RESUMO
In this paper, a methodology for the development of a multimodal chromatography process is presented that is aimed at removal of under-conjugated antibody-drug conjugate (ADC) species. Two ADCs are used as case studies: One ADC results from site-directed conjugation to inserted cysteine residues and has a drug-to-antibody ratio (DAR) of two, the other is the product of conjugation to interchain disulfide bonds with a DAR of eight. First, filter plate screening studies are designed for the unconjugated antibody and the ADCs. Different metrics for the analysis of these data sets are presented and discussed. From this analysis, the selected process conditions are then carried out using a benchtop chromatography system to confirm the separations observed in the filter plate studies while simultaneously generating data to estimate steric mass-action isotherm and mass transport parameters for process simulation. This column model is then employed to develop separation processes in-silico for the removal of the unconjugated parent antibody and under-conjugated product variants. The optimized process conditions identified using the model are then verified experimentally. The methodology presented in this work utilizes multimodal chromatography for ADC purification and provides the framework for a streamlined systematic approach to process development.
Assuntos
Química Farmacêutica , Cromatografia Líquida , Imunoconjugados , Química Farmacêutica/métodos , Simulação por Computador , Cisteína , Ensaios de Triagem em Larga Escala , Imunoconjugados/isolamento & purificaçãoRESUMO
BACKGROUND: We prepared an anti-p21Ras scFv which could specifically bind with mutant and wild-type p21Ras. However, it cannot penetrate the cell membrane, which prevents it from binding to p21Ras in the cytoplasm. Here, the RGD4C peptide was used to mediate the scFv penetration into tumor cells and produce antitumor effects. METHODS: RGD4C-EGFP and RGD4C-p21Ras-scFv recombinant expression plasmids were constructed to express fusion proteins in E. coli, then the fusion proteins were purified with HisPur Ni-NTA. RGD4C-EGFP was used as reporter to test the factors affecting RGD4C penetration into tumor cell. The immunoreactivity of RGD4C-p21Ras-scFv toward p21Ras was identified by ELISA and western blotting. The ability of RGD4C-p21Ras-scFv to penetrate SW480 cells and colocalization with Ras protein was detected by immunocytochemistry and immunofluorescence. The antitumor activity of the RGD4C-p21Ras-scFv was assessed with the MTT, TUNEL, colony formation and cell migration assays. Chloroquine (CQ) was used an endosomal escape enhancing agent to enhance endosomal escape of RGD4C-scFv. RESULTS: RGD4C-p21Ras-scFv fusion protein were successfully expressed and purified. We found that the RGD4C fusion protein could penetrate into tumor cells, but the tumor cell entry of was time and concentration dependent. Endocytosis inhibitors and a low temperature inhibited RGD4C fusion protein endocytosis into cells. The change of the cell membrane potential did not affect penetrability. RGD4C-p21Ras-scFv could penetrate SW480 cells, effectively inhibit the growth, proliferation and migration of SW480 cells and promote this cells apoptosis. In addition, chloroquine (CQ) could increase endosomal escape and improve antitumor activity of RGD4C-scFv in SW480 cells. CONCLUSION: The RGD4C peptide can mediate anti-p21Ras scFv entry into SW480 cells and produce an inhibitory effect, which indicates that RGD4C-p21Ras-scFv may be a potential therapeutic antibody for the treatment of ras-driven cancers.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Neoplasias do Colo/tratamento farmacológico , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Recombinantes de Fusão/farmacologia , Antineoplásicos Imunológicos/isolamento & purificação , Antineoplásicos Imunológicos/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Humanos , Imunoconjugados/genética , Imunoconjugados/isolamento & purificação , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/uso terapêutico , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/isolamento & purificação , Anticorpos de Cadeia Única/farmacologia , Anticorpos de Cadeia Única/uso terapêuticoRESUMO
B7-H3, also referred to as CD276, is a member of the B7 family of immune regulatory proteins. B7-H3 is overexpressed on many solid cancers, including prostate cancer, renal cell carcinoma, melanoma, squamous cell carcinoma of the head and neck, non-small cell lung cancer, and breast cancer. Overexpression of B7-H3 is associated with disease severity, risk of recurrence and reduced survival. In this article, we report the preclinical development of MGC018, an antibody-drug conjugate targeted against B7-H3. MGC018 is comprised of the cleavable linker-duocarmycin payload, valine-citrulline-seco duocarmycin hydroxybenzamide azaindole (vc-seco-DUBA), conjugated to an anti-B7-H3 humanized IgG1/kappa mAb through reduced interchain disulfides, with an average drug-to-antibody ratio of approximately 2.7. MGC018 exhibited cytotoxicity toward B7-H3-positive human tumor cell lines, and exhibited bystander killing of target-negative tumor cells when cocultured with B7-H3-positive tumor cells. MGC018 displayed potent antitumor activity in preclinical tumor models of breast, ovarian, and lung cancer, as well as melanoma. In addition, antitumor activity was observed toward patient-derived xenograft models of breast, prostate, and head and neck cancer displaying heterogeneous expression of B7-H3. Importantly, MGC018 exhibited a favorable pharmacokinetic and safety profile in cynomolgus monkeys following repeat-dose administration. The antitumor activity observed preclinically with MGC018, together with the positive safety profile, provides evidence of a potentially favorable therapeutic index and supports the continued development of MGC018 for the treatment of solid cancers. GRAPHICAL ABSTRACT: http://mct.aacrjournals.org/content/molcanther/19/11/2235/F1.large.jpg.
Assuntos
Antígenos B7/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , Inibidores de Checkpoint Imunológico/farmacologia , Imunoconjugados/farmacologia , Neoplasias/tratamento farmacológico , Animais , Antígenos B7/genética , Antígenos B7/metabolismo , Efeito Espectador , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Monitoramento de Medicamentos , Técnicas de Silenciamento de Genes , Humanos , Inibidores de Checkpoint Imunológico/química , Inibidores de Checkpoint Imunológico/isolamento & purificação , Imunoconjugados/química , Imunoconjugados/isolamento & purificação , Camundongos , Neoplasias/metabolismo , Neoplasias/patologia , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Antibody-drug conjugates (ADCs) are hybrid molecules based on monoclonal antibodies (mAbs) with covalently attached cytotoxic small-molecule drugs. Due to their potential for targeted cancer therapy, they form part of the diversifying pipeline of various biopharmaceutical companies, in addition to currently seven commercial ADCs. With other new modalities, ADCs contribute to the increasing complexity of biopharmaceutical development in times of growing costs and competition. Another challenge is the implementation of quality by design (QbD), which receives a lot of attention. In order to answer these challenges, mechanistic models are gaining interest as tools for enhanced process understanding and efficient process development. The drug-to-antibody ratio (DAR) is a critical quality attribute (CQA) of ADCs. After the conjugation reaction, the DAR can still be adjusted by including a hydrophobic interaction chromatography (HIC) step. In this work, we developed a mechanistic model for the preparative separation of cysteine-engineered mAbs with different degrees of conjugation with a non-toxic surrogate drug. The model was successfully validated for varying load compositions with linear and optimized step gradient runs, applying conditions differing from the calibration runs. In two in silico studies, we then present scenarios for how the model can be applied profitably to ensure a more robust achievement of the target DAR and for the efficient characterization of the design space. For this, we also used the model in a linkage study with a kinetic reaction model developed by us previously. The combination of the two models effectively widens system boundaries over two adjacent process steps. We believe this work has great potential to help advance the incorporation of digital tools based on mechanistic models in ADC process development by illustrating their capabilities for efficient process development and increased robustness. Mechanistic models can support the implementation of QbD and eventually might be the basis for digital process twins able to represent multiple unit operations.
Assuntos
Anticorpos Monoclonais/química , Cromatografia Líquida/métodos , Imunoconjugados/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Cisteína/química , Cisteína/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Imunoconjugados/isolamento & purificação , Imunoconjugados/metabolismo , Cinética , Modelos QuímicosRESUMO
Antibody drug conjugates are cytotoxic pharmaceuticals, designed to destroy malignant cells. A cytotoxic molecule is attached to an antibody that binds specific to a cancer-cell surface. Given the high toxicity of the drugs, strict safety standards have to be kept. For this reason, an antibody drug conjugates model was developed with fluorescein 5-isothiocyanate as the nontoxic payload surrogate. Due to the similar hydrophobicity, this model is used to establish a suitable purification process and characterization method for antibody drug conjugates. Because of the pH dependent solubility of fluorescein, the hydrophobicity of conjugates can be modulated by the pH value. Based on the complex heterogeneity and hydrophobicity of the conjugates a chromatographic purification is challenging. Hydrophobic interaction chromatography is used for analytical as well as for preparative separation. Because of the increased hydrophobicity of the conjugates compared to native antibody, hydrophobic interaction chromatography often suffer from resolution and recovery problems. Conjugates were separated differing on the number of payloads attached to the antibody. For this matter, the drug-antibody ratio is determined and used as a quantitative term. The conjugates are purified at high recoveries and resolution by step gradients using suitable resins, allowing the separation of the target drug-antibody ratio.
Assuntos
Imunoconjugados/isolamento & purificação , Modelos Químicos , Resinas Sintéticas/química , Cromatografia Líquida de Alta Pressão , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunoconjugados/químicaRESUMO
Protein semisynthesis is a powerful tool for studying proteins and has contributed to a better understanding of protein structure and function and also driven innovations in protein science. Expressed protein ligation (EPL) is a widely used method to generate chemically modified proteins. However, EPL has some limitations, particularly relevant to modify challenging proteins such as antibodies. The method termed streamlined expressed protein ligation (SEPL) overcomes some of the problems of EPL, and other methods of protein semisynthesis, to generate challenging modified proteins such as antibody-drug conjugates (ADCs). ADCs targeting highly cytotoxic molecules to cancer cells, offer an attractive strategy to selectively eliminate tumor cells with improved therapeutic index than the antibodies or cytotoxic molecules themselves. Despite the potential of ADCs, the development of such complex molecules is challenging. We provide here protocols to prepare site-specifically modified ADCs by streamlined expressed protein ligation (SEPL), which does not require the incorporation of unnatural modifications into the antibody. Therefore, fully native antibodies, with only the desired cytotoxic molecules attached, can be generated.
Assuntos
Antineoplásicos Imunológicos/química , Clonagem Molecular/métodos , Citotoxinas/química , Imunoconjugados/química , Imunoglobulina G/química , Engenharia de Proteínas/métodos , Aminobenzoatos/síntese química , Aminobenzoatos/química , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida/métodos , Cromatografia de Fase Reversa/métodos , Escherichia coli/genética , Expressão Gênica , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunoconjugados/isolamento & purificação , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , Inteínas , Oligopeptídeos/síntese química , Oligopeptídeos/química , Plasmídeos/genética , Processamento de Proteína , Receptor ErbB-2/imunologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
Antibodies bearing engineered cysteine residues (termed THIOMAB™ antibodies) enable the site-selective attachment of a drug, label or other payload for specific delivery to certain tissues (e.g., tumors). This Chapter describes detailed methods we have developed and optimized for the conjugation, purification and analysis of THIOMAB™ antibody drug conjugates (TDCs).
Assuntos
Anticorpos Monoclonais/química , Cisteína/química , Desenvolvimento de Medicamentos , Imunoconjugados/química , Bioengenharia , Cromatografia , Engenharia Genética , Humanos , Imunoconjugados/isolamento & purificação , Imunoconjugados/farmacologiaRESUMO
Interchain disulfide bonds of antibodies can be reduced by agents such as TCEP or DTT to form reactive cysteine residues. These endogenous cysteines are used for conjugation to biologically active drugs either directly or via linkers to prepare antibody drug conjugates (ADCs). The anti-notch 3 ADC described here is being evaluated in the early clinical development program as a potential treatment for a variety of cancers. The ADC is composed of an IgG1 mAb that is conjugated by endogenous cysteines to a cytotoxic microtubulin inhibitor via a maleimide-containing linker. The endogenous cysteine residues are produced by partial reduction of the mAb with TCEP reducing agent. The conjugation results in the formation of a mixture of 2, 4, 6, and 8 loaded ADC species. In addition to the desired product, several product-related impurities such as aggregates are generated during the conjugation reaction. The product- and process-related impurities are separated from the monomeric ADC by column chromatography and ultrafiltration-diafiltration techniques. The temperature of TCEP reduction step has an impact on the level of aggregates produced in the reaction. The temperature also impacts the isomeric composition of the 4 loaded ADC species.
Assuntos
Anticorpos Monoclonais/química , Cisteína/química , Desenvolvimento de Medicamentos , Imunoconjugados/química , Imunoconjugados/farmacologia , Anticorpos Monoclonais/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Biomarcadores , Cromatografia Líquida de Alta Pressão , Humanos , Imunoconjugados/isolamento & purificação , Relação Estrutura-AtividadeRESUMO
For therapeutic and diagnostic applications, site-specific antibody conjugates have proven superior for both the ease of characterization as well as for optimal biophysical and therapeutic properties. Screening multiple antibodies on multiple sites with multiple linker-drugs can become very tedious and time-consuming. As solid-phase reactions are best suited to simplify multistep reactions, readily available protein A/L agarose beads can be utilized to generate site-specific, antibody -drug conjugates on engineered cysteines. Multiple site-specific labels on an antibody with either fluorophore or other-linker drugs is highly desired to evaluate antibody trafficking or payload-synergy for therapeutics. Utilizing solid-phase conjugation, a simple method to generate dual-labeled, site-specific antibody and Fab conjugates from antibody with engineered cysteine is also been described.
Assuntos
Aminoácidos/química , Anticorpos Monoclonais/química , Desenvolvimento de Medicamentos , Ensaios de Triagem em Larga Escala , Imunoconjugados/química , Fragmentos Fab das Imunoglobulinas/química , Coloração e Rotulagem , Desenvolvimento de Medicamentos/métodos , Humanos , Imunoconjugados/isolamento & purificação , Imunoconjugados/farmacologia , Coloração e Rotulagem/métodosRESUMO
Preparation of antibody-drug conjugates (ADCs) with a highly homogeneous drug loading in general requires site-selective conjugation of a cytotoxic payload. Typically, functionality utilized for attachment of the payload is achieved through engineering of suitable chemical handles or by enzymatic modification of the antibody. Relatively few methods to produce ADCs with homogeneous drug loading via endogenous amino acid conjugation have been developed. Herein we describe a robust method for the conjugation of antibodies using a cysteine rebridging approach to produce ADCs with highly homogeneous drug-to-antibody ratios (DAR) at the native interchain disulfides, called ThioBridge®. The process described relies upon an elegant cascade of addition-elimination reactions carried out under mild aqueous conditions that can be readily applied to wild-type antibodies without the need for prior modification via recombinant or enzymatic means. Using this method, conversions to a conserved DAR ADC are typically in the range of 70-95% and overall process yields of >70% are readily achieved.
Assuntos
Anticorpos Monoclonais/química , Cisteína/química , Desenvolvimento de Medicamentos , Imunoconjugados/química , Anticorpos Monoclonais Humanizados/química , Cromatografia , Dissulfetos/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunoconjugados/isolamento & purificação , Imunoconjugados/farmacologia , Relação Estrutura-AtividadeRESUMO
From a structural point of view, the complete characterization of ADCs is a challenging task due to their high complexity. ADCs combine the heterogeneity of the initial antibody to the variability associated with the conjugation strategy, the manufacturing process, and the storage. Given the inherent complexity of these biomolecules, online comprehensive two-dimensional liquid chromatography (LC × LC) is an attractive technique to address the challenges associated with ADC characterization. Compared to conventional one-dimensional liquid chromatography techniques (1D-LC), LC × LC combines two different and complementary separation systems. In the context of ADC analysis, LC × LC has been proven to be a rapid and efficient analytical tool: (1) to provide a higher resolving power by increasing the overall peak capacity and thus allowing to gain more information within a single run and (2) to allow mass spectrometry (MS) coupling with some chromatographic techniques that are not MS-compatible and hence to facilitate the structural elucidation of ADCs. In this chapter, we present the coupling of different chromatographic techniques including hydrophobic interaction chromatography (HIC), reversed phase liquid chromatography (RPLC), size exclusion chromatography (SEC), ion exchange chromatography (IEX), and hydrophilic liquid chromatography (HILIC). The interest of HIC × SEC, SEC × SEC, HIC × RPLC, IEX × RPLC, RPLC × RPLC, and HILIC × RPLC, all hyphenated to high-resolution mass spectrometry (HRMS), is discussed in the context of the characterization of ADCs.
Assuntos
Cromatografia Líquida , Imunoconjugados/análise , Imunoconjugados/química , Espectrometria de Massas , Aminoácidos/química , Anticorpos Monoclonais/química , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida/métodos , Interações Hidrofóbicas e Hidrofílicas , Imunoconjugados/isolamento & purificação , Espectrometria de Massas/métodosRESUMO
Affinity agents coupled to a functional moiety play an ever-increasing role in modern medicine, ranging from radiolabeled selective binders in diagnosis to antibody-drug conjugates in targeted therapies. In biomedical research, protein coupling to fluorophores, surfaces and nanoparticles has become an integral part of many procedures. In addition to antibodies, small scaffold proteins with similar target binding properties are being widely explored as alternative targeting moieties. To label these binders of interest with different functional moieties, conventional chemical coupling methods can be employed, but often result in heterogeneously modified protein products. In contrast, enzymatic labeling methods are highly site-specific and efficient. Protein farnesyltransferase (PFTase) catalyzes the transfer of an isoprenoid moiety from farnesyl diphosphate (FPP) to a cysteine residue in a C-terminal CaaX motif at the C-terminus of a protein substrate. The addition of only four amino acid residues minimizes the influence on the native protein structure. In addition, a variety of isoprenoid analogs containing different bioorthogonal functional groups, including azides, alkynes, and aldehydes, have been developed to enable conjugation to various cargos after being incorporated onto the target protein by PFTase. In this protocol, we present a detailed procedure for labeling Designed Ankyrin Repeat Proteins (DARPins) engineered with a C-terminal CVIA sequence using an azide-containing FPP analog by yeast PFTase (yPFTase). In addition, procedures to subsequently conjugate the labeled DARPins to a TAMRA fluorophore using strained-promoted alkyne-azide cycloaddition (SPAAC) reactions as well as the sample preparation to evaluate the target binding ability of the conjugates by flow cytometry are described.
Assuntos
Alquil e Aril Transferases/química , Pesquisa Biomédica/métodos , Coloração e Rotulagem/métodos , Alquil e Aril Transferases/genética , Alcinos/química , Repetição de Anquirina/genética , Azidas/química , Catálise , Imunoconjugados/química , Imunoconjugados/isolamento & purificação , Nanopartículas/química , Prenilação de Proteína , Rodaminas/química , Especificidade por SubstratoRESUMO
Antibody-drug conjugates (ADCs) are highly potent targeted anticancer therapies. They rely on the linking of a selectively targeting antibody moiety with potent cytotoxic payloads to effect antitumoral activity. In recent years, one focus in the ADC field was to create novel methods for site-specifically conjugating payloads to antibodies. The method presented here is based on the S. aureus sortase A-mediated transpeptidation reaction. This method requires antibodies to be engineered in such a way that they possess the sortase recognition pentapeptide motif LPETG on the C-terminus of the immunoglobulin heavy and/or light chains. In addition, the toxin must contain an oligoglycine motif in order to make it a suitable substrate for sortase A. Here we describe a detailed method to conjugate a pentaglycine-modified toxin to the C-termini of LPETG-tagged antibody heavy and light chains using sortase-mediated antibody conjugation (SMAC-Technology™). Highly homogenous, site-specifically conjugated ADCs with controlled drug to antibody ratio and improved overall properties can be obtained with this method.
Assuntos
Aminoaciltransferases/química , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Proteínas de Bactérias/química , Cisteína Endopeptidases/química , Imunoconjugados/química , Imunoconjugados/farmacologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Composição de Medicamentos , Humanos , Imunoconjugados/imunologia , Imunoconjugados/isolamento & purificação , Estrutura MolecularRESUMO
Antibody-drug conjugates (ADCs) are a relatively young class of cancer therapeutics that combine the superior selectivity of monoclonal antibodies (mAbs) with the high potency of cytotoxic agents. In the first generation of ADCs, the toxic payload is attached to the mAb via chemical conjugation to endogenous lysine or cysteine residues providing only limited control over site specificity and drug-to-antibody ratio (DAR). The resulting product is a heterogeneous population of different ADC species, each with individual characteristics concerning pharmacokinetics, toxicology, and efficacy. Such diverse ADC mixtures are not only difficult to develop but are potentially also accompanied by a suboptimal therapeutic window. To overcome these limitations, alternative conjugation technologies have been developed that allow the production of tailor-made homogeneous ADCs. Due to its high specificity and robust applicability, microbial transglutaminase (mTG), a protein-glutamine γ-glutamyltransferase isolated from Streptomyces mobaraensis, emerged as a versatile tool for ADC manufacturing. Herein, we report a protocol for the site-specific, mTG-mediated modification of antibodies that allows the production of homogeneous and defined ADCs. Moreover, analytical methods for ADC characterization are provided.
Assuntos
Imunoconjugados/química , Transglutaminases/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Catálise , Cromatografia , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunoconjugados/isolamento & purificação , Estrutura Molecular , Mutagênese Sítio-Dirigida , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Immunoconjugates are essential tools in diagnostics for the detection and quantification of proteins and in cell biology for the characterization of different cell populations as well as for tracking intracellular pathways. In recent years, antibody-drug conjugates (ADCs) have emerged as promising therapeutics to treat cancer and have moved into the focus of interest of the pharmaceutical industry. Here we describe a conjugation method for the generation of antibody conjugates that relies on the formation of a spontaneous isopeptide bond between two peptide tags referred to as SpyTag and KTag. This reaction is catalyzed by SpyLigase, an engineered cell surface protein obtained from Streptococcus pyogenes. We describe the preparation of SpyLigase by expression from E. coli cells, chemical solid-phase synthesis of the KTag peptide and its coupling to reporter molecules and cytotoxins as well as the transient expression from mammalian cells to produce Spy-tagged antibodies. Furthermore, we describe the purification and analytics of the formed conjugates.
Assuntos
Anticorpos/química , Ligases/química , Sequência de Aminoácidos , Animais , Anticorpos/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cromatografia de Afinidade , Humanos , Imunoconjugados/química , Imunoconjugados/isolamento & purificação , Espectrometria de Massas , Peptídeos/síntese química , Peptídeos/química , Engenharia de Proteínas , Proteínas Recombinantes/química , Relação Estrutura-AtividadeRESUMO
An Antibody-Drug Conjugate (armed antibody) is a vectorized chemotherapy that results from the grafting of a cytotoxic agent on a monoclonal antibody via a judiciously designed spacer arm. ADCs have made considerable progress in 10 years. In 2009, only gemtuzumab ozogamicin (Mylotarg®) was used clinically. In 2019, 4 other ADCs have been approved and more than 80 others are in active clinical trials. The second part of this review will focus on new emerging strategies to address ADCs drawbacks and attempt to broaden their therapeutic window. Finally, combinations with conventional chemotherapy or checkpoint inhibitors will be discussed, in the pursuit to make Antibody-Drug Conjugates the embodiment of Paul Ehrlich's dream of the magic bullet.
TITLE: Les immunoconjugués en oncologie - Les nouvelles stratégies en développement. ABSTRACT: Un anticorps armé (ADC, antibody-drug conjugate en anglais) est une chimiothérapie vectorisée qui résulte du greffage d'un agent cytotoxique sur un anticorps monoclonal par l'intermédiaire d'un bras espaceur judicieusement construit. Les anticorps armés ont fait des progrès considérables en 10 ans. En 2009, seul le gemtuzumab ozogamicine (Mylotarg®) était utilisé en clinique. En 2019, 4 autres ADC ont été approuvés par la Food and Drug Administration et plus de 80 autres sont en études cliniques actives. La seconde partie de cette revue sera focalisée sur les nouvelles stratégies émergentes pour faire face aux limitations des ADC actuels et pour tenter d'élargir leur fenêtre thérapeutique. Enfin, les combinaisons avec la chimiothérapie classique ou les inhibiteurs de points de contrôles seront discutées, pour tenter de faire des anticorps armés la magic bullet dont rêvait Paul Ehrlich.
Assuntos
Desenvolvimento de Medicamentos/métodos , Imunoconjugados , Oncologia , Neoplasias/tratamento farmacológico , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos Imunológicos/classificação , Antineoplásicos Imunológicos/isolamento & purificação , Antineoplásicos Imunológicos/uso terapêutico , Desenvolvimento de Medicamentos/tendências , História do Século XXI , Humanos , Imunoconjugados/classificação , Imunoconjugados/isolamento & purificação , Imunoconjugados/uso terapêutico , Oncologia/métodos , Oncologia/tendências , Neoplasias/patologiaRESUMO
There is considerable interest in the development of chemical methods for the precise, site-selective modification of antibodies for therapeutic applications. In this protocol, we describe a strategy for the irreversible and selective modification of cysteine residues on antibodies, using functionalized carbonylacrylic reagents. This protocol is based on a thiol-Michael-type addition of native or engineered cysteine residues to carbonylacrylic reagents equipped with functional compounds such as cytotoxic drugs. This approach is a robust alternative to the conventional maleimide technique; the reaction is irreversible and uses synthetically accessible reagents. Complete conversion to the conjugates, with improved quality and homogeneity, is often achieved using a minimal excess (typically between 5 and 10 equiv.) of the carbonylacrylic reagent. Potential applications of this method cover a broad scope of cysteine-tagged antibodies in various formats (full-length IgGs, nanobodies) for the site-selective incorporation of cytotoxic drugs without loss of antigen-binding affinity. Both the synthesis of the carbonylacrylic reagent armed with a synthetic molecule of interest and the subsequent preparation of the chemically defined, homogeneous antibody conjugate can be achieved within 48 h and can be easily performed by nonspecialists. Importantly, the conjugates formed are stable in human plasma. The use of liquid chromatography-mass spectrometry (LC-MS) analysis is recommended for monitoring the progression of the bioconjugation reactions on protein and antibody substrates with accurate resolution.
Assuntos
Acrilatos/química , Métodos Analíticos de Preparação de Amostras , Cisteína/química , Imunoconjugados/química , Imunoglobulinas/química , Anticorpos de Domínio Único/química , Cromatografia Líquida , Reagentes de Ligações Cruzadas/química , Humanos , Imunoconjugados/isolamento & purificação , Imunoglobulinas/isolamento & purificação , Maleimidas/química , Anticorpos de Domínio Único/isolamento & purificação , Espectrometria de Massas em TandemRESUMO
The recombinant murine IgG2a antibody TA99, directed against a melanoma antigen, was used to study combination modalities that potentiate antibody-dependent cell cytotoxicity. As previously reported, IgG2a(TA99) was extremely efficacious in preventing the growth of B16 lung metastases. However, the same antibody mediated only minimal tumor growth retardation when used to treat established neoplastic masses. The therapeutic activity of IgG2a(TA99) could be substantially enhanced by co-administration with an antibody-cytokine fusion (TA99-murine tumor necrosis factor [mTNF]), consisting of the TA99 antibody in single-chain variable fragment format fused to murine TNF. This fusion protein efficiently killed endothelial cells in vitro and displayed only minimal activity against B16 melanoma cells. In vivo, TA99-mTNF boosted the influx of natural killer cells and macrophages into B16 melanoma lesions. Therapy studies with two different administration schedules showed that the combination of TA99-mTNF and IgG2a(TA99) was superior to the individual products used as single agents. The combination treatment converted most of the tumor mass into a necrotic lesion, but a vital tumor rim eventually regrew, even when dacarbazine was included in the therapeutic regimen. The treatment modality described in this article may be applicable to the treatment of melanoma patients, given the specificity of the gp75 antigen and its conservation across species.
Assuntos
Antígenos de Neoplasias/imunologia , Imunoconjugados/administração & dosagem , Melanoma Experimental/tratamento farmacológico , Glicoproteínas de Membrana/imunologia , Oxirredutases/imunologia , Neoplasias Cutâneas/tratamento farmacológico , Animais , Anticorpos Monoclonais Murinos/administração & dosagem , Anticorpos Monoclonais Murinos/genética , Anticorpos Monoclonais Murinos/isolamento & purificação , Células CHO , Linhagem Celular Tumoral/transplante , Cricetulus , Esquema de Medicação , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Imunoconjugados/genética , Imunoconjugados/isolamento & purificação , Imunoglobulina G/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Fator de Necrose Tumoral alfa/administração & dosagem , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/isolamento & purificaçãoRESUMO
Receptor tyrosine kinase-like orphan receptor 2 (ROR2) has been identified as a highly relevant tumor-associated antigen in a variety of cancer indications of high unmet medical need, including renal cell carcinoma and osteosarcoma, making it an attractive target for targeted cancer therapy. Here, we describe the de novo discovery of fully human ROR2-specific antibodies and potent antibody drug conjugates (ADCs) derived thereof by combining antibody discovery from immune libraries of human immunoglobulin transgenic animals using the Transpo-mAb mammalian cell-based IgG display platform with functional screening for internalizing antibodies using a secondary ADC assay. The discovery strategy entailed immunization of transgenic mice with the cancer antigen ROR2, harboring transgenic IgH and IgL chain gene loci with limited number of fully human V, D, and J gene segments. This was followed by recovering antibody repertoires from the immunized animals, expressing and screening them as full-length human IgG libraries by transposon-mediated display in progenitor B lymphocytes ("Transpo-mAb Display") for ROR2 binding. Individual cellular "Transpo-mAb" clones isolated by single cell sorting and capable of expressing membrane-bound as well as secreted human IgG were directly screened during antibody discovery, not only for high affinity binding to human ROR2, but also functionally as ADCs using a cytotoxicity assay with a secondary anti-human IgG-toxin-conjugate. Using this strategy, we identified and validated 12 fully human, monoclonal anti-human ROR2 antibodies with nanomolar affinities that are highly potent as ADCs and could be promising candidates for the therapy of human cancer. The screening for functional and internalizing antibodies during the early phase of antibody discovery demonstrates the utility of the mammalian cell-based Transpo-mAb Display platform to select for functional binders and as a powerful tool to improve the efficiency for the development of therapeutically relevant ADCs.
Assuntos
Anticorpos Monoclonais Humanizados/isolamento & purificação , Anticorpos Monoclonais/isolamento & purificação , Imunoconjugados/isolamento & purificação , Neoplasias/terapia , Células Precursoras de Linfócitos B/fisiologia , Animais , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Humanos , Imunização , Imunoconjugados/farmacologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Imunotoxinas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias/imunologia , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/imunologia , Análise de Célula Única , Éxons VDJ/genéticaRESUMO
Antibody-drug conjugates (ADCs) form a rapidly growing class of biopharmaceuticals which attracts a lot of attention throughout the industry due to its high potential for cancer therapy. They combine the specificity of a monoclonal antibody (mAb) and the cell-killing capacity of highly cytotoxic small molecule drugs. Site-specific conjugation approaches involve a multi-step process for covalent linkage of antibody and drug via a linker. Despite the range of parameters that have to be investigated, high-throughput methods are scarcely used so far in ADC development. In this work an automated high-throughput platform for a site-specific multi-step conjugation process on a liquid-handling station is presented by use of a model conjugation system. A high-throughput solid-phase buffer exchange was successfully incorporated for reagent removal by utilization of a batch cation exchange step. To ensure accurate screening of conjugation parameters, an intermediate UV/Vis-based concentration determination was established including feedback to the process. For conjugate characterization, a high-throughput compatible reversed-phase chromatography method with a runtime of 7â¯min and no sample preparation was developed. Two case studies illustrate the efficient use for mapping the operating space of a conjugation process. Due to the degree of automation and parallelization, the platform is capable of significantly reducing process development efforts and material demands and shorten development timelines for antibody-drug conjugates.