RESUMO
Staphylococcus aureus is considered to be an extracellular pathogen. However, survival of S. aureus within host cells may provide a reservoir relatively protected from antibiotics, thus enabling long-term colonization of the host and explaining clinical failures and relapses after antibiotic therapy. Here we confirm that intracellular reservoirs of S. aureus in mice comprise a virulent subset of bacteria that can establish infection even in the presence of vancomycin, and we introduce a novel therapeutic that effectively kills intracellular S. aureus. This antibody-antibiotic conjugate consists of an anti-S. aureus antibody conjugated to a highly efficacious antibiotic that is activated only after it is released in the proteolytic environment of the phagolysosome. The antibody-antibiotic conjugate is superior to vancomycin for treatment of bacteraemia and provides direct evidence that intracellular S. aureus represents an important component of invasive infections.
Assuntos
Antibacterianos/farmacologia , Bacteriemia , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Espaço Intracelular/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Vancomicina/farmacologia , Animais , Antibacterianos/uso terapêutico , Bacteriemia/tratamento farmacológico , Bacteriemia/microbiologia , Portador Sadio/tratamento farmacológico , Portador Sadio/microbiologia , Desenho de Fármacos , Feminino , Imunoconjugados/química , Espaço Intracelular/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Camundongos , Testes de Sensibilidade Microbiana , Fagossomos/efeitos dos fármacos , Fagossomos/metabolismo , Fagossomos/microbiologia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/patologia , Staphylococcus aureus/patogenicidade , Vancomicina/uso terapêuticoRESUMO
Infection of host tissues by Staphylococcus aureus and S. epidermidis requires an unusual family of staphylococcal adhesive proteins that contain long stretches of serine-aspartate dipeptide-repeats (SDR). The prototype member of this family is clumping factor A (ClfA), a key virulence factor that mediates adhesion to host tissues by binding to extracellular matrix proteins such as fibrinogen. However, the biological siginificance of the SDR-domain and its implication for pathogenesis remain poorly understood. Here, we identified two novel bacterial glycosyltransferases, SdgA and SdgB, which modify all SDR-proteins in these two bacterial species. Genetic and biochemical data demonstrated that these two glycosyltransferases directly bind and covalently link N-acetylglucosamine (GlcNAc) moieties to the SDR-domain in a step-wise manner, with SdgB appending the sugar residues proximal to the target Ser-Asp repeats, followed by additional modification by SdgA. GlcNAc-modification of SDR-proteins by SdgB creates an immunodominant epitope for highly opsonic human antibodies, which represent up to 1% of total human IgG. Deletion of these glycosyltransferases renders SDR-proteins vulnerable to proteolysis by human neutrophil-derived cathepsin G. Thus, SdgA and SdgB glycosylate staphylococcal SDR-proteins, which protects them against host proteolytic activity, and yet generates major eptopes for the human anti-staphylococcal antibody response, which may represent an ongoing competition between host and pathogen.
Assuntos
Proteínas de Bactérias/imunologia , Glicosiltransferases/imunologia , Interações Hospedeiro-Patógeno/imunologia , Staphylococcus aureus Resistente à Meticilina/fisiologia , Infecções Estafilocócicas/imunologia , Staphylococcus epidermidis/fisiologia , Fatores de Virulência/imunologia , Animais , Anticorpos Antibacterianos/genética , Anticorpos Antibacterianos/imunologia , Aderência Bacteriana/genética , Aderência Bacteriana/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Catepsina G/genética , Catepsina G/imunologia , Catepsina G/metabolismo , Linhagem Celular Tumoral , Parede Celular/enzimologia , Parede Celular/genética , Parede Celular/imunologia , Epitopos/genética , Epitopos/imunologia , Epitopos/metabolismo , Feminino , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Interações Hospedeiro-Patógeno/genética , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Masculino , Camundongos , Sequências Repetitivas de Aminoácidos , Infecções Estafilocócicas/enzimologia , Infecções Estafilocócicas/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
The treatment of chronic lymphocytic leukaemia (CLL) has been improved by introduction of monoclonal antibodies (mAbs) that exert their effect through secondary effector mechanisms. CLL cells are characterized by expression of CD5 and CD23 along with CD19 and CD20, hence anti-CD5 Abs that engage secondary effector functions represent an attractive opportunity for CLL treatment. Here, a repertoire of mAbs against human CD5 was generated and tested for ability to induce complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) both as single mAbs and combinations of two mAbs against non-overlapping epitopes on human CD5. The results demonstrated that combinations of two mAbs significantly increased the level of CDC compared to the single mAbs, while no enhancement of ADCC was seen with anti-CD5 mAb combinations. High levels of CDC and ADCC correlated with low levels of Ab-induced CD5 internalization and degradation. Importantly, an anti-CD5 mAb combination enhanced CDC of CLL cells when combined with the anti-CD20 mAbs rituximab and ofatumumab as well as with the anti-CD52 mAb alemtuzumab. These results suggest that an anti-CD5 mAb combination inducing CDC and ADCC may be effective alone, in combination with mAbs against other targets or combined with chemotherapy for CLL and other CD5-expressing haematological or lymphoid malignancies.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos CD5/metabolismo , Proteínas do Sistema Complemento/imunologia , Citotoxicidade Imunológica/efeitos dos fármacos , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Alemtuzumab , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos CD20/imunologia , Antígenos CD20/metabolismo , Antígenos CD5/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Aberrações Cromossômicas , Sinergismo Farmacológico , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Masculino , Pessoa de Meia-IdadeRESUMO
Characterization of conformational and dynamic changes associated with protein interactions can be done by hydrogen/deuterium exchange mass spectrometry (HDX-MS) by comparing the deuterium uptake in the bound and unbound state of the proteins. Investigation of local hydrogen/deuterium exchange in heteromultimeric protein complexes poses a challenge for the method due to the increased complexity of the mixture of peptides originating from all interaction partners in the complex. Previously, interference of peptides from one interaction partner has been removed by immobilizing the intact protein on beads prior to the HDX-MS experiment. However, when studying protein complexes of more than two proteins, immobilization can possibly introduce steric limitations to the interactions. Here, we present a method based on the high affinity biotin-streptavidin interaction that allows selective capture of biotinylated proteins even under the extreme conditions for hydrogen/deuterium exchange quenching i.e. pH 2.5 and 0 °C. This biotin-streptavidin capture strategy allows hydrogen/deuterium exchange to occur in proteins in solution and enables characterization of specific proteins in heteromultimeric protein complexes without interference of peptides originating from other interaction partners in the complex. The biotin-streptavidin strategy has been successfully implemented in a model system with two recombinant monoclonal antibodies that target nonoverlapping epitopes on the human epidermal growth factor receptor (EGFR). We present a workflow for biotinylation and characterization of recombinant antibodies and demonstrate affinity capture of biotinylated antibodies under hydrogen/deuterium exchange quench conditions by the biotin-streptavidin strategy.
Assuntos
Biotinilação , Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , Multimerização Proteica , Proteínas/metabolismo , Animais , Biotina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Estrutura Quaternária de Proteína , Proteínas/química , Estreptavidina/metabolismo , TemperaturaRESUMO
Despite development of effective SARS-CoV-2 vaccines, a sub-group of vaccine non-responders depends on therapeutic antibodies or small-molecule drugs in cases of severe disease. However, perpetual viral evolution has required continuous efficacy monitoring as well as exploration of new therapeutic antibodies, to circumvent resistance mutations arising in the viral population. We performed SARS-CoV-2-specific B cell sorting and subsequent single-cell sequencing on material from 15 SARS-CoV-2 convalescent participants. Through screening of 455 monoclonal antibodies for SARS-CoV-2 variant binding and virus neutralization, we identified a cluster of activated B cells highly enriched for SARS-CoV-2 neutralizing antibodies. Epitope binning and Cryo-EM structure analysis identified the majority of neutralizing antibodies having epitopes overlapping with the ACE2 receptor binding motif (class 1 binders). Extensive functional antibody characterization identified two potent neutralizing antibodies, one retaining SARS-CoV-1 neutralizing capability, while both bind major common variants of concern and display prophylactic efficacy in vivo. The transcriptomic signature of activated B cells harboring broadly binding neutralizing antibodies with therapeutic potential identified here, may be a guide in future efforts of rapid therapeutic antibody discovery.
Assuntos
Antígenos de Grupos Sanguíneos , COVID-19 , Humanos , SARS-CoV-2 , Vacinas contra COVID-19 , Anticorpos Antivirais , Anticorpos Neutralizantes , EpitoposRESUMO
Discovery of therapeutic antibodies is a field of intense development, where immunization of rodents remains a major source of antibody candidates. However, high orthologue protein sequence homology between human and rodent species disfavors generation of antibodies against functionally conserved binding epitopes. Chickens are phylogenetically distant from mammals. Since chickens generate antibodies from a restricted set of germline genes, the possibility of adapting the Symplex antibody discovery platform to chicken immunoglobulin genes and combining it with high-throughput humanization of antibody frameworks by "mass complementarity-determining region grafting" was explored. Hence, wild type chickens were immunized with an immune checkpoint inhibitor programmed cell death 1 (PD1) antigen, and a repertoire of 144 antibodies was generated. The PD1 antibody repertoire was successfully humanized, and we found that most humanized antibodies retained affinity largely similar to that of the parental chicken antibodies. The lead antibody Sym021 blocked PD-L1 and PD-L2 ligand binding, resulting in elevated T-cell cytokine production in vitro. Detailed epitope mapping showed that the epitope recognized by Sym021 was unique compared to the clinically approved PD1 antibodies pembrolizumab and nivolumab. Moreover, Sym021 bound human PD1 with a stronger affinity (30 pM) compared to nivolumab and pembrolizumab, while also cross-reacting with cynomolgus and mouse PD1. This enabled direct testing of Sym021 in the syngeneic mouse in vivo cancer models and evaluation of preclinical toxicology in cynomolgus monkeys. Preclinical in vivo evaluation in various murine and human tumor models demonstrated a pronounced anti-tumor effect of Sym021, supporting its current evaluation in a Phase 1 clinical trial. Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; CD, cluster of differentiation; CDC, complement-dependent cytotoxicity; CDR, complementarity determining region; DC, dendritic cell; ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence activated cell sorting; FR, framework region; GM-CSF, granulocyte-macrophage colony-stimulating factor; HRP, horseradish peroxidase; IgG, immunoglobulin G; IL, interleukin; IFN, interferon; mAb, monoclonal antibody; MLR, mixed lymphocyte reaction; NK, natural killer; PBMC, peripheral blood mono-nuclear cell; PD1, programmed cell death 1; PDL1, programmed cell death ligand 1; RT-PCR, reverse transcription polymerase chain reaction; SEB, Staphylococcus Enterotoxin B; SPR, surface Plasmon Resonance; VL, variable part of light chain; VH, variable part of heavy chain.
Assuntos
Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais/genética , Proteínas Aviárias/genética , Galinhas/fisiologia , Engenharia de Proteínas/métodos , Linfócitos T/imunologia , Animais , Antígeno B7-H1/metabolismo , Células Cultivadas , Citocinas/metabolismo , Mapeamento de Epitopos , Humanos , Epitopos Imunodominantes/genética , Ativação Linfocitária , Macaca fascicularis , Camundongos , Camundongos Endogâmicos C57BL , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/imunologia , Ligação ProteicaRESUMO
IMPORTANCE: Acquired resistance to anti-EGFR therapy (epidermal growth factor receptor) is frequently due to RAS and EGFR extracellular domain (ECD) mutations in metastatic colorectal cancer (mCRC). Some anti-EGFR-refractory patients retain tumor EGFR dependency potentially targetable by agents such as Sym004, which is a mixture of 2 nonoverlapping monoclonal antibodies targeting EGFR. OBJECTIVE: To determine if continuous blockade of EGFR by Sym004 has survival benefit. DESIGN, SETTING, AND PARTICIPANTS: Multicenter, phase 2, randomized, clinical trial comparing 2 regimens of Sym004 with investigator's choice from March 6, 2014, through October 15, 2015. Circulating tumor DNA (ctDNA) was analyzed for biomarker and tracking clonal dynamics during treatment. Participants had wild-type KRAS exon 2 mCRC refractory to standard chemotherapy and acquired resistance to anti-EGFR monoclonal antibodies. INTERVENTIONS: Participants were randomly assigned in a 1:1:1 ratio to Sym004, 12 mg/kg/wk (arm A), Sym004, 9 mg/kg loading dose followed by 6 mg/kg/wk (arm B), or investigator's choice of treatment (arm C). MAIN OUTCOMES AND MEASURES: Overall survival (OS). Secondary end points included preplanned exploratory biomarker analysis in ctDNA. RESULTS: A total of 254 patients were randomized (intent-to-treat [ITT] population) (median age, 63 [range, 34-91] years; 63% male; n = 160). Median OS in the ITT population was 7.9 months (95% CI, 6.5-9.9 months), 10.3 months (95% CI, 9.0-12.9 months), and 9.6 months (95% CI, 8.3-12.2 months) for arms A, B, and C, respectively (hazard ratio [HR], 1.31; 95% CI, 0.92-1.87 for A vs C; and HR, 0.97; 95% CI, 0.68-1.40 for B vs C). The ctDNA revealed high intrapatient genomic heterogeneity following anti-EGFR therapy. Sym004 effectively targeted EGFR ECD-mutated cancer cells, and a decrease in EGFR ECD ctDNA occurred in Sym004-treated patients. However, this did not translate into clinical benefit in patients with EGFR ECD mutations, likely owing to co-occurring resistance mechanisms. A subgroup of patients was defined by ctDNA (RAS/BRAF/EGFR ECD-mutation negative) associated with improved OS in Sym004-treated patients in arm B compared with arm C (median OS, 12.8 and 7.3 months, respectively). CONCLUSIONS AND RELEVANCE: Sym004 did not improve OS in an unselected population of patients with mCRC and acquired anti-EGFR resistance. A prospective clinical validation of Sym004 efficacy in a ctDNA molecularly defined subgroup of patients with refractory mCRC is warranted. TRIAL REGISTRATION: clinicaltrialsregister.eu Identifier: 2013-003829-29.
Assuntos
Anticorpos Monoclonais/uso terapêutico , DNA Tumoral Circulante/sangue , Neoplasias Colorretais/sangue , Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Seleção de Pacientes , Inibidores de Proteínas Quinases/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , DNA Tumoral Circulante/análise , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Análise de Sobrevida , Resultado do TratamentoRESUMO
Purpose: Activation of the receptor tyrosine kinase MET is associated with poor clinical outcome in certain cancers. To target MET more effectively, we developed an antagonistic antibody mixture, Sym015, consisting of two humanized mAbs directed against nonoverlapping epitopes of MET.Experimental Design/Results: We screened a large panel of well-annotated human cancer cell lines and identified a subset with highly elevated MET expression. In particular, cell lines of lung cancer and gastric cancer origin demonstrated high MET expression and activation, and Sym015 triggered degradation of MET and significantly inhibited growth of these cell lines. Next, we tested Sym015 in patient- and cell line-derived xenograft models with high MET expression and/or MET exon 14 skipping alterations, and in models harboring MET amplification as a mechanism of resistance to EGFR-targeting agents. Sym015 effectively inhibited tumor growth in all these models and was superior to an analogue of emibetuzumab, a monoclonal IgG4 antibody against MET currently in clinical development. Sym015 also induced antibody-dependent cellular cytotoxicity (ADCC) in vitro, suggesting that secondary effector functions contribute to the efficacy of Sym015.Retrospectively, all responsive, high MET-expressing models were scored as highly MET-amplified by in situ hybridization, pointing to MET amplification as a predictive biomarker for efficacy. Preclinical toxicology studies in monkeys showed that Sym015 was well tolerated, with a pharmacokinetic profile supporting administration of Sym015 every second or third week in humans.Conclusions: The preclinical efficacy and safety data provide a clear rationale for the ongoing clinical studies of Sym015 in patients with MET-amplified tumors. Clin Cancer Res; 23(19); 5923-35. ©2017 AACR.
Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas c-met/genética , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados/imunologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Linhagem Celular Tumoral , Epitopos/imunologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Amplificação de Genes/genética , Humanos , Camundongos , Mutação , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Increased MET activity is linked with poor prognosis and outcome in several human cancers currently lacking targeted therapies. Here, we report on the characterization of Sym015, an antibody mixture composed of two humanized IgG1 antibodies against nonoverlapping epitopes of MET. Sym015 was selected by high-throughput screening searching for antibody mixtures with superior growth-inhibitory activity against MET-dependent cell lines. Synergistic inhibitory activity of the antibodies comprising Sym015 was observed in several cancer cell lines harboring amplified MET locus and was confirmed in vivo Sym015 was found to exert its activity via multiple mechanisms. It disrupted interaction of MET with the HGF ligand and prompted activity-independent internalization and degradation of the receptor. In addition, Sym015 induced high levels of CDC and ADCC in vitro The importance of these effector functions was confirmed in vivo using an Fc-effector function-attenuated version of Sym015. The enhanced effect of the two antibodies in Sym015 on both MET degradation and CDC and ADCC is predicted to render Sym015 superior to single antibodies targeting MET. Our results demonstrate strong potential for use of Sym015 as a therapeutic antibody mixture for treatment of MET-driven tumors. Sym015 is currently being tested in a phase I dose escalation clinical trial (NCT02648724). Mol Cancer Ther; 16(12); 2780-91. ©2017 AACR.
Assuntos
Epitopos/genética , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Nus , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Approved anti-EGFR antibodies cetuximab and panitumumab provide significant clinical benefit in patients with metastatic colorectal cancer (MCRC). However, patients ultimately develop disease progression, often driven by acquisition of mutations in the extracellular domain (ECD) of EGFR. Sym004 is a novel 1:1 mixture of two nonoverlapping anti-EGFR mAbs that recently showed promising clinical activity in a phase I trial in MCRC. Our aim was to determine the efficacy of Sym004 to circumvent cetuximab resistance driven by EGFR ECD mutations. EXPERIMENTAL DESIGN: Functional studies were performed to assess drug-receptor binding as well as ligand-dependent activation of individual EGFR mutants in the presence of cetuximab, panitumumab, and Sym004. Cell viability and molecular effects of the drugs were assayed in cetuximab-resistant cell lines and in tumor xenograft models. Efficacy of Sym004 was evaluated in patients progressing to cetuximab that harbored EGFR mutation in the post-cetuximab tumor sample. RESULTS: Contrary to cetuximab and panitumumab, Sym004 effectively bound and abrogated ligand-induced phosphorylation of all individual EGFR mutants. Cells resistant to cetuximab harboring mutations in EGFR maintained sensitivity to Sym004, which was consistent with an effective suppression of EGFR downstream signaling, translating into profound and sustained tumor regression in the xenograft model. As proof-of-principle, a patient with a tumor harboring an EGFR mutation (G465R) following cetuximab therapy benefited from Sym004 therapy. CONCLUSIONS: Sym004 is an active drug in MCRC resistant to cetuximab/panitumumab mediated by EGFR mutations. EGFR mutations are potential biomarkers of response to Sym004 to be evaluated in ongoing large clinical trials. Clin Cancer Res; 22(13); 3260-7. ©2016 AACR.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Cetuximab/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Células 3T3 , Animais , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/genética , Receptores ErbB/imunologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Panitumumabe , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Human bone marrow is a major repository for maturated antibody-secreting plasma cells, which produce the majority of the antibodies found in serum, making it an attractive source for generating human immune antibody libraries. Unfortunately, bone marrow is not always readily available and, although human immune libraries can be generated from circulating B cells, the low frequency of antigen-specific B cells in the circulation yield few monoclonal antibodies of interest. We used a pre-selection strategy to enrich for antigen-specific B cells prior to library generation, and applied this approach to evaluate, at a molecular level, the nature of the human anti-HIV-1 gp120 repertoire encoded by circulating B cells. IgG antibody phage display libraries were generated from HIV-1 seropositive individuals using either affinity-selected anti-gp120 IgG-bearing circulating B cells, predominantly exhibiting memory/activated B cell phenotype, or unselected PBMCs. These libraries were selected against HIV-1 gp120, resulting in isolation of a panel of gp120-specific antibodies. Whereas only 2 gp120-specific antibodies were retrieved from the non-pre-selected HIV-1 library, 9 gp120-specific antibodies were retrieved from the 10-fold smaller library generated from the pre-selected B cells, demonstrating the feasibility of the approach. The anti-gp120 antibodies derived from the circulating B cells of HIV-1 donors generally resembles those from bone marrow plasma cells with respect to epitope specificity, affinity and neutralization ability. They exhibit high affinity for gp120, are directed against a variety of epitopes, but rarely exhibit the ability to neutralize HIV-1.
Assuntos
Linfócitos B/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Imunoglobulina G/imunologia , Biblioteca de Peptídeos , Sequência de Aminoácidos , Afinidade de Anticorpos , Mapeamento de Epitopos , Anticorpos Anti-HIV/genética , Humanos , Epitopos Imunodominantes , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/genética , Dados de Sequência Molecular , Testes de NeutralizaçãoRESUMO
HER2 plays an important role in the development and maintenance of the malignant phenotype of several human cancers. As such, it is a frequently pursued therapeutic target and two antibodies targeting HER2 have been clinically approved, trastuzumab and pertuzumab. It has been suggested that optimal inhibition of HER2 is achieved when utilizing two or more antibodies targeting nonoverlapping epitopes. Superior clinical activity of the trastuzumab plus pertuzumab combination in metastatic breast cancer supports this hypothesis. Because trastuzumab and pertuzumab were not codeveloped, there may be potential for further optimizing HER2 targeting. The study herein evaluated functional activity of anti-HER2 antibody combinations identifying optimal epitope combinations that provide efficacious HER2 inhibition. High-affinity antibodies to all four extracellular domains on HER2 were identified and tested for ability to inhibit growth of different HER2-dependent tumor cell lines. An antibody mixture targeting three HER2 subdomains proved to be superior to trastuzumab, pertuzumab, or a combination in vitro and to trastuzumab in two in vivo models. Specifically, the tripartite antibody mixture induced efficient HER2 internalization and degradation demonstrating increased sensitivity in cell lines with HER2 amplification and high EGFR levels. When compared with individual and clinically approved mAbs, the synergistic tripartite antibody targeting HER2 subdomains I, II, and IV demonstrates superior anticancer activity.
Assuntos
Anticorpos Monoclonais/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptor ErbB-2/metabolismo , Trastuzumab/farmacologia , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Receptores ErbB/metabolismo , Feminino , Humanos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
PURPOSE: Accumulating evidence indicates a high degree of plasticity and compensatory signaling within the human epidermal growth factor receptor (HER) family, leading to resistance upon therapeutic intervention with HER family members. EXPERIMENTAL DESIGN/RESULTS: We have generated Pan-HER, a mixture of six antibodies targeting each of the HER family members EGFR, HER2, and HER3 with synergistic pairs of antibodies, which simultaneously remove all three targets, thereby preventing compensatory tumor promoting mechanisms within the HER family. Pan-HER induces potent growth inhibition in a range of cancer cell lines and xenograft models, including cell lines with acquired resistance to therapeutic antibodies. Pan-HER is also highly efficacious in the presence of HER family ligands, indicating that it is capable of overcoming acquired resistance due to increased ligand production. All three target specificities contribute to the enhanced efficacy, demonstrating a distinct benefit of combined HER family targeting when compared with single-receptor targeting. CONCLUSIONS: Our data show that simultaneous targeting of three receptors provides broader efficacy than targeting a single receptor or any combination of two receptors in the HER family, especially in the presence of HER family ligands. Pan-HER represents a novel strategy to deal with primary and acquired resistance due to tumor heterogeneity and plasticity in terms of HER family dependency and as such may be a viable alternative in the clinic.
Assuntos
Anticorpos Monoclonais Humanizados/química , Receptores ErbB/química , Receptor ErbB-2/química , Receptor ErbB-3/química , Animais , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Resistencia a Medicamentos Antineoplásicos/imunologia , Receptores ErbB/imunologia , Regulação Neoplásica da Expressão Gênica , Humanos , Ligantes , Camundongos , Camundongos Nus , Neoplasias Experimentais/tratamento farmacológico , Receptor ErbB-2/imunologia , Receptor ErbB-3/imunologia , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The epidermal growth factor receptor (EGFR) is frequently dysregulated in human malignancies and a validated target for cancer therapy. Two monoclonal anti-EGFR antibodies (cetuximab and panitumumab) are approved for clinical use. However, the percentage of patients responding to treatment is low and many patients experiencing an initial response eventually relapse. Thus, the need for more efficacious treatments remains. Previous studies have reported that mixtures of antibodies targeting multiple distinct epitopes are more effective than single mAbs at inhibiting growth of human cancer cells in vitro and in vivo. The current work describes the rational approach that led to discovery and selection of a novel anti-EGFR antibody mixture Sym004, which is currently in Phase 2 clinical testing. Twenty-four selected anti-EGFR antibodies were systematically tested in dual and triple mixtures for their ability to inhibit cancer cells in vitro and tumor growth in vivo. The results show that targeting EGFR dependent cancer cells with mixtures of antibodies is superior at inhibiting their growth both in vitro and in vivo. In particular, antibody mixtures targeting non-overlapping epitopes on domain III are efficient and indeed Sym004 is composed of two monoclonal antibodies targeting this domain. The superior growth inhibitory activity of mixtures correlated with their ability to induce efficient EGFR degradation.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/uso terapêutico , Receptores ErbB/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Antineoplásicos/química , Antineoplásicos/imunologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Mapeamento de Epitopos , Epitopos/química , Epitopos/imunologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Ressonância de Plasmônio de Superfície , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Epidermal growth factor receptor (EGFR) is a validated therapeutic target in cancer and EGFR antagonists with greater effectiveness than existing clinical agents remain of interest. Here, we report a novel approach based on Sym004, a mixture of two anti-EGFR monoclonal antibodies directed against distinct nonoverlapping epitopes in EGFR extracellular domain III. Like anti-EGFR monoclonal antibodies in current clinical use, Sym004 inhibits cancer cell growth and survival by blocking ligand-binding receptor activation and phosphorylation and downstream receptor signaling. However, unlike the other antibodies, Sym004 induces rapid and efficient removal of the receptor from the cancer cell surface by triggering EGFR internalization and degradation. Compared with reference anti-EGFR monoclonal antibodies, Sym004 exhibited more pronounced growth inhibition in vitro and superior efficacy in vivo. Together, these findings illustrate a strategy to target EGFR more effectively than existing clinical antibodies.
Assuntos
Anticorpos Monoclonais/farmacologia , Receptores ErbB/antagonistas & inibidores , Neoplasias/terapia , Animais , Anticorpos Monoclonais/imunologia , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação para Baixo , Sinergismo Farmacológico , Epitopos/imunologia , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/enzimologia , Fosforilação , Estrutura Terciária de Proteína , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
HIV-1-associated thrombocytopenia (HIV-1-ITP) is a common complication of HIV-1 infection, frequently caused by increased peripheral platelet destruction mediated by antiplatelet antibodies (Abs) and/or platelet-bound immune complexes. Little is known about the specificity of the antiplatelet Abs at a molecular level. Here, we used immunoglobulin G (IgG) phage-display libraries generated from 3 HIV-1-ITP patients to isolate a large panel of human monoclonal antiplatelet Abs by selection on unfixed platelets. The platelet antigen recognized by all the cloned Abs was identified to be the talin head domain (talin-H), a cleavage product of talin that can be generated by platelet activation or HIV-1 protease. Talin-H was found in HIV-1-ITP-circulating immune complexes, and antitalin Abs were detected in HIV-1-ITP sera but not in controls. The cloned anti-talin-H IgGs were highly somatically mutated, indicative of an antigen-driven, affinity-matured response. These findings suggest that talin-H Ab may be a marker of HIV-1-ITP elicited due to exposure of immunodominant epitopes on talin-H as a result of a disease-related process. Abs to talin-H and related immune complexes (ICs) may contribute to HIV-1-ITP.