RESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Movimiento Celular/efectos de los fármacos , Quimiocina CXCL12/metabolismo , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Unión Competitiva , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Proteínas del Sistema Complemento/inmunología , Modelos Animales de Enfermedad , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/inmunología , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Unión Proteica , Multimerización de Proteína , Receptores CXCR4/química , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Arrestina beta 2/metabolismoRESUMEN
Antibody-drug conjugates (ADCs) are becoming a major class of oncology therapeutics. They combine monoclonal antibody specificity for over-expressed tumor antigens and the high cytoxicity of small molecular drugs (SMDs) and can therefore selectively kill tumor cells while minimizing toxicity to normal cells. Nevertheless, the premature deconjugation of ADCs in the circulation may trigger off target toxicity in patients. The released free drug level must be low in circulation for an extended period of time as well as the de-conjugation rate to ensure an acceptable therapeutic window. As a result, the assessment of the stability of the linker between payload and mAb in the systemic circulation is of paramount importance before entering in clinical trial. Here we report a new universal method to immunocapture and analyze by LC-MS the stability and distribution of ADCs in sera from relevant preclinical species (mouse, rat and cynomolgus monkey). Furthermore we demonstrated that this workflow can be applied to both ADCs with cleavable and non cleavable linkers. Last but not least, the results obtained in cynomolgus serum using immunoprecipitation and LC-MS analysis were cross validated using an ELISA orthogonal method. As the ligand used for immunoprecipitation is targeting the Fc part of mAb (CaptureSelect™ Human IgG-Fc PK Biotin), this protocol can be applied to analyze the stability of virtually all ADCs in sera for preclinical studies without the need to prepare specific molecular tools.
Asunto(s)
Anticuerpos Monoclonales/sangre , Inmunoconjugados/sangre , Animales , Cromatografía Liquida/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Macaca fascicularis , Espectrometría de Masas/métodos , Ratones , Ratones Desnudos , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: In preclinical studies, monoclonal antibodies (mAbs) are traditionally assayed by ligand-binding-assays. Recently, quantitative liquid chromatography mass spectrometry (MS)-based assays have emerged which circumvent a number of challenges. These assays may also be multiplex, making them potentially compatible with pharmacokinetic assays for combined antibody therapies. MATERIALS & METHODS: We combined a quantitative MS-based approach with the protein standard for absolute quantification (PSAQ™) strategy to simultaneously quantify three mAb variants presenting minor sequence differences. Stable isotopically labeled mAbs were produced and used as quantification standards. Titration curves were performed to assess the analytical performances of the method. LC-MS/MS and ELISA data were cross-compared. RESULTS: The approach presented provides similar accuracy and precision than ELISA, while being multiplex and faster to develop. It has applications at all stages of the pharmaceutical development.
Asunto(s)
Anticuerpos Monoclonales/sangre , Cromatografía Liquida/métodos , Inmunoglobulina G/sangre , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina G/química , Datos de Secuencia Molecular , RatasRESUMEN
Antibody-drug conjugates (ADCs) are becoming a major class of oncology therapeutics. Because ADCs combine the monoclonal antibody specificity with the high toxicity of a drug, they can selectively kill tumor cells while minimizing toxicity to normal cells. Most of the current ADCs in clinical trials are controlled, but heterogeneous mixtures of isomers and isoforms. Very few protocols on ADC characterization at the peptide level have been published to date. Here, we report on the improvement of an ADC peptide mapping protocol to characterize the drug-loaded peptides by LC-MS analysis. These methods were developed on brentuximab vedotin (Adcetris), a commercial ADC with an average of four drugs linked to interchain cysteine residues of its antibody component. Because of the drug hydrophobicity, all the steps of this protocol including enzymatic digestion were improved to maintain the hydrophobic drug-loaded peptides in solution, allowing their unambiguous identification by LC-MS. For the first time, the payloads positional isomers observed by RP-HPLC after IdeS-digestion and reduction of the ADC were also characterized.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Cisteína/química , Inmunoconjugados/química , Espectrometría de Masas/métodos , Fragmentos de Péptidos/química , Mapeo Peptídico/métodos , Isomerismo , Fragmentos de Péptidos/análisisRESUMEN
The World Antibody-Drug Conjugate (WADC) Summits organized by Hanson Wade are currently the largest meetings fully dedicated to ADCs. The first global ADC Summit was organized in Boston in October 2010. Since 2011, two WADC are held every year in Frankfurt and San Francisco, respectively. The 2013 WADC San Francisco event was structured around plenary sessions with keynote speakers from AbbVie, Agensys, ImmunoGen, Immunomedics, Genentech, Pfizer and Seattle Genetics. Parallel tracks were also organized addressing ADC discovery, development and optimization of chemistry, manufacturing and control (CMC) issues. Discovery and process scientists, regulatory experts (US Food and Drug Administration), academics and clinicians were present, including representatives from biotechnology firms (Concortis, CytomX Therapeutics, Glykos, Evonik, Igenica, Innate Pharma, Mersana Therapeutics, Polytherics, Quanta Biodesign, Redwood Bioscience, Sutro Biopharma, SynAffix), pharmaceutical companies (Amgen, Genmab, Johnson and Johnson, MedImmune, Novartis, Progenics, Takeda) and contract research or manufacturing organizations (Baxter, Bayer, BSP Pharmaceuticals, Fujifilm/Diosynth, Lonza, Pierre Fabre Contract Manufacturing, Piramal, SAFC, SafeBridge).
Asunto(s)
Anticuerpos Antineoplásicos/uso terapéutico , Antineoplásicos/uso terapéutico , Aprobación de Drogas , Sistemas de Liberación de Medicamentos , Inmunoconjugados/uso terapéutico , Neoplasias/tratamiento farmacológico , Congresos como Asunto , HumanosRESUMEN
Antibodies and related products represent one of the fastest growing areas of new drug development within the pharmaceutical industry. Monoclonal antibodies (mAbs) undergo many posttranslational modifications (PTMs) that must be extensively characterized. Here we described a rapid mass spectrometry (MS) method for the characterization of cetuximab glycosylation. The reported analytical technique is based on the use of a cystein protease, immunoglobulin-degrading enzyme of Streptococcus pyogenes that allows a fast limited proteolysis of the mAb with low material consumption. The resulting large fragments are analyzed by ultrahigh-performance liquid chromatography combined to an electrospray ionization mass spectrometer and a time-of-flight analyzer (ESI-TOF). Cetuximab is a potent chimeric mouse/human antibody worldwide approved for the treatment of colon and head and neck cancers. This antibody, produced by SP2/0 murine myeloma cells, is N-glycosylated both in the Fc and Fab moieties, which have been shown to impact on safety and PK/PD and considered as a critical quality attribute. The method can also be applied for biosimilars, biobetters, and next-generation antibodies and Fc-fusion proteins.
Asunto(s)
Anticuerpos Monoclonales Humanizados/química , Proteínas Bacterianas/química , Cisteína Endopeptidasas/química , Polisacáridos/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales Humanizados/aislamiento & purificación , Tampones (Química) , Conformación de Carbohidratos , Secuencia de Carbohidratos , Cetuximab , Cromatografía Líquida de Alta Presión , Ditiotreitol/química , Glicosilación , Humanos , Fragmentos Fc de Inmunoglobulinas , Ratones , Datos de Secuencia Molecular , Neuraminidasa/química , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/química , Polisacáridos/aislamiento & purificación , Procesamiento Proteico-Postraduccional , Proteolisis , Sustancias Reductoras/químicaRESUMEN
The 101-residue long Tat protein of primary isolate 133 of the human immunodeficiency virus type 1 (HIV-1), wt-Tat(133) displays a high transactivation activity in vitro, whereas the mutant thereof, STLA-Tat(133), a vaccine candidate for HIV-1, has none. These two proteins were chemically synthesized and their biological activity was validated. Their structural properties were characterized using circular dichroism (CD), fluorescence emission, gel filtration, dynamic light scattering, and small angle X-ray scattering (SAXS) techniques. SAXS studies revealed that both proteins were extended and belong to the family of intrinsically unstructured proteins. CD measurements showed that wt-Tat(133) or STLA-Tat(133) underwent limited structural rearrangements when complexed with specific fragments of antibodies. Crystallization trials have been performed on the two forms, assuming that the Tat(133) proteins might have a better propensity to fold in supersaturated conditions, and small crystals have been obtained. These results suggest that biologically active Tat protein is natively unfolded and requires only a limited gain of structure for its function.
Asunto(s)
VIH-1/química , Proteínas Mutantes/química , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/química , Vacunas contra el SIDA/química , Vacunas contra el SIDA/genética , Vacunas contra el SIDA/inmunología , Secuencia de Aminoácidos , Cromatografía en Gel , Dicroismo Circular , Cristalografía por Rayos X , VIH-1/genética , VIH-1/aislamiento & purificación , Interacciones Hidrofóbicas e Hidrofílicas , Fragmentos de Inmunoglobulinas , Luz , Metilaminas , Datos de Secuencia Molecular , Pliegue de Proteína , Dispersión de Radiación , Dispersión del Ángulo Pequeño , Espectrofotometría Ultravioleta , Trifluoroetanol , Agua , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Monoclonal antibodies (mAb) are attractive biologic drugs because of their specificity and well understood mechanisms of action. So far, most mAb have been developed for treating cancers or immunological disorders. However, the antibiotic resistance crisis, emerging viral diseases and bioterrorism have increased the development of anti-infectious mAb, for which more than twenty clinical trials are in progress to evaluate their safety and efficacy. The synergies obtained using combinations of anti-infectious mAb and small molecule drugs will certainly offer new opportunities for the treatment of infectious diseases.
Asunto(s)
Antiinfecciosos/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Enfermedades Transmisibles/tratamiento farmacológico , Adulto , Anciano , Infecciones Bacterianas/tratamiento farmacológico , Infecciones Bacterianas/inmunología , Ensayos Clínicos como Asunto , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Humanos , Huésped Inmunocomprometido , Recién Nacido , Enfermedades del Prematuro/tratamiento farmacológico , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Infecciones por Virus Sincitial Respiratorio/inmunologíaRESUMEN
Despite technological advances, detection of deamidation in large proteins remains a challenge and the use of orthogonal methods is needed for unequivocal assignment. By a combination of cation-exchange separation, papain digestion, and a panel of mass spectrometry techniques we identified asparagine deamidation in light chain complementarity determining region 1 (CDR1) of a humanized IgG1 monoclonal antibody. The reaction yields both Asp and isoAsp, which were assigned by Edman degradation and by isoAsp detection using protein isoaspartate methyltransferase. The deamidated antibody variants were less potent in antigen binding compared to the nondegraded antibody. Changes in near-UV CD spectra, susceptibility to papain cleavage in an adjacent CDR2 loop, and the tendency of the newly formed isoAsp to undergo isomerization suggest local perturbations in the structure of the isoAsp-containing antibody.
Asunto(s)
Asparagina/análisis , Regiones Determinantes de Complementariedad/química , Inmunoglobulina G/química , Cadenas Ligeras de Inmunoglobulina/química , Antígenos/inmunología , Asparagina/química , Rastreo Diferencial de Calorimetría , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Regiones Determinantes de Complementariedad/inmunología , Regiones Determinantes de Complementariedad/metabolismo , Cristalografía por Rayos X , Desaminación , Humanos , Concentración de Iones de Hidrógeno , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Cadenas Ligeras de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/metabolismo , Isomerismo , Espectrometría de Masas , Modelos Moleculares , Estructura Molecular , Papaína/metabolismoRESUMEN
Monoclonal antibodies (MAbs) are the fastest growing class of human pharmaceuticals. More than 20 MAbs have been approved and several hundreds are in clinical trials in various therapeutic indications including oncology, inflammatory diseases, organ transplantation, cardiology, viral infection, allergy, and tissue growth and repair. Most of the current therapeutic antibodies are humanized or human Immunoglobulins (IgGs) and are produced as recombinant glycoproteins in eukaryotic cells. Many alternative production systems and improved constructs are also being actively investigated. IgGs glycans represent only an average of around 3% of the total mass of the molecule. Despite this low percentage, particular glycoforms are involved in essential immune effector functions. On the other hand, glycoforms that are not commonly biosynthesized in human may be allergenic, immunogenic and accelerate the plasmatic clearance of the linked antibody. These glyco-variants have to be identified, controlled and limited for therapeutic uses. Glycosylation depends on multiple factors like production system, selected clonal population, manufacturing process and may be genetically or chemically engineered. The present account reviews the glycosylation patterns observed for the current approved therapeutic antibodies produced in mammalian cell lines, details classical and state-of-the-art analytical methods used for the characterization of glycoforms and discusses the expected benefits of manipulating the carbohydrate components of antibodies by bio- or chemical engineering as well as the expected advantages of alternative biotechnological production systems developed for new generation of therapeutic antibodies and Fc-fusion proteins.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Carbohidratos/inmunología , Diseño de Fármacos , Industria Farmacéutica/tendencias , Fragmentos Fc de Inmunoglobulinas/inmunología , Ingeniería de Proteínas/tendencias , Anticuerpos Monoclonales/genética , Carbohidratos/genética , Glicosilación , Humanos , Fragmentos Fc de Inmunoglobulinas/genéticaRESUMEN
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.
Asunto(s)
Inmunoterapia/métodos , Péptidos/inmunología , Péptidos/uso terapéutico , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Antígenos Virales/química , Antígenos Virales/genética , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/inmunología , Epítopos de Linfocito B/genética , Epítopos de Linfocito T/genética , Humanos , Ratones , Modelos Inmunológicos , Modelos Moleculares , Datos de Secuencia Molecular , Mapeo Peptídico , Vacunas contra Virus Sincitial Respiratorio/genética , Vacunas contra Virus Sincitial Respiratorio/inmunología , Virus Sincitial Respiratorio Humano/genética , Virus Sincitial Respiratorio Humano/inmunologíaRESUMEN
7H2HM is a new humanized recombinant monoclonal antibody (MAb) directed against insulin-like growth factor-1 receptor and produced in CHO cells. Homogeneity of intact antibody, reduced light and heavy chains, Fab and Fc fragments were investigated by analytical methods based on mass (SDS-PAGE, SEC), charge (IEF, C-IEX) and hydrophobicity differences (RP-HPLC, HIC) and compared side-by-side with A2CHM, produced in NS0 cells. Primary structures and disulfide bridge pairing were analyzed by microsequencing (Edman degradation), mass spectrometry (MALDI-TOF, ES-TOF) and peptide mapping after enzymatic digestion (Trypsin, endoprotease Lys-C, papain). The light chains demonstrated the expected sequences. The heavy chains yielded post-translational modifications previously reported for other recombinant humanized or human IgG1 such as N-terminal pyroglutamic acid, C-terminal lysine clipping and N-glycosylation for asparagine 297. More surprisingly, two-thirds of the 7H2HM heavy chains were shown to contain an additional 24-amino-acid sequence, corresponding to the translation of an intron located between the variable and the constant domains. Taken together these data suggest that 7H2HM is a mixture of three families of antibodies corresponding (i) to the expected structure (17%; 14,9297 Da; 1330 amino acids), (ii) a variant with a translated intron in one heavy chains (33%; 15,2878 Da; 1354 amino acids) and (iii) a variant with translated introns in two heavy chains (50%; 15,4459 Da; 1378 amino acids), respectively. RP-HPLC is not a commonly used chromatographic method to assess purity of monoclonal antibodies but unlike to SEC and SDS-PAGE, was able to show and to quantify the family of structures present in 7H2HM, which were also identified by peptide mapping, mass spectrometry and microsequencing.
Asunto(s)
Anticuerpos Monoclonales/análisis , Cromatografía Líquida de Alta Presión/métodos , Factor I del Crecimiento Similar a la Insulina/inmunología , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Células CHO , Cricetinae , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Mapeo Peptídico , Procesamiento Proteico-PostraduccionalRESUMEN
Human respiratory syncytial virus (hRSV) is one of the most common causes of respiratory infection in infants and the elderly. Previous attempts to vaccinate children against RSV failed and the induction of an aberrant Th2-type immune response was shown to induce severe to fatal pulmonary disease characterised in part by eosinophilia. BBG2Na is a promising human RSV subunit vaccine candidate which successfully passed phase II clinical trials in adults in association with Adju-Phos((R)). However, this formulation is not the most suitable for use in children since aluminium salts are known to induce a Th2-based immune response. In this study, we describe a potent and safe adjuvant formulation for BBG2Na in dimethyldioctadecylammonium bromide (DDA) that induces a mixed Th1/Th2 immune response in BALB/c mice. Furthermore, BBG2Na showed the same protective efficacy against RSV challenge when formulated either in DDA or in alum in mice and cotton rats.