RESUMO
Targeted protein degradation is an innovative therapeutic strategy to selectively eliminate disease-causing proteins. Exemplified by proteolysis-targeting chimeras (PROTACs), they have shown promise in overcoming drug resistance and targeting previously undruggable proteins. However, PROTACs face challenges, such as low oral bioavailability and limited selectivity. The recently published PROxAb Shuttle technology offers a solution enabling the targeted delivery of PROTACs using antibodies fused with PROTAC-binding domains derived from camelid single-domain antibodies (VHHs). Here, a modular approach to quickly generate PROxAb Shuttles by enzymatically coupling PROTAC-binding VHHs to off-the-shelf antibodies was developed. The resulting conjugates retained their target binding and internalization properties, and incubation with BRD4-targeting PROTACs resulted in formation of defined PROxAb-PROTAC complexes. These complexes selectively induced degradation of the BRD4 protein, resulting in cytotoxicity specifically to cells expressing the antibody's target. The chemoenzymatic approach described herein provides a versatile and efficient solution for generating antibody-VHH conjugates for targeted protein degradation applications, but it could also be used to combine antibodies and VHH binders to generate bispecific antibodies for further applications.
Assuntos
Anticorpos Biespecíficos , Proteólise , Humanos , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/imunologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/imunologia , Proteínas de Ciclo Celular/imunologia , Proteínas de Ciclo Celular/metabolismo , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Proteínas que Contêm BromodomínioRESUMO
The toolbox of modern antibody engineering allows the design of versatile novel functionalities exceeding nature's repertoire. Many bispecific antibodies comprise heterodimeric Fc portions recently validated through the approval of several bispecific biotherapeutics. While heterodimerization methodologies have been established for low-throughput large-scale production, few approaches exist to overcome the bottleneck of large combinatorial screening efforts that are essential for the identification of the best possible bispecific antibody. This report presents a novel, robust and miniaturized heterodimerization process based on controlled Fab-arm exchange (cFAE), which is applicable to a variety of heterodimeric formats and compatible with automated high-throughput screens. Proof of applicability was shown for two therapeutic molecule classes and two relevant functional screening read-outs. First, the miniaturized production of biparatopic anti-c-MET antibody-drug conjugates served as a proof of concept for their applicability in cytotoxic screenings on tumor cells with different target expression levels. Second, the automated workflow enabled a large unbiased combinatorial screening of biparatopic antibodies and the identification of hits mediating potent c-MET degradation. The presented workflow utilizes standard equipment and may serve as a facile, efficient and robust method for the discovery of innovative therapeutic agents in many laboratories worldwide.
Assuntos
Anticorpos Biespecíficos , Imunoconjugados , Anticorpos Biespecíficos/uso terapêutico , Imunoconjugados/farmacologiaRESUMO
The exposition of cancer cells to cytotoxic doses of payload is fundamental for the therapeutic efficacy of antibody drug conjugates (ADCs) in solid cancers. To maximize payload exposure, tissue penetration can be increased by utilizing smaller-sized drug conjugates which distribute deeper into the tumor. Our group recently explored small human epidermal growth factor receptor 2 (HER2) targeting Fc antigen binding fragments (Fcabs) for ADC applications in a feasibility study. Here, we expand this concept using epidermal growth factor receptor (EGFR) targeting Fcabs for the generation of site-specific auristatin-based drug conjugates. In contrast to HER2-targeting Fcabs, we identified novel conjugation sites in the EGFR-targeting Fcab scaffold that allowed for higher DAR enzymatic conjugation. We demonstrate feasibility of resultant EGFR-targeting Fcab-drug conjugates that retain binding to half-life prolonging neonatal Fc receptor (FcRn) and EGFR and show high serum stability as well as target receptor mediated cell killing at sub-nanomolar concentrations. Our results emphasize the applicability of the Fcab format for the generation of drug conjugates designed for increased penetration of solid tumors and potential FcRn-driven antibody-like pharmacokinetics.
Assuntos
Antineoplásicos , Imunoconjugados , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Humanos , Imunoconjugados/química , Imunoconjugados/metabolismo , Imunoconjugados/farmacologia , Recém-Nascido , Ligação ProteicaRESUMO
BACKGROUND: Expansion of the genetic code is a frequently employed approach for the modification of recombinant protein properties. It involves reassignment of a codon to another, e.g., unnatural, amino acid and requires the action of a pair of orthogonal tRNA and aminoacyl tRNA synthetase modified to recognize only the desired amino acid. This approach was applied for the production of trastuzumab IgG carrying p-azido-L-phenylalanine (pAzF) in the industrial yeast Pichia pastoris. Combining the knowledge of protein folding and secretion with bioreactor cultivations, the aim of the work was to make the production of monoclonal antibodies with an expanded genetic code cost-effective on a laboratory scale. RESULTS: Co-translational transport of proteins into the endoplasmic reticulum through secretion signal prepeptide change and overexpression of lumenal chaperones Kar2p and Lhs1p improved the production of trastuzumab IgG and its Fab fragment with incorporated pAzF. In the case of Fab, a knockout of vacuolar targeting for protein degradation further increased protein yield. Fed-batch bioreactor cultivations of engineered P. pastoris strains increased IgG and IgGpAzF productivity by around 50- and 20-fold compared to screenings, yielding up to 238 mg L-1 and 15 mg L-1 of fully assembled tetrameric protein, respectively. Successful site-specific incorporation of pAzF was confirmed by mass spectrometry. CONCLUSIONS: Pichia pastoris was successfully employed for cost-effective laboratory-scale production of a monoclonal antibody with an unnatural amino acid. Applying the results of this work in glycoengineered strains, and taking further steps in process development opens great possibilities for utilizing P. pastoris in the development of antibodies for subsequent conjugations with, e.g., bioactive payloads.
Assuntos
Aminoácidos , Pichia , Aminoácidos/metabolismo , Anticorpos Monoclonais/metabolismo , Formação de Anticorpos , Imunoglobulina G , Pichia/metabolismo , Proteínas Recombinantes , Saccharomyces cerevisiae/metabolismo , Saccharomycetales , Trastuzumab/metabolismoRESUMO
Site-specific bioconjugation technologies are frequently employed to generate homogeneous antibody-drug conjugates (ADCs) and are generally considered superior to stochastic approaches like lysine coupling. However, most of the technologies developed so far require undesired manipulation of the antibody sequence or its glycan structures. Herein, we report the successful engineering of microbial transglutaminase enabling efficient, site-specific conjugation of drug-linker constructs to position HC-Q295 of native, fully glycosylated IgG-type antibodies. ADCs generated via this approach demonstrate excellent stability in vitro as well as strong efficacy in vitro and in vivo. As it employs different drug-linker structures and several native antibodies, our study additionally proves the broad applicability of this approach.
Assuntos
Imunoconjugados/metabolismo , Engenharia de Proteínas , Transglutaminases/genética , Transglutaminases/metabolismo , Sítios de Ligação , Streptomyces/enzimologia , Transglutaminases/químicaRESUMO
Multivalent ligands of death receptors hold particular promise as tumor cell-specific therapeutic agents because they induce an apoptotic cascade in cancerous cells. Herein, we present a modular approach to generate death receptorâ 5 (DR5) binding constructs comprising multiple copies of DR5 targeting peptide (DR5TP) covalently bound to biomolecular scaffolds of peptidic nature. This strategy allows for efficient oligomerization of synthetic DR5TP-derived peptides in different spatial orientations using a set of enzyme-promoted conjugations or recombinant production. Heptameric constructs based on a short (60-75 residues) scaffold of a C-terminal oligomerization domain of human C4b binding protein showed remarkable proapoptotic activity (EC50=3â nm) when DR5TP was ligated to its carboxy terminus. Our data support the notion that inter-ligand distance, relative spatial orientation and copy number of receptor-binding modules are key prerequisites for receptor activation and cell killing.
Assuntos
Apoptose , Peptídeos/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , HumanosRESUMO
Cystine-knot peptides sharing a common fold but displaying a notably large diversity within the primary structure of flanking loops have shown great potential as scaffolds for the development of therapeutic and diagnostic agents. In this study, we demonstrated that the cystine-knot peptide MCoTI-II, a trypsin inhibitor from Momordica cochinchinensis, can be engineered to bind to cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), an inhibitory receptor expressed by T lymphocytes, that has emerged as a target for the treatment of metastatic melanoma. Directed evolution was used to convert a cystine-knot trypsin inhibitor into a CTLA-4 binder by screening a library of variants using yeast surface display. A set of cystine-knot peptides possessing dissociation constants in the micromolar range was obtained; the most potent variant was synthesized chemically. Successive conjugation with neutravidin, fusion to antibody Fc domain or the oligomerization domain of C4b binding protein resulted in oligovalent variants that possessed enhanced (up to 400-fold) dissociation constants in the nanomolar range. Our data indicate that display of multiple knottin peptides on an oligomeric scaffold protein is a valid strategy to improve their functional affinity with ramifications for applications in diagnostics and therapy.
Assuntos
Antígeno CTLA-4/metabolismo , Ciclotídeos/genética , Ciclotídeos/farmacologia , Avidina/metabolismo , Antígeno CTLA-4/química , Ciclotídeos/química , Humanos , Modelos Moleculares , Biblioteca de Peptídeos , Ligação Proteica , Engenharia de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
Based on the crystal structure of a natural protein substrate for microbial transglutaminase, an enzyme that catalyzes protein crosslinking, a recognition motif for site-specific conjugation was rationally designed. Conformationally locked by an intramolecular disulfide bond, this structural mimic of a native conjugation site ensured efficient conjugation of a reporter cargo to the therapeutic monoclonal antibody cetuximab without erosion of its binding properties.
Assuntos
Cetuximab/química , Transglutaminases/química , Animais , Células CHO , Linhagem Celular Tumoral , Cetuximab/metabolismo , Cricetulus , Dissulfetos/química , Dissulfetos/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , Transglutaminases/metabolismoRESUMO
Antibody-drug conjugates (ADCs) are a rapidly expanding class of anticancer therapeutics, with 14 ADCs already approved worldwide. We developed unique linker technologies for the bioconjugation of drug molecules with controlled-release applications. We synthesized cathepsin-cleavable ADCs using a dimeric prodrug system based on a self-immolative dendritic scaffold, resulting in a high drug-antibody ratio (DAR) with the potential to reach 16 payloads due to its dendritic structure, increased stability in the circulation and efficient release profile of a highly cytotoxic payload at the targeted site. Using our novel cleavable linker technologies, we conjugated the anti-human epidermal growth factor receptor 2 (anti-HER2) antibody, trastuzumab, with topoisomerase I inhibitors, exatecan or belotecan. The newly synthesized ADCs were tested in vitro on mammary carcinoma cells overexpressing human HER2, demonstrating a substantial inhibitory effect on the proliferation of HER2-positive cells. Importantly, a single dose of our trastuzumab-based ADCs administered in vivo to mice bearing HER2-positive tumors, showed a dose-dependent inhibition of tumor growth and survival benefit, with the most potent antitumor effects observed at 10 mg/kg, which resulted in complete tumor regression and survival of 100% of the mice. Overall, our novel dendritic technologies using the protease-cleavable Val-Cit linker present an opportunity for the development of highly selective and potent controlled-released therapeutic payloads. This strategy could potentially lead to the development of novel and effective ADC technologies for patients diagnosed with HER2-positive cancers. Moreover, our proposed ADC linker technology can be implemented in additional medical conditions such as other malignancies as well as autoimmune diseases that overexpress targets, other than HER2.
Assuntos
Antineoplásicos , Imunoconjugados , Humanos , Camundongos , Animais , Inibidores da Topoisomerase I/uso terapêutico , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/farmacologia , Linhagem Celular Tumoral , Trastuzumab/química , Antineoplásicos/química , Receptor ErbB-2/metabolismo , Imunoconjugados/uso terapêutico , Imunoconjugados/químicaRESUMO
BACKGROUND: Site-specific coupling of toxin entities to antibodies has become a popular method of synthesis of antibody-drug conjugates (ADCs), as it leads to a homogenous product and allows a free choice of a convenient site for conjugation. METHODS: We introduced a short motif, containing a single cysteine surrounded by aromatic residues, into the N-terminal FG-loop of the CH2 domain of two model antibodies, cetuximab and trastuzumab. The extent of conjugation with toxic payload was examined with hydrophobic interaction chromatography and mass spectrometry and the activity of resulting conjugates was tested on antigen-overexpressing cell lines. RESULTS: Antibody mutants were amenable for rapid coupling with maleimide-based linker endowed toxin payload and the modifications did not impair their reactivity with target cell lines or negatively impact their biophysical properties. Without any previous reduction, up to 50% of the antibody preparation was found to be coupled with two toxins per molecule. After the isolation of this fraction with preparative hydrophobic interaction chromatography, the ADC could elicit a potent cytotoxic effect on the target cell lines. CONCLUSION: By fine-tuning the microenvironment of the reactive cysteine residue, this strategy offers a simplified protocol for production of site-selectively coupled ADCs. GENERAL SIGNIFICANCE: Our unique approach allows the generation of therapeutic ADCs with controlled chemical composition, which facilitates the optimization of their pharmacological activity. This strategy for directional coupling could in the future simplify the construction of ADCs with double payloads ("dual warheads") introduced with orthogonal techniques.
Assuntos
Antineoplásicos , Imunoconjugados , Antineoplásicos/farmacologia , Cisteína/química , Imunoconjugados/química , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Espectrometria de Massas , Trastuzumab/farmacologiaRESUMO
Appropriate selection of conjugation sites and conjugation technologies is now widely accepted as crucial for the success of antibody-drug conjugates (ADCs). Herein, we present ADCs conjugated by different conjugation methods to different conjugation positions being systematically characterized by multiple in vitro assays as well as in vivo pharmacokinetic (PK) analyses in transgenic Tg276 mice. Conjugation to cysteines, genetically introduced at positions N325, L328, S239, D265, and S442, was compared to enzymatic conjugation via microbial transglutaminase (mTG) either to C-terminal light (LC) or heavy chain (HC) recognition motifs or to endogenous position Q295 of a native antibody. All conjugations yielded homogeneous DAR 2 ADCs with similar hydrophobicity, thermal stability, human neonatal Fc receptor (huFcRn) binding, and serum stability properties, but with pronounced differences in their PK profiles. mTG-conjugated ADC variants conjugated either to Q295 or to LC recognition motifs showed superior PK behavior. Within the panel of engineered cysteine variants L328 showed a similar PK profile compared to previously described S239 but superior PK compared to S442, D265, and N325. While all positions were first tested with trastuzumab, L328 and mTG LC were further evaluated with additional antibody scaffolds derived from clinically evaluated monoclonal antibodies (mAb). Based on PK analyses, this study confirms the newly described position L328 as favorable site for cysteine conjugation, comparable to the well-established engineered cysteine position S239, and emphasizes the favorable position Q295 of native antibodies and the tagged LC antibody variant for enzymatic conjugations via mTG. In addition, hemizygous Tg276 mice are evaluated as an adequate model for ADC pharmacokinetics, facilitating the selection of suitable ADC candidates early in the drug discovery process.
Assuntos
Antineoplásicos Imunológicos , Imunoconjugados , Animais , Anticorpos Monoclonais/química , Antineoplásicos Imunológicos/química , Cisteína/química , Imunoconjugados/química , Camundongos , Trastuzumab/químicaRESUMO
Anti-hapten antibodies are widely used as specific immunochemical detection tools in a variety of clinical and environmental analyses. The sensitivity, however, is limited due to the resulting antibody affinities to the haptens which, in turn, leads to a high demand for specific affinity reagents. A well-established path for the generation of high-affinity antibodies is the immunization of animals with the target antigen. However, the generation of anti-hapten antibodies via immunization remains challenging as small molecule haptens usually possess low immunogenicity and, therefore, must be coupled to an immunogenic and high molecular weight carrier to provoke an immune response.Consequently, antibodies are primarily raised against the carrier molecule or structural features of the hapten-linker fused to the carrier protein. This turns the generation of antibodies which bind exclusively to the hapten structure into a search for the needle in a haystack. In the following chapter, we describe how yeast surface display and high-throughput fluorescence-activated cell sorting can be used to isolate anti-hapten antibodies from a large, yeast-displayed B-cell receptor gene library derived from immunized animals. For this, we describe in detail the preparation of protein-hapten conjugates, the immunization procedure, and the subsequent screening process. Moreover, we provide a simple flow cytometry protocol that allows for a rapid analysis of the enriched clones toward free hapten binding.
Assuntos
Anticorpos Monoclonais , Haptenos , Biblioteca de Peptídeos , Receptores de Antígenos de Linfócitos B , Saccharomyces cerevisiae , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Camelídeos Americanos , Galinhas , Haptenos/química , Haptenos/imunologia , Camundongos , Coelhos , Receptores de Antígenos de Linfócitos B/biossíntese , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , OvinosRESUMO
Antibody-drug conjugates (ADCs) have been proven to be a successful therapeutic concept, allowing targeted delivery of highly potent active pharmaceutical ingredients (HPAPIs) selectively to tumor tissue. So far, HPAPIs have been mainly attached to the antibody via a chemical reaction of the payload with lysine or cysteine side chains of the antibody backbone. However, these conventional conjugation technologies result in formation of rather heterogeneous products with undesired properties. To overcome the limitations of heterogeneous ADC mixtures, several site-specific conjugation technologies have been developed over the last years. Originally pioneered by scientist from Genentech with their work on THIOMABs, several engineered cysteine mAb ADCs (ECM-ADCs) are now investigated in clinical trials. Here, we describe in detail how to engineer additional cysteines into antibodies and efficiently use them as highly site-specific conjugation sites for HPAPIs.
Assuntos
Anticorpos Monoclonais Humanizados/genética , Citotoxinas/genética , Imunoconjugados/genética , Engenharia de Proteínas , Animais , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos Imunológicos , Proliferação de Células/efeitos dos fármacos , Cisteína/química , Cisteína/genética , Citotoxinas/química , Citotoxinas/imunologia , Citotoxinas/farmacologia , Humanos , Imunoconjugados/química , Imunoconjugados/imunologia , Imunoconjugados/farmacologia , Camundongos , Mutagênese Sítio-Dirigida , Compostos de Sulfidrila/química , Trastuzumab/química , Trastuzumab/genética , Trastuzumab/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Conventional chemotherapy is associated with therapy-limiting side effects, which might be alleviated by targeted chemotherapeutics such as immunoliposomes. The targeting ligands of immunoliposomes are commonly attached by unspecific chemical conjugation, bearing risk of structural heterogeneity and therewith related biological consequences. Chemoenzymatic methods may mitigate such risks through site-specific conjugation. METHODS: The formulation parameters for pentaglycine-modified, doxorubicin-loaded liposomes and the reaction conditions for a site-specific, Sortase-A mediated conjugation with monoclonal antibodies were thoroughly evaluated. The cytotoxicity of such sortagged, epidermal growth factor receptor (EGFR)-specific immunoliposomes was tested on human breast cancer cells. RESULTS: Sortaggable liposomes with a defined size (140â¯nm, PDIâ¯<â¯0.25) and high encapsulation efficiency (>90%) were obtained after manufacturing optimization. A ratio of 1.0-2.5⯵M mAb/100⯵M pentaglycine yielded stable dispersions and circumvented carrier precipitation during ligand grafting. The cytotoxicity on EGFR+ MDA-MB-468 was up to threefold higher for EGFR-specific immunoliposomes than for the nontargeted controls. CONCLUSIONS: Sortase-A is suitable to generate immunoliposomes with a site-specific ligand-carrier linkage and hence improves chemical homogeneity of targeted therapeutics. However, the sweet spot for manufacturability utilizing mAbs with two Sortase-A recognition sites is narrow, making mono-reactive binders such as scFvs or Fab's preferable for a further development. Despite this, the immunoliposomes demonstrated a targeted delivery of doxorubicin, indicating the potential to increase the therapeutic window during the treatment of EGFR+ tumors.
Assuntos
Aminoaciltransferases/administração & dosagem , Antibióticos Antineoplásicos/administração & dosagem , Proteínas de Bactérias/administração & dosagem , Cisteína Endopeptidases/administração & dosagem , Citotoxinas/administração & dosagem , Doxorrubicina/análogos & derivados , Sistemas de Liberação de Medicamentos/métodos , Aminoaciltransferases/farmacocinética , Antibióticos Antineoplásicos/farmacocinética , Proteínas de Bactérias/farmacocinética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Cisteína Endopeptidases/farmacocinética , Citotoxinas/farmacocinética , Relação Dose-Resposta a Droga , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacocinética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Feminino , Humanos , Lipossomos , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/farmacocinéticaRESUMO
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
Anti-idiotypic binders which specifically recognize the variable region of monoclonal antibodies have proven to be robust tools for pharmacokinetic studies of antibody therapeutics and for the development of cancer vaccines. In the present investigation, we focused on the identification of anti-idiotypic, shark-derived IgNAR antibody variable domains (vNARs) targeting the therapeutic antibodies matuzumab and cetuximab for the purpose of developing specific capturing ligands. Using yeast surface display and semi-synthetic, CDR3-randomized libraries, we identified several highly specific binders targeting both therapeutic antibodies in their corresponding variable region, without applying any counter selections during screening. Importantly, anti-idiotypic vNAR binders were not cross-reactive towards cetuximab or matuzumab, respectively, and comprised good target recognition in the presence of human and mouse serum. When coupled to magnetic beads, anti-idiotypic vNAR variants could be used as efficient capturing tools. Moreover, a two-step procedure involving vNAR-functionalized beads was employed for the enrichment of potentially bispecific cetuximab × matuzumab antibody constructs. In conclusion, semi-synthetic and CDR3-randomized vNAR libraries in combination with yeast display enable the fast and facile identification of anti-idiotypic vNAR domains targeting monoclonal antibodies primarily in an anti-idiotypic manner.
Assuntos
Anticorpos Anti-Idiotípicos/metabolismo , Anticorpos Monoclonais Humanizados/metabolismo , Antineoplásicos Imunológicos/metabolismo , Técnicas de Visualização da Superfície Celular , Cetuximab/metabolismo , Anticorpos de Domínio Único/metabolismo , Anticorpos Anti-Idiotípicos/genética , Anticorpos Monoclonais Humanizados/isolamento & purificação , Antineoplásicos Imunológicos/isolamento & purificação , Cetuximab/isolamento & purificação , Separação Imunomagnética , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Anticorpos de Domínio Único/genéticaRESUMO
Bispecific IgG-like antibodies can simultaneously interact with two epitopes on the same or on different antigens. Therefore, these molecules facilitate novel modes of action, which cannot be addressed by conventional monospecific IgGs. However, the generation of such antibodies still appears to be demanding due to their specific architecture comprising four different polypeptide chains that need to assemble correctly. This review focusses on different strategies to circumvent this issue or to enforce a correct chain association with a focus on common-chain bispecific antibodies.
Assuntos
Anticorpos Biespecíficos/metabolismo , Engenharia de Proteínas/métodos , Animais , Humanos , Cadeias Pesadas de Imunoglobulinas , Cadeias Leves de Imunoglobulina/metabolismo , Multimerização ProteicaRESUMO
Over the past decade, DNA and RNA aptamers have attracted keen research interest due to their ability to specifically bind targets of therapeutic relevance. However, their application is often hampered by a short serum half-life and missing effector functions. Conjugation of aptamers to antibody Fc fragments could improve pharmacokinetics, enable immune effector mechanisms, and provide an option for the introduction of desired payloads (e.g., toxins or fluorescent dyes). We developed a modular scaffold-supported system based on human IgG1 Fc fragments, which allows for its dual functionalization with moieties of interest. In our approach, two bioorthogonal, enzyme-mediated reactions were used in combination with oxime ligation and self-assembly based on PNA-DNA base pairing. Thus, an engineered synthetic peptide nucleic acid (PNA) oligomer was coupled to the C-termini of the Fc dimer upon sequence-specific sortase A-mediated transpeptidation. Hybridization of the resulting Fc-PNA conjugate with a tailored DNA aptamer that binds cancer-related hepatocyte growth factor receptor (c-MET) led to a hybrid construct which showed strong and specific binding to c-MET and was readily internalized by c-MET-overexpressing cells. To install an additional orthogonally addressable site, aldehyde tag technology was applied followed by oxime ligation with an aminooxy-bearing fluorescent dye as model cargo. Delivery of fluorescent probe specifically to c-MET-overexpressing cells was confirmed by flow cytometry. Our approach can provide access to engineered aptamer-Fc conjugates with desired target specificity and cytotoxic payloads.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Sistemas de Liberação de Medicamentos , Imunoconjugados/química , Fragmentos Fc das Imunoglobulinas/química , Ácidos Nucleicos Peptídicos/química , Proteínas Proto-Oncogênicas c-met/metabolismo , Sequência de Aminoácidos , Aptâmeros de Nucleotídeos/química , Linhagem Celular Tumoral , Corantes Fluorescentes/administração & dosagem , Células HEK293 , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismoRESUMO
In this present study, we engineered hypervariable loop 2 (HV2) of the IgNAR variable domain in a way that it solely facilitates antigen binding, potentially functioning as an autonomous paratope. For this, the surface-exposed loop corresponding to HV2 was diversified and antigen-specific variable domain of IgNAR antibody (vNAR) molecules were isolated by library screening using yeast surface display (YSD) as platform technology. An epithelial cell adhesion molecule (EpCAM)-specific vNAR was used as starting material, and nine residues in HV2 were randomized. Target-specific clones comprising a new HV2-mediated paratope were isolated against cluster of differentiation 3ε (CD3ε) and human Fcγ while retaining high affinity for EpCAM. Essentially, we demonstrate that a new paratope comprising moderate affinities against a given target molecule can be engineered into the vNAR scaffold that acts independent of the original antigen-binding site, composed of complementarity-determining region 3 (CDR3) and CDR1.