RESUMO
Monoclonal antibodies (mAbs) have established themselves as the leading biopharmaceutical therapeutic modality. Once the developability of a mAb drug candidate has been assessed, an important step is to check its in vivo stability through pharmacokinetics (PK) studies. The gold standard is ligand-binding assay (LBA) and liquid chromatography-mass spectrometry (LC-MS) performed at the peptide level (bottom-up approach). However, these analytical techniques do not allow to address the different mAb proteoforms that can arise from biotransformation. In recent years, top-down and middle-down mass spectrometry approaches have gained popularity to characterize proteins at the proteoform level but are not yet widely used for PK studies. We propose here a workflow based on an automated immunocapture followed by top-down and middle-down liquid chromatography-tandem mass spectrometry (LC-MS/MS) approaches to characterize mAb proteoforms spiked in mouse plasma. We demonstrate the applicability of our workflow on a large concentration range using pembrolizumab as a model. We also compare the performance of two state-of-the-art Orbitrap platforms (Tribrid Eclipse and Exploris 480) for these studies. The added value of our workflow for an accurate and sensitive characterization of mAb proteoforms in mouse plasma is highlighted.
Assuntos
Peptídeos , Espectrometria de Massas em Tandem , Animais , Camundongos , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Plasma , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacocinéticaRESUMO
Cabazitaxel is a semisynthetic taxane approved for the treatment of patients with hormone-refractory metastatic prostate cancer (now known as metastatic castration-resistant prostate cancer) treated previously with a docetaxel-containing treatment regimen. The human plasma pharmacokinetics of cabazitaxel have been described previously, but detailed analyses of the metabolism and excretion pathways of cabazitaxel have not yet been published. Metabolite profiling, quantification, and identification as well as excretion analyses were carried out on samples from patients with advanced solid tumors who received an intravenous infusion of 25 mg/m [C]-cabazitaxel (50 µCi, 1.85 MBq) over 1 h. In plasma, cabazitaxel was the main circulating compound. Seven metabolites were detected, but with each accounting for 5% or less of the parent drug exposure, none were considered relevant metabolites. In excreta, 76.0% of the administered dose was recovered in feces within 2 weeks and 3.7% of the dose was excreted in urine within 1 week. Approximately 20 metabolites were detected in excreta; the main metabolites corresponded to combined mono-O-demethyl or di-O-demethyl derivatives on the taxane ring, with hydroxyl or cyclized derivatives on the lateral chain. Docetaxel (di-O-demethyl-cabazitaxel) was only detected at trace levels in excreta. These results suggest an extensive hepatic metabolism and biliary excretion of cabazitaxel in humans.
Assuntos
Antineoplásicos/farmacocinética , Taxoides/metabolismo , Taxoides/farmacocinética , Antineoplásicos/uso terapêutico , Radioisótopos de Carbono/farmacocinética , Neoplasias Esofágicas/tratamento farmacológico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/tratamento farmacológico , Sarcoma/tratamento farmacológico , Neoplasias de Tecidos Moles/tratamento farmacológico , Taxoides/sangue , Taxoides/uso terapêuticoRESUMO
Natural killer cell engagers (NKCEs), a treatment that stimulates innate immunity, have lately gained attention owing to their favorable safety profile, and their efficacy. Natural killer (NK) cell activation is driven by immune synapse formation between drugs, NK cells, and tumor cells. However, no clear translational modeling approach has been reported for first-in-human (FIH) dose estimation of humanized NKCEs. We developed the first translational mechanistic synapse-driven pharmacokinetic/pharmacodynamic (PK/PD) model for a trifunctional NKp46/CD16a-CD123 (CD123-NKCE) by integrating (i) in vitro target cell cytotoxicity in MOLM-13 tumor cell lines at varying effector-to-tumor cell ratios and incubation intervals; (ii) nonhuman primate PK and profiles of CD123+ cells and NKP46+ NK cells; and (iii) healthy human or patients with acute myeloid leukemia system-specific parameters. To depict direct tumor cell killing by the innate immunity, no transit compartment was included in PK/PD model structures. Model predictions suggested an intrapatient dose escalation of 10/30/100 µg/kg twice weekly to be selected as the starting dose in the FIH trial. However, sensitivity analyses revealed that CD123+ cell growth rate constant and maximal tumor killing rate constant were the key uncertainties to the recommended active dose. This novel translational model structure can be used as the basis to predict clinical PK/PD data for CD123-NKCE, and the translational strategy may serve as a foundation for future advancements of NKCEs.
Assuntos
Subunidade alfa de Receptor de Interleucina-3 , Leucemia Mieloide Aguda , Animais , Humanos , Subunidade alfa de Receptor de Interleucina-3/metabolismo , Subunidade alfa de Receptor de Interleucina-3/uso terapêutico , Células Matadoras Naturais , Linhagem Celular Tumoral , Imunidade Inata , Leucemia Mieloide Aguda/tratamento farmacológicoRESUMO
In antibody-based drug research, a complete characterization of antibody proteoforms covering both the amino acid sequence and all posttranslational modifications remains a major concern. The usual mass spectrometry-based approach to achieve this goal is bottom-up proteomics, which relies on the digestion of antibodies but does not allow the diversity of proteoforms to be assessed. Middle-down and top-down approaches have recently emerged as attractive alternatives but are not yet mastered and thus used in routine by many analytical chemistry laboratories. The work described here aims at providing guidelines to achieve the best sequence coverage for the fragmentation of intact light and heavy chains generated from a simple reduction of intact antibodies using Orbitrap mass spectrometry. Three parameters were found crucial to this aim: the use of an electron-based activation technique, the multiplex selection of precursor ions of different charge states, and the combination of replicates.
Assuntos
Anticorpos Monoclonais , Espectrometria de Massas em Tandem , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Processamento de Proteína Pós-TraducionalRESUMO
We recently described C18 fatty acid acylated peptides as a new class of potent long-lasting single-chain RXFP1 agonists that displayed relaxin-like activities in vivo. Early pharmacokinetics and toxicological studies of these stearic acid acylated peptides revealed a relevant oxidative metabolism occurring in dog and minipig, and also seen at a lower extent in monkey and rat. Mass spectrometry combined to NMR spectroscopy studies revealed that the oxidation occurred, unexpectedly, on the stearic acid chain at ω-1, ω-2 and ω-3 positions. Structure-metabolism relationship studies on acylated analogues with different fatty acids lengths (C15-C20) showed that the extent of oxidation was higher with longer chains. The oxidized metabolites could be generated in vitro using liver microsomes and engineered bacterial CYPs. These systems were correlating poorly with in vivo metabolism observed across species; however, the results suggest that this biotransformation pathway might be catalyzed by some unknown CYP enzymes.
Assuntos
Sistema Enzimático do Citocromo P-450 , Ácidos Graxos , Animais , Cães , Ratos , Sistema Enzimático do Citocromo P-450/metabolismo , Ácidos Graxos/metabolismo , Redes e Vias Metabólicas , Microssomos Hepáticos/metabolismo , Oxirredução , Ácidos Esteáricos , Suínos , Porco Miniatura/metabolismo , HaplorrinosRESUMO
Monoclonal antibodies (mAbs) are among the fastest growing and most effective therapies for myriad diseases. Multispecific antibodies are an emerging class of novel therapeutics that can target more than one tumor- or immune-associated modulators per molecule. The combination of different binding affinities and target classes, such as soluble or membrane-bound antigens, within multispecific antibodies confers unique pharmacokinetic (PK) properties. Numerous factors affect an antibody's PK, with affinity to the neonatal Fc receptor (FcRn) a key determinant of half-life. Recent work has demonstrated the potential for humanized FcRn transgenic mice to predict the PK of mAbs in humans. However, such work has not been extended to multispecific antibodies. We engineered mAbs and multispecific antibodies with various Fc modifications to enhance antibody performance. PK analyses in humanized FcRn transgenic mouse (homozygous Tg32 and Tg276) and non-human primate (NHP) models showed that FcRn-binding mutations improved the plasma half-lives of the engineered mAbs and multispecific antibodies, while glycan engineering to eliminate effector function did not affect the PK compared with wild-type controls. Furthermore, results suggest that the homozygous Tg32 mouse model can replace NHP models to differentiate PK of variants during lead optimization, not only for wild-type mAbs but also for Fc-engineered mAbs and multispecific antibodies. This Tg32-mouse model would enable prediction of half-life and linear clearance of mAbs and multispecific antibodies in NHPs to guide the design of further pharmacology/safety studies in this species. The allometric exponent for clearance scaling from Tg32 mice to NHPs was estimated to be 0.91 for all antibodies.
Assuntos
Anticorpos Monoclonais , Antígenos de Histocompatibilidade Classe I/imunologia , Receptores Fc/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/farmacologia , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Macaca fascicularis , Camundongos , Camundongos Transgênicos , Receptores Fc/genéticaRESUMO
Antibodies mediate effector functions through Fcγ receptor (FcγR) interactions and complement activation, causing cytokine release, degranulation, phagocytosis, and cell death. They are often undesired for development of therapeutic antibodies where only antigen binding or neutralization would be ideal. Effector elimination has been successful with extensive mutagenesis, but these approaches can potentially lead to manufacturability and immunogenicity issues. By switching the native glycosylation site from position 297 to 298, we created alternative antibody glycosylation variants in the receptor interaction interface as a novel strategy to eliminate the effector functions. The engineered glycosylation site at Asn298 was confirmed by SDS-PAGE, mass spectrometry, and X-ray crystallography (PDB code 6X3I). The lead NNAS mutant (S298N/T299A/Y300S) shows no detectable binding to mouse or human FcγRs by surface plasmon resonance analyses. The effector functions of the mutant are completely eliminated when measured in antibody-dependent cell-meditated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays. In vivo, the NNAS mutant made on an antibody against a human lymphocyte antigen does not deplete T cells or B cells in transgenic mice, in contrast to wild-type antibody. Structural study confirms the successful glycosylation switch to the engineered Asn298 site. The engineered glycosylation would clash with approaching FcγRs based on reported Fc-FcγR co-crystal structures. In addition, the NNAS mutants of multiple antibodies retain binding to antigens and neonatal Fc receptor, exhibit comparable purification yields and thermal stability, and display normal circulation half-life in mice and non-human primate. Our work provides a novel approach for generating therapeutic antibodies devoid of any ADCC and CDC activities with potentially lower immunogenicity.
Assuntos
Substituição de Aminoácidos , Ativação do Complemento , Citotoxicidade Imunológica , Antígenos de Histocompatibilidade Classe I/imunologia , Fragmentos Fc das Imunoglobulinas , Mutação de Sentido Incorreto , Receptores Fc/imunologia , Animais , Células CHO , Cricetulus , Glicosilação , Células HEK293 , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Receptores Fc/genéticaRESUMO
The neonatal Fc receptor (FcRn) promotes antibody recycling through rescue from normal lysosomal degradation. The binding interaction is pH-dependent with high affinity at low pH, but not under physiological pH conditions. Here, we combined rational design and saturation mutagenesis to generate novel antibody variants with prolonged half-life and acceptable development profiles. First, a panel of saturation point mutations was created at 11 key FcRn-interacting sites on the Fc region of an antibody. Multiple variants with slower FcRn dissociation kinetics than the wildtype (WT) antibody at pH 6.0 were successfully identified. The mutations were further combined and characterized for pH-dependent FcRn binding properties, thermal stability and the FcγRIIIa and rheumatoid factor binding. The most promising variants, YD (M252Y/T256D), DQ (T256D/T307Q) and DW (T256D/T307W), exhibited significantly improved binding to FcRn at pH 6.0 and retained similar binding properties as WT at pH 7.4. The pharmacokinetics in human FcRn transgenic mice and cynomolgus monkeys demonstrated that these properties translated to significantly prolonged plasma elimination half-life compared to the WT control. The novel variants exhibited thermal stability and binding to FcγRIIIa in the range comparable to clinically validated YTE and LS variants, and showed no enhanced binding to rheumatoid factor compared to the WT control. These engineered Fc mutants are promising new variants that are widely applicable to therapeutic antibodies, to extend their circulation half-life with obvious benefits of increased efficacy, and reduced dose and administration frequency.