RESUMO
Mesothelin (MSLN) is a cell surface protein overexpressed in a number of cancer types. Several antibody- and cellular-based MSLN targeting agents have been tested in clinical trials where their therapeutic efficacy has been moderate at best. Previous studies using antibody and Chimeric Antigen Receptor-T cells (CAR-T) strategies have shown the importance of particular MSLN epitopes for optimal therapeutic response, while other studies have found that certain MSLN-positive tumors can produce proteins that can bind to subsets of IgG1-type antibodies and suppress their immune effector activities. In an attempt to develop an improved anti-MSLN targeting agent, we engineered a humanized divalent anti-MSLN/anti-CD3ε bispecific antibody that avoids suppressive factors, can target a MSLN epitope proximal to the tumor cell surface, and is capable of effectively binding, activating, and redirecting T cells to the surface of MSLN-positive tumor cells. NAV-003 has shown significantly improved tumor cell killing against lines producing immunosuppressive proteins in vitro and in vivo. Moreover, NAV-003 demonstrated good tolerability in mice and efficacy against patient-derived mesothelioma xenografts co-engrafted with human peripheral blood mononuclear cells. Together these data support the potential for NAV-003 clinical development and human proof-of-concept studies in patients with MSLN-expressing cancers.
Assuntos
Leucócitos Mononucleares , Mesotelina , Humanos , Animais , Camundongos , Proteínas Ligadas por GPI , Epitopos , Linhagem Celular Tumoral , Modelos Animais de DoençasRESUMO
C1q-engagement with IgG and IgM type antibodies is the initiating step of classical complement-mediated immunity. The tumor shed antigen CA125 has been reported to have immunosuppressive effects on host tumor responses as well as commercially approved and experimental monoclonal antibody (mAb)-based therapeutic agents. To better understand this effect, molecular and cellular studies were carried out testing the ability of CA125 to perturb the classical complement pathway. Here, we show that patient-derived CA125 inhibits IgG1, IgG3, and IgM-mediated complement-dependent cytotoxicity (CDC) by perturbing antibody-Fc interaction with the C1q complement-initiating protein only in those mAbs that are directly bound by CA125. This mechanism was found to impact naturally generated IgM antibodies as well as experimental and clinically approved mAbs, such as farletuzumab and rituximab, respectively. These data support a role for CA125 in humoral immune suppression and as a potential mechanism by which tumors may possibly avoid host immune responses.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Neoplasias/imunologia , Antígeno Ca-125/imunologia , Complemento C1q/imunologia , Neoplasias/imunologia , Animais , Anticorpos Monoclonais Humanizados/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Células CHO , Ativação do Complemento/imunologia , Cricetulus , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Rituximab/imunologiaRESUMO
Farletuzumab is a humanized monoclonal antibody that binds to folate receptor alpha and elicits an anti-tumor response via immune effector activity. Recent studies from a global phase 3 trial in ovarian cancer patients treated with carboplatin/taxane plus farletuzumab found that the tumor-produced CA125 protein can suppress farletuzumab function via perturbing its engagement to the activating Fc-γ receptors CD32a (FCGR2A) and CD16a (FCGR3A). Previous reports have indicated that naturally occurring polymorphisms in both of these receptors may play a role in their ability to engage therapeutic antibodies and elicit an optimal immune response via antibody-dependent cellular cytotoxicity (ADCC). In light of the importance of farletuzumab ADCC function for optimal tumor cell killing, we evaluated the frequency of FCGR2A-131H/R and FCGR3A-158V/F polymorphisms in 461 consenting patients from this global clinical study and their association with clinical outcome to placebo versus farletuzumab treatment. Here, we show that farletuzumab has enhanced binding to FCGR3A-158V high-affinity receptor and has an enhanced clinical outcome in patients with low baseline CA125 levels and at least 1 high-affinity allele of FCGR2A or FCGR3A.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Antígeno Ca-125/sangue , Proteínas de Membrana/sangue , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/terapia , Receptores de IgG/genética , Feminino , Genótipo , Humanos , Estimativa de Kaplan-Meier , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/imunologia , Recidiva Local de Neoplasia/terapia , Neoplasias Ovarianas/imunologia , Polimorfismo de Nucleotídeo Único , Resultado do TratamentoRESUMO
The use of microbial transglutaminase (MTG) to produce site-specific antibody-drug conjugates (ADCs) has thus far focused on the transamidation of engineered acyl donor glutamine residues in an antibody based on the hypothesis that the lower specificity of MTG for acyl acceptor lysines may result in the transamidation of multiple native lysine residues, thereby yielding heterogeneous products. We investigated the utilization of native IgG lysines as acyl acceptor sites for glutamine-based acyl donor substrates. Of the approximately 80 lysines in multiple recombinant IgG monoclonal antibodies (mAbs), none were transamidated. Because recombinant mAbs lack the C-terminal Lys447 due to cleavage by carboxypeptidase B in the production cell host, we explored whether blocking the cleavage of Lys447 by the addition of a C-terminal amino acid could result in transamidation of Lys447 by a variety of acyl donor substrates. MTG efficiently transamidated Lys447 in the presence of any nonacidic, nonproline amino acid residue at position 448. Lysine scanning mutagenesis throughout the antibody further revealed several transamidation sites in both the heavy- and light-chain constant regions. Additionally, scanning mutagenesis of the hinge region in a Fab' fragment revealed sites of transamidation that were not reactive in the context of the full-length mAb. Here, we demonstrate the utility of single lysine substitutions and the C-terminal Lys447 for engineering efficient acyl acceptor sites suitable for site-specific conjugation to a range of glutamine-based acyl donor substrates.
Assuntos
Substituição de Aminoácidos , Imunoconjugados/metabolismo , Imunoglobulina G/metabolismo , Lisina/metabolismo , Streptomyces/enzimologia , Transglutaminases/metabolismo , Sequência de Aminoácidos , Células HEK293 , Humanos , Imunoconjugados/química , Imunoconjugados/genética , Imunoglobulina G/química , Imunoglobulina G/genética , Lisina/química , Lisina/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Engenharia de Proteínas , Especificidade por SubstratoRESUMO
Subsets of tumor-produced cell surface and secreted proteins can bind to IgG1 type antibodies and suppress their immune-effector activities. As they affect antibody and complement-mediated immunity, we call these proteins humoral immuno-oncology (HIO) factors. Antibody-drug conjugates (ADCs) use antibody targeting to bind cell surface antigens, internalize into the cell, then kill target cells upon liberation of the cytotoxic payload. Binding of the ADC antibody component by a HIO factor may potentially hamper ADC efficacy due to reduced internalization. To determine the potential effects of HIO factor ADC suppression, we evaluated the efficacy of a HIO-refractory, mesothelin-directed ADC (NAV-001) and a HIO-bound, mesothelin-directed ADC (SS1). The HIO factor MUC16/CA125 binding to SS1 ADC was shown to have a negative effect on internalization and tumor cell killing. The MUC16/CA125 refractory NAV-001 ADC was shown to have robust killing of MUC16/CA125 expressing and non-expressing tumor cells in vitro and in vivo at single, sub-mg/kg dosing. Moreover, NAV-001-PNU, which contains the PNU-159682 topoisomerase II inhibitor, demonstrated good stability in vitro and in vivo as well as robust bystander activity of resident cells while maintaining a tolerable safety profile in vivo. Single-dose NAV-001-PNU demonstrated robust tumor regression of a number of patient-derived xenografts from different tumor types regardless of MUC16/CA125 expression. These findings suggest that identification of HIO-refractory antibodies to be used in ADC format may improve therapeutic efficacy as observed by NAV-001 and warrants NAV-001-PNU's advancement to human clinical trials as a monotherapy to treat mesothelin-positive cancers.
Assuntos
Antineoplásicos , Imunoconjugados , Humanos , Anticorpos , Antineoplásicos/farmacologia , Antígeno Ca-125/metabolismo , Linhagem Celular Tumoral , Imunoconjugados/farmacologia , Proteínas de Membrana/metabolismo , MesotelinaRESUMO
The tumor-shed antigen CA125 has recently been found to bind certain monoclonal antibodies (mAbs) and suppress immune-effector mediated killing through perturbation of the Fc domain with CD16a and CD32a Fc-γ activating receptors on immune-effector cells. Amatuximab is a mAb targeting mesothelin whose mechanism of action utilizes in part antibody-dependent cellular cytotoxicity (ADCC). It is being tested for its therapeutic activity in patients with mesothelioma in combination with first line standard-of-care. To determine if CA125 has immunosuppressive effects on amatuximab ADCC and associated clinical outcomes, post hoc subgroup analysis of patients from a Phase 2 study with primary diagnosed stage III/IV unresectable mesothelioma treated with amatuximab plus cisplatin and pemetrexed were conducted. Analysis found patients with baseline CA125 levels no greater than 57 U/m (â¼3X the upper limit of normal) had a 2 month improvement in progression free survival (HR = 0.43, p = 0.0062) and a 7 month improvement in overall survival (HR = 0.40, p = 0.0022) as compared to those with CA125 above 57 U/mL. In vitro studies found that CA125 was able to bind amatuximab and perturb ADCC activity via decreased Fc-γ-receptor engagement. These data suggest that clinical trial designs of antibody-based drugs in cancers producing CA125, including mesothelioma, should consider stratifying patients on baseline CA125 levels for mAbs that are experimentally determined to be bound by CA125.
Assuntos
Anticorpos Monoclonais/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Antígeno Ca-125/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Proteínas de Membrana/metabolismo , Mesotelioma/tratamento farmacológico , Neoplasias Pleurais/tratamento farmacológico , Idoso , Anticorpos Monoclonais/uso terapêutico , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Antígeno Ca-125/sangue , Antígeno Ca-125/genética , Antígeno Ca-125/imunologia , Linhagem Celular Tumoral , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Ensaios Clínicos Fase II como Assunto , Feminino , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Proteínas de Membrana/sangue , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Mesotelina , Mesotelioma/sangue , Mesotelioma/mortalidade , Mesotelioma/patologia , Mesotelioma Maligno , Pessoa de Meia-Idade , Pemetrexede/farmacologia , Pemetrexede/uso terapêutico , Neoplasias Pleurais/sangue , Neoplasias Pleurais/mortalidade , Neoplasias Pleurais/patologia , Intervalo Livre de Progressão , RNA Interferente Pequeno/metabolismo , Receptores de IgG/imunologia , Receptores de IgG/metabolismoRESUMO
The highly restricted distribution of human folate receptor-alpha (FRalpha) in normal tissues and its high expression in some tumors, along with its putative role in tumor cell transformation, make this antigen a suitable target for antigen-specific, monoclonal antibody-based immunotherapy for oncology indications. We have developed a therapeutic humanized monoclonal antibody with high affinity for FRalpha, named MORAb-003, which was derived from the optimization of the LK26 antibody using a whole cell genetic evolution platform. Here we show that MORAb-003 possesses novel, growth-inhibitory functions on cells overexpressing FRalpha. In addition, MORAb-003 elicited robust antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in vitro, and inhibited growth of human ovarian tumor xenografts in nude mice. Because of its multimodal activity in vitro and its safe toxicology profile in non-human primates, MORAb-003 development has recently been advanced to clinical trials involving ovarian cancer patients.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/imunologia , Imunoterapia/métodos , Neoplasias Ovarianas/tratamento farmacológico , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/imunologia , Animais , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/farmacocinética , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Células CHO , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cricetinae , Cricetulus , Avaliação Pré-Clínica de Medicamentos , Feminino , Receptores de Folato com Âncoras de GPI , Humanos , Cinética , Camundongos , Camundongos Nus , Proteínas de Neoplasias/imunologia , Primatas/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Novel therapeutic agents that are safe and effective are needed for the treatment of pancreatic, ovarian, lung adenocarcinomas and mesotheliomas. Mesothelin is a glycosyl-phosphatidyl inositol (GPI)-linked membrane protein of 40 kDa over-expressed in all pancreatic adenocarcinoma and mesothelioma, in >70% of ovarian adenocarcinoma, and in non-small cell lung and colorectal cancers. The biological functions of mesothelin are not known, although it appears to be involved in cell adhesion via its interaction with MUC16. We have recently developed MORAb-009, a mouse-human chimeric IgG1kappa monoclonal antibody with an affinity of 1.5 nM for human mesothelin. Here we provide evidence that MORAb-009 prevents adhesion of mesothelin-bearing tumor cells to MUC16 positive cells and can elicit cell-mediated cytotoxicity on mesothelin-bearing tumor cells. Treatment that included MORAb-009 in combination with chemotherapy led to a marked reduction in tumor growth of mesothelin-expressing tumors in nude mice compared to chemotherapy or MORAb-009 treatment alone. No adverse effects of MORAb-009 were noted during toxicology studies conducted in non-human primates. The preclinical data obtained from our studies warrants pursuing clinical testing of MORAb-009. We have in fact initiated a Phase I clinical study enrolling patients with mesothelin-positive pancreatic, mesothelioma, non-small cell lung and ovarian cancers.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antineoplásicos/imunologia , Anticorpos Antineoplásicos/uso terapêutico , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/imunologia , Neoplasias/tratamento farmacológico , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/farmacologia , Anticorpos Antineoplásicos/biossíntese , Anticorpos Antineoplásicos/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Antineoplásicos/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Endocitose/efeitos dos fármacos , Proteínas Ligadas por GPI , Humanos , Mesotelina , Camundongos , Camundongos Nus , Neoplasias/imunologiaRESUMO
The prevailing techniques used to generate antibody-drug conjugates (ADCs) involve random conjugation of the linker-drug to multiple lysines or cysteines in the antibody. Engineering natural and non-natural amino acids into an antibody has been demonstrated to be an effective strategy to produce homogeneous ADC products with defined drug-to-antibody ratios. We recently reported an efficient residue-specific conjugation technology (RESPECT) where thiol-reactive payloads can be efficiently conjugated to a native unpaired cysteine in position 80 (C80) of rabbit light chains. Deimmunizing the rabbit variable domains through humanization is necessary to reduce the risk of anti-drug antibody responses in patients. However, we found that first-generation humanized RESPECT ADCs showed high levels of aggregation and low conjugation efficiency. We correlated these negative properties to the phenylalanine at position 83 present in most human variable kappa frameworks. When position 83 was substituted with selected amino acids, conjugation was restored and aggregation was reduced to levels similar to the chimeric ADC. This engineering strategy allows for development of second-generation humanized RESPECT ADCs with desirable biopharmaceutical properties.
RESUMO
The conjugation of toxins, dyes, peptides, or proteins to monoclonal antibodies is often performed via free thiol groups generated by either partial reduction methods or engineering free cysteine residues into the antibody sequence. Antibodies from the rabbit Oryctolagus cuniculus have an additional intrachain disulfide bond, whereby the light chain variable kappa domain is bridged to the constant kappa region between cysteine residues at positions 80 and 171, respectively. Chimerization of rabbit antibodies with human constant domains allows for the generation of a free thiol group at the light chain position 80 (C80) that can be used for site-specific conjugation. An efficient process for the purification and simultaneous removal of cysteinylation at the C80 site was developed. The unpaired C80 was shown to be efficiently conjugated using several different maleimido-based ligands. REsidue SPEcific Conjugation Technology (RESPECT) antibody-drug conjugates prepared using rabbit-human chimeric anti-human mesothelin rabbit antibodies and maleimido-PEG2-auristatin conjugated to C80 were shown to be highly potent and specific in vitro and effective in vivo in reduction of tumor growth in a highly aggressive mesothelin-expressing xenograft tumor model.
Assuntos
Anticorpos Monoclonais/imunologia , Imunoconjugados/imunologia , Neoplasias/tratamento farmacológico , Aminobenzoatos/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/uso terapêutico , Cisteína/química , Cisteína/imunologia , Humanos , Imunoconjugados/uso terapêutico , Mesotelina , Camundongos , Neoplasias/imunologia , Oligopeptídeos/imunologia , Coelhos , Trastuzumab/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Single-cell based amplification of immunoglobulin variable regions is a rapid and powerful technique for cloning antigen-specific monoclonal antibodies (mAbs) for purposes ranging from general laboratory reagents to therapeutic drugs. From the initial screening process involving small quantities of hundreds or thousands of mAbs through in vitro characterization and subsequent in vivo experiments requiring large quantities of only a few, having a robust system for generating mAbs from cloning through stable cell line generation is essential. A protocol was developed to decrease the time, cost, and effort required by traditional cloning and expression methods by eliminating bottlenecks in these processes. Removing the clonal selection steps from the cloning process using a highly efficient ligation-independent protocol and from the stable cell line process by utilizing bicistronic plasmids to generate stable semi-clonal cell pools facilitated an increased throughput of the entire process from plasmid assembly through transient transfections and selection of stable semi-clonal cell pools. Furthermore, the time required by a single individual to clone, express, and select stable cell pools in a high-throughput format was reduced from 4 to 6months to only 4 to 6weeks.
Assuntos
Anticorpos Monoclonais/genética , Clonagem Molecular/métodos , Genes de Cadeia Pesada de Imunoglobulina , Genes de Cadeia Leve de Imunoglobulina , Sequenciamento de Nucleotídeos em Larga Escala , Transfecção/métodos , Antibacterianos/farmacologia , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Citomegalovirus/genética , Vírus da Encefalomiocardite/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos , Células HEK293 , Humanos , Plasmídeos/genética , Poli A/genética , Regiões Promotoras Genéticas , Vírus 40 dos Símios/genética , Fatores de Tempo , Fluxo de TrabalhoRESUMO
Prolactin (PRL)-dependent signaling occurs as the result of ligand-induced homodimerization of the PRL receptor (PRLr). To date, short, intermediate, and long human PRLr isoforms have been characterized. To investigate the expression of other possible human PRLr isoforms, RT-PCR was performed on mRNA isolated from the breast carcinoma cell line T47D. A 1.5-kb PCR fragment was isolated, subcloned, and sequenced. The PCR product exhibited a nucleotide sequence 100% homologous to the human long isoform except bp 71-373 were deleted, which code for the S1 motif of the extracellular domain. Therefore, this isoform was designated the deltaS1 PRLr. Northern analysis revealed variable deltaS1 PRLr mRNA expression in a variety of tissues. Transfection of Chinese hamster ovary cells with deltaS1 cDNA showed the isoform is expressed at the protein level on the cell surface with a molecular mass of approximately 70 kDa. Kinetic studies indicated the deltaS1 isoform bound ligand at a lower affinity than wild-type receptor. The deltaS1 PRLr was also shown to activate the proximal signaling molecule Jak2 upon addition of ligand to transfected cells, and, unlike the long PRLr, high concentrations of ligand did not function as a self-antagonist to signaling during intervals of PRL serum elevation, i.e. stress and pregnancy. Given its apparent widespread expression, this PRLr isoform may contribute to PRL action. Furthermore, the functionality of this receptor raises interesting questions regarding the minimal extracellular domain necessary for ligand-induced receptor signaling.
Assuntos
Proteínas Proto-Oncogênicas , Receptores da Prolactina/genética , Receptores da Prolactina/metabolismo , Animais , Sequência de Bases , Neoplasias da Mama/genética , Células CHO , Clonagem Molecular , Cricetinae , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Hormônios/metabolismo , Humanos , Janus Quinase 2 , Dados de Sequência Molecular , Prolactina/metabolismo , Isoformas de Proteínas , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Valores de Referência , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Because of its high mortality rate, ovarian cancer is a leading cause of death among women and a highly unmet medical need. New therapeutic agents that are effective and well tolerated are needed and cancer antigen-specific monoclonal antibodies that have direct pharmacologic effects or can stimulate immunological responses represent a promising class of agents for the treatment of this disease. The human folate receptor α (FOLR1), which is overexpressed in ovarian cancer but largely absent in normal tissues, appears to play a role in the transformed phenotype in ovarian cancer, cisplatin sensitivity, and growth in depleted folate conditions and therefore has potential as a target for passive immunotherapy. The anti-FOLR1 monoclonal antibody MORAb-003 (farletuzumab) was previously shown to elicit antibody dependent cellular cytotoxicity (ADCC) and inhibit tumor growth of human tumor xenografts in nude mice. Because of its promising preclinical profile, farletuzumab has been evaluated in clinical trials as a potential therapeutic agent for ovarian cancer. In this report, we demonstrated that farletuzumab's antitumor effect against an experimental model of ovarian cancer is mediated by its ADCC activity.