RESUMO
c-Met is a prototypic member of a sub-family of RTKs. Inappropriate c-Met activation plays a crucial role in tumor formation, proliferation and metastasis. Using a key c-Met dimerization assay, a set of 12 murine whole IgG1 monoclonal antibodies was selected and a lead candidate, m224G11, was humanized by CDR-grafting and engineered to generate a divalent full antagonist humanized IgG1 antibody, hz224G11. Neither m224G11 nor hz224G11 bind to the murine c-Met receptor. Their antitumor activity was investigated in vitro in a set of experiments consistent with the reported pleiotropic effects mediated by c-Met and, in vivo, using several human tumor xenograft models. Both m224G11 and hz224G11 exhibited nanomolar affinities for the receptor and inhibited HGF binding, c-Met phosphorylation, and receptor dimerization in a similar fashion, resulting in a profound inhibition of all c-Met functions in vitro. These effects were presumably responsible for the inhibition of c-Met's major functions including cell proliferation, migration, invasion scattering, morphogenesis and angiogenesis. In addition to these in vitro properties, hz224G11 dramatically inhibits the growth of autocrine, partially autophosphorylated and c-Met amplified cell lines in vivo. Pharmacological studies performed on Hs746T gastric cancer xenografts demonstrate that hz224G11 strongly downregulates c-Met expression and phosphorylation. It also decreases the tumor mitotic index (Ki67) and induces apoptosis. Taken together, the in vitro and in vivo data suggest that hz224G11 is a promising candidate for the treatment of tumors. This antibody, now known as ABT-700 and currently in Phase I clinical trials, may provide a novel therapeutic approach to c-Met-expressing cancers.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Murinos/farmacologia , Neoplasias/terapia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/imunologia , Células A549 , Animais , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Murinos/imunologia , Células CHO , Linhagem Celular Tumoral , Cricetulus , Feminino , Fator de Crescimento de Hepatócito/imunologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Imunoglobulina G/imunologia , Ligantes , Células MCF-7 , Masculino , Camundongos , Camundongos Nus , Camundongos SCID , Neoplasias/imunologia , Proteínas Proto-Oncogênicas c-met/biossíntese , Distribuição Aleatória , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The insulin-like growth factor type 1 receptor (IGF-1R) is important in tumorigenesis, and its overexpression occurs in numerous tumor tissues. To date, therapeutic approaches based on mAbs and tyrosine kinase inhibitors targeting IGF-1R have only shown clinical benefit in specific patient populations. We report a unique IGF-1R-targeted antibody-drug conjugate (ADC), W0101, designed to deliver a highly potent cytotoxic auristatin derivative selectively to IGF-1R overexpressing tumor cells. The mAb (hz208F2-4) used to prepare the ADC was selected for its specific binding properties to IGF-1R compared with the insulin receptor, and for its internalization properties. Conjugation of a novel auristatin derivative drug linker to hz208F2-4 did not alter its binding and internalization properties. W0101 induced receptor-dependent cell cytotoxicity in vitro when applied to various cell lines overexpressing IGF-1R, but it did not affect normal cells. Efficacy studies were conducted in several mouse models expressing different levels of IGF-1R to determine the sensitivity of the tumors to W0101. W0101 induced potent tumor regression in certain mouse models. Interestingly, the potency of W0101 correlated with the expression level of IGF-1R evaluated by IHC. In an MCF-7 breast cancer model with high-level IGF-1R expression, a single injection of W0101 3 mg/kg led to strong inhibition of tumor growth. W0101 provides a potential new therapeutic option for patients overexpressing IGF-1R. A first-in-human trial of W0101 is currently ongoing to address clinical safety.
Assuntos
Imunoconjugados/uso terapêutico , Neoplasias/tratamento farmacológico , Receptor IGF Tipo 1/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Imunoconjugados/farmacologia , Camundongos , Camundongos Nus , Neoplasias/patologiaRESUMO
Junctional adhesion molecule-A (JAM-A) is an adherens and tight junction protein expressed by endothelial and epithelial cells and associated with cancer progression. We present here the extensive characterization of immune complexes involving JAM-A antigen and three monoclonal antibodies (mAbs), including hz6F4-2, a humanized version of anti-tumoral 6F4 mAb identified by a functional and proteomic approach in our laboratory. A specific workflow that combines orthogonal approaches has been designed to determine binding stoichiometries along with JAM-A epitope mapping determination at high resolution for these three mAbs. Native mass spectrometry experiments revealed different binding stoichiometries and affinities, with two molecules of JAM-A being able to bind to hz6F4-2 and F11 Fab, while only one JAM-A was bound to J10.4. Surface plasmon resonance indirect competitive binding assays suggested epitopes located in close proximity for hz6F4-2 and F11. Finally, hydrogen-deuterium exchange mass spectrometry was used to precisely identify epitopes for all mAbs. The results obtained by orthogonal biophysical approaches showed a clear correlation between the determined epitopes and JAM-A binding characteristics, allowing the basis for molecular recognition of JAM-A by hz6F4-2 to be definitively established for the first time. Taken together, our results highlight the power of MS-based structural approaches for epitope mapping and mAb conformational characterization.
Assuntos
Anticorpos Monoclonais/imunologia , Moléculas de Adesão Celular/imunologia , Epitopos/imunologia , Espectrometria de Massas/métodos , Receptores de Superfície Celular/imunologia , Ressonância de Plasmônio de Superfície/métodos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Ligação Competitiva/imunologia , Medição da Troca de Deutério , Mapeamento de Epitopos , Epitopos/química , Epitopos/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica/imunologia , Conformação ProteicaRESUMO
The type IV C-X-C-motif chemokine receptor (CXCR4) is expressed in a large variety of human cancers, including hematologic malignancies, and this receptor and its ligand, stromal cell-derived factor-1 (SDF-1), play a crucial role in cancer progression. We generated a humanized immunoglobulin G1 mAb, hz515H7, which binds human CXCR4, efficiently competes for SDF-1 binding, and induces a conformational change in CXCR4 homodimers. Furthermore, it inhibits both CXCR4 receptor-mediated G-protein activation and ß-arrestin-2 recruitment following CXCR4 activation. The binding of the hz515H7 antibody to CXCR4 inhibits the SDF-1-induced signaling pathway, resulting in reduced phosphorylation of downstream effectors, such as Akt, Erk1/2, p38, and GSK3ß. Hz515H7 also strongly inhibits cell migration and proliferation and, while preserving normal blood cells, induces both antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against neoplastic cells. In mouse xenograft models, hz515H7 displays antitumor activities with multiple hematologic tumor cell lines, with its Fc-mediated effector functions proving essential in this context. Furthermore, hz515H7 binds to primary tumor cells from acute myeloid leukemia and multiple myeloma patients. Collectively, our results demonstrate two major mechanisms of action, making hz515H7 unique in this regard. Its potential as a best-in-class molecule is currently under investigation in a phase I clinical trial. Mol Cancer Ther; 15(8); 1890-9. ©2016 AACR.
Assuntos
Anticorpos Monoclonais/farmacologia , Movimento Celular/efeitos dos fármacos , Quimiocina CXCL12/metabolismo , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Ligação Competitiva , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Proteínas do Sistema Complemento/imunologia , Modelos Animais de Doenças , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Ligação Proteica , Multimerização Proteica , Receptores CXCR4/química , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , beta-Arrestina 2/metabolismoRESUMO
Antibody-drug conjugates (ADCs) are biochemotherapeutics consisting of a cytotoxic chemical drug linked covalently to a monoclonal antibody. Two main classes of ADCs, namely cysteine and lysine conjugates, are currently available on the market or involved in clinical trials. The complex structure and heterogeneity of ADCs makes their biophysical characterization challenging. For cysteine conjugates, hydrophobic interaction chromatography is the gold standard technique for studying drug distribution, the naked antibody content, and the average drug to antibody ratio (DAR). For lysine ADC conjugates on the other hand, which are not amenable to hydrophobic interaction chromatography because of their higher heterogeneity, denaturing mass spectrometry (MS) and UV/Vis spectroscopy are the most powerful approaches. We report here the use of native MS and ion mobility (IM-MS) for the characterization of trastuzumab emtansine (T-DM1, Kadcyla(®)). This lysine conjugate is currently being considered for the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer, and combines the anti-HER2 antibody trastuzumab (Herceptin(®)), with the cytotoxic microtubule-inhibiting maytansine derivative, DM1. We show that native MS combined with high-resolution measurements and/or charge reduction is beneficial in terms of the accurate values it provides of the average DAR and the drug load profiles. The use of spectral deconvolution is discussed in detail. We report furthermore the use of native IM-MS to directly determine DAR distribution profiles and average DAR values, as well as a molecular modeling investigation of positional isomers in T-DM1.
Assuntos
Anticorpos Monoclonais Humanizados/química , Antineoplásicos/química , Imunoconjugados/química , Lisina/química , Maitansina/análogos & derivados , Ado-Trastuzumab Emtansina , Humanos , Isomerismo , Espectrometria de Massas , Maitansina/química , Modelos Moleculares , TrastuzumabRESUMO
Surface plasmon resonance (SPR) is a key technology to evaluate IgG effector functions in vitro involving binding to Fcgamma receptors (FcγR). We describe here the chemical coupling of protein A to a sensor chip. IgGs prepared either from purified antibodies or from crude cell media supernatants are captured by the protein A and are used as the ligand. Soluble forms of FcγRs are injected at different concentrations and are used as the analyte. This setup allows the definition of the kinetic rates of binding of the FcγRI (high affinity) or the FcγRIIIa (low affinity) on the Fc domain of IgGs.
Assuntos
Proteínas Imobilizadas/química , Imunoglobulina G/química , Proteína Estafilocócica A/química , Ressonância de Plasmônio de Superfície/métodos , Anticorpos Monoclonais Humanizados/química , Humanos , Cinética , Análise Serial de Proteínas/métodos , Ligação Proteica , Receptores de IgG/química , TrastuzumabRESUMO
Peptides are essential tools for discovery and pre-clinical and pharmaceutical development of viral and cancer vaccines ('active immunotherapies') as well as for therapeutic antibodies ('passive immunotherapies'). They help to trigger and analyze immune responses at a molecular level (B-cell, T-helper and CTL epitopes). They contribute largely to the design of new vaccine candidates and to the generation of monoclonal antibodies. They are also valuable analytical reference compounds for the structural characterisation by liquid chromatography and mass spectrometry of recombinant proteins used as biopharmaceuticals. As for other therapeutic applications, formulation, solubilisation, batch consistency and stability, issues have to be addressed to allow the pre-clinical and clinical development of this class of compounds as immunotherapeutic drugs. In the present review, three case studies dealing with (i) the design and the characterisation of Respiratory Syntycial Virus subunit vaccines, (ii) peptide-based melanoma vaccines, and (iii) therapeutic monoclonal antibodies, all investigated in clinical trials, are reported and discussed.
Assuntos
Imunoterapia/métodos , Peptídeos/imunologia , Peptídeos/uso terapêutico , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Antígenos Virais/química , Antígenos Virais/genética , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Epitopos de Linfócito B/genética , Epitopos de Linfócito T/genética , Humanos , Camundongos , Modelos Imunológicos , Modelos Moleculares , Dados de Sequência Molecular , Mapeamento de Peptídeos , Vacinas contra Vírus Sincicial Respiratório/genética , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/imunologiaRESUMO
Most conventional vaccines consist of killed organisms or purified antigenic proteins. Such molecules are generally poorly immunogenic and need to be coupled to carrier proteins. We have identified a new carrier molecule, BB, derived from the G protein of Streptococcus strain G148. We show that BB is able to induce strong antibody responses when conjugated to peptides or polysaccharides. In order to localize T and B cell epitopes in BB and match them with the albumin-binding region of the molecule, we immunized mice with BB, performed B and T pepscan analyses, and compared the results with pepscan done with sera and cells from humans. Our results indicate that BB has two distinct T helper epitopes, seven linear B-cell epitopes, and one conformational B-cell epitope in BALB/c mice. Four linear B-cell epitopes were identified from human sera, three of which overlapped mouse B-cell epitopes. Finally, three human T-cell epitopes were detected on the BB protein. One of these T-cell epitopes is common to BALB/c mice and humans and was localized in the region that contains the albumin-binding site. These data are of interest for the optimization of new carrier molecules derived from BB.
Assuntos
Proteínas de Bactérias/imunologia , Epitopos de Linfócito B/química , Epitopos de Linfócito T/química , Sequência de Aminoácidos , Animais , Proteínas de Transporte/administração & dosagem , Proteínas de Transporte/química , Proteínas de Transporte/imunologia , Mapeamento de Epitopos , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Humanos , Imunização , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/imunologia , Polissacarídeos/química , Polissacarídeos/imunologia , Proteínas Virais/administração & dosagem , Proteínas Virais/química , Proteínas Virais/imunologiaRESUMO
The protective mechanisms induced in the mouse upper respiratory tract (URT) after intraperitoneal immunization with G2Na, a recombinant respiratory syncytial virus (RSV) G protein fragment (amino acid residues 130 to 230), were investigated. This protection was recently shown to be mediated by CD4(+) T cells and to be critically dependent on the cysteines and amino acids 193 and 194 (H. Plotnicky-Gilquin, A. Robert, L. Chevalet, J.-F. Haeuw, A. Beck, J.-Y. Bonnefoy, C. Brandt, C.-A. Siegrist, T. N. Nguyen, and U. F. Power, J. Virol. 74:3455-3463, 2000). On G2Na, we identified a domain (amino acid residues 182 to 198) responsible for the T-helper-cell activity. This region coincided with a peptide designed AICK (residues 184 to 198) which includes the previously identified murine and human T-helper-cell epitope on the native G protein (P. W. Tebbey, M. Hagen, and G. E. Hancock, J. Exp. Med. 188:1967-1972, 1998). Immunization with AICK, in alum or complete Freund's adjuvant, significantly reduced nasal RSV titers in normal BALB/c mice. However, although lung protection was induced, in contrast to the case with live RSV, neither AICK nor G2Na was able to prevent nasal infection in gamma interferon (IFN-gamma)-knockout mice. Anti-IFN-gamma neutralizing antibodies partially inhibited URT protection after administration to G2Na-immunized BALB/c mice. Furthermore, while purified CD4(+) T cells from BALB/c mice immunized with G2Na or AICK significantly reduced lung and nasal infection of naive recipient mice after adoptive transfer, the cells from IFN-gamma-knockout mice had no effect. Together, these results demonstrated for the first time that the T-helper-cell epitope of RSV G protein induces URT protection in mice after parenteral immunization through a Th1-type, IFN-gamma-dependent mechanism.
Assuntos
Epitopos de Linfócito T/imunologia , Imunidade Materno-Adquirida/imunologia , Interferon gama/imunologia , Pulmão/imunologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Vacinas Sintéticas/imunologia , Proteínas Virais/imunologia , Transferência Adotiva , Sequência de Aminoácidos , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos SCID , Dados de Sequência Molecular , Testes de Neutralização , Fragmentos de Peptídeos/imunologia , Sistema Respiratório/imunologia , Células Th1/imunologia , VacinaçãoRESUMO
Respiratory syncytial virus (RSV) is responsible for severe low respiratory tract infections in young infants and the elderly. To investigate whether BBG2Na, a recombinant subunit vaccine comprising aa 130-230 of the RSV G protein, induced protective Abs in subjects over 60 years during phase II clinical trial, pre- and postimmunization sera of individuals immunized with BBG2Na or placebo were transferred into SCID mice before RSV challenge. These sera dose-dependently reduced lung RSV titers. However at some points of serial dilutions, postimmunization sera of BBG2Na-immunized subjects only were significantly more efficient than the corresponding preimmunization sera, in agreement with the induction of an increased Ab response against multiple epitopes on RSV-A G protein. Thus, BBG2Na is immunogenic in the elderly and confers passive protection in mice after serum transfer. To our knowledge, this is the first description of protective Abs induced by a subunit vaccine in human.