CD3 and anti-CD19 single-chain Fv portions in K(D) ranges of 10(-7) and 10(-9) M, respectively. Cell lysis by bscCD19xCD3 was predominantly mediated by the population of CD8/CD45RO-positive T cells. Both immortalized CD4- and CD8-positive human T-cell clones were highly active effector cells as well. Cell lysis by bscCD19xCD3 was rapid and specific. The respective parental monoclonal antibodies inhibited cell lysis and CD19-negative cells were not harmed by T cells in the presence of high amounts of bscCD19xCD3. The potent T-cell stimulus IL-2 could not markedly augment the activity of bscCD19xCD3-stimulated T cells. In conclusion, bscCD19xCD3 could redirect unstimulated cytotoxic T cells against CD19-positive cells in an unexpectedly potent, rapid and specific fashion.
Assuntos
Anticorpos Biespecíficos/metabolismo , Antígenos CD19/metabolismo , Complexo CD3/metabolismo , Linfoma/metabolismo , Linfócitos T Citotóxicos/metabolismo , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Murinos , Células CHO , Separação Celular , Cricetinae , Relação Dose-Resposta Imunológica , Citometria de Fluxo , Humanos , Interleucina-2/metabolismo , Cinética , Ativação Linfocitária , Camundongos , Rituximab , Fatores de Tempo , Células Tumorais CultivadasRESUMO
In our study, a novel, fully human, recombinant monoclonal antibody of the IgG1 isotype, called MT201, was characterized for its binding properties, complement-dependent (CDC) and antibody-dependent cellular cytotoxicity (ADCC), as well as for its in vivo antitumor activity in a nude mouse model. MT201 was found to bind its target, the epithelial cell adhesion molecule (Ep-CAM; also called 17-1A antigen, KSA, EGP-2, GA733-2), with low affinity in a range similar to that of the clinically validated, murine monoclonal IgG2a antibody edrecolomab (Panorex(R)). MT201 exhibited Ep-CAM-specific CDC with a potency similar to that of edrecolomab. However, the efficacy of ADCC of MT201, as mediated by human immune effector cells, was by 2 orders of magnitude higher than that of edrecolomab. Addition of human serum reduced the ADCC of MT201 while it essentially abolished ADCC of edrecolomab within the concentration range tested. In a nude mouse xenograft model, growth of tumors derived from the human colon carcinoma line HT-29 was significantly and comparably suppressed by MT201 and edrecolomab. The fully human nature and the improved ADCC of MT201 with human effector cells will make MT201 a promising candidate for the clinical development of a novel pan-carcinoma antibody that is superior to edrecolomab.