RESUMO
Hijacking the ubiquitin proteasome system to elicit targeted protein degradation (TPD) has emerged as a promising therapeutic strategy to target and destroy intracellular proteins at the post-translational level. Small molecule-based TPD approaches, such as proteolysis-targeting chimeras (PROTACs) and molecular glues, have shown potential, with several agents currently in clinical trials. Biological PROTACs (bioPROTACs), which are engineered fusion proteins comprised of a target-binding domain and an E3 ubiquitin ligase, have emerged as a complementary approach for TPD. Here, we describe a new method for the evolution and design of bioPROTACs. Specifically, engineered binding scaffolds based on the third fibronectin type III domain of human tenascin-C (Tn3) were installed into the E3 ligase tripartite motif containing-21 (TRIM21) to redirect its degradation specificity. This was achieved via selection of naïve yeast-displayed Tn3 libraries against two different oncogenic proteins associated with B-cell lymphomas, mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) and embryonic ectoderm development protein (EED), and replacing the native substrate-binding domain of TRIM21 with our evolved Tn3 domains. The resulting TRIM21-Tn3 fusion proteins retained the binding properties of the Tn3 as well as the E3 ligase activity of TRIM21. Moreover, we demonstrated that TRIM21-Tn3 fusion proteins efficiently degraded their respective target proteins through the ubiquitin proteasome system in cellular models. We explored the effects of binding domain avidity and E3 ligase utilization to gain insight into the requirements for effective bioPROTAC design. Overall, this study presents a versatile engineering approach that could be used to design and engineer TRIM21-based bioPROTACs against therapeutic targets.
Assuntos
Complexo de Endopeptidases do Proteassoma , Proteínas , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteólise , Ubiquitinação , Ubiquitina/metabolismoRESUMO
Bispecific antibodies combine the specificity of two antibodies into one molecule. During the past two decades, advancement in protein engineering enabled the development of more than 100 bispecific formats, three of which are approved by the FDA for clinical use. In parallel to protein engineering methods, advancement in conjugation chemistries have spurred the use of chemical engineering approaches to generate bispecific antibodies. Herein, we review selected chemical strategies employed to generate bispecific antibodies that cannot be made using protein engineering methods.
Assuntos
Anticorpos BiespecíficosRESUMO
Synthetic gene delivery systems employ multiple functions to enable safe and effective transport of DNA to target cells. Here, we describe metabolite-based poly(l-lysine) (PLL) modifiers that improve transfection by imparting both pH buffering and nanoparticle stabilization functions within a single molecular unit. PLL modifiers were based on morpholine (M), morpholine and niacin (MN), or thiomorpholine (TM). PLL modification with (MN) or (TM) imparted buffering function over the pH range of 5-7 both in solution and live cells and enhanced the stability of PLL DNA nanoparticles, which exhibited higher resistance to polyanion exchange and prolonged blood circulation. These properties translated into increased transfection efficiency in vitro coupled with reduced toxicity compared to unmodified PLL and PLL(M). Furthermore, PEG-PLL(MN) DNA nanoparticles transfected muscle tissue in vivo for >45 days following intramuscular injection. These polymer modifiers demonstrate the successful design of multifunctional units that improve transfection of synthetic gene delivery systems while maintaining biocompatibility.
Assuntos
Técnicas de Transferência de Genes , Polilisina , DNA/genética , Terapia Genética , Polietilenoglicóis , TransfecçãoRESUMO
First-generation cysteine-based site-specific antibody-drug conjugates (ADCs) are limited to one drug per cysteine. However, certain applications require a high drug to antibody ratio (DAR), such as when low-potency payloads are used. Higher drug load can be achieved using classical cysteine conjugation methods, but these result in heterogeneity, suboptimal efficacy and pharmacokinetics. Here, we describe the design, synthesis and validation of heterobifunctional linkers that can be used for the preparation of ADCs with a DAR of two, three and four in a site-specific manner per single cysteine conjugation site, resulting in site-specific ADCs with a DAR of four, six and eight. The designed linkers carry a sulfhydryl-specific iodoacetyl reactive group, and multiple cyclic diene moieties which can efficiently react with maleimide-carrying payloads through the Diels-Alder reaction. As a proof of concept, we synthesized site-specific DAR four, six and eight ADCs carrying tubulysin (AZ13601508) using engineered antibodies with a cysteine inserted after position 239 in the antibody CH2 domain. We evaluated and compared the in vitro cytotoxicity of ADCs obtained via the site-specific platform described herein, with ADCs prepared using classical cysteine conjugation. Our data validated a novel cysteine-based conjugation platform for the preparation of site-specific ADCs with high drug load for therapeutic applications.
Assuntos
Antineoplásicos Imunológicos , Antineoplásicos , Neoplasias da Mama/tratamento farmacológico , Imunoconjugados , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Cisteína/química , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Imunoconjugados/química , Imunoconjugados/farmacologiaRESUMO
Pathways of human epidermal growth factor (EGF) receptors are activated upon ligand-dependent or -independent homo- or heterodimerization and their subsequent transphosphorylation. Overexpression of these receptors positively correlates with transphosphorylation rates and increased tumor growth rates. MEDI4276, an anti-human epidermal growth factor receptor 2 (HER2) biparatopic antibody-drug conjugate, has two paratopes within each antibody arm. One, 39S, is aiming at the HER2 site involved in receptor dimerization and the second, single chain fragment (scFv), mimicking trastuzumab. Here we present the cocrystal structure of the 39S Fab-HER2 complex and, along with biophysical and functional assays, determine the corresponding epitope of MEDI4276 and its underlying mechanism of action. Our results reveal that MEDI4276's uniqueness is based first on the ability of its 39S paratope to block HER2 homo- or heterodimerization and second on its ability to cluster the receptors on the surface of receptor-overexpressing cells.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Neoplasias da Mama/tratamento farmacológico , Multimerização Proteica , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Trastuzumab/farmacologia , Sequência de Aminoácidos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Cristalografia por Raios X , Feminino , Humanos , Modelos Moleculares , Fosforilação , Conformação Proteica , Homologia de Sequência , Células Tumorais CultivadasRESUMO
The normal electron-demand Diels-Alder (DA) cycloaddition is a classic transformation routinely used in synthesis; however, applications in biological systems are limited. Here, we report a spiro[2.4]hepta-4,6-diene-containing noncanonical amino acid (SCpHK) capable of efficient incorporation into antibodies and subsequent coupling with maleimide via a DA reaction. SCpHK was stable throughout protein expression in mammalian cells and enabled covalent attachment of maleimide drug-linkers yielding DA antibody-drug conjugates (DA-ADCs) with nearly quantitative conversion in a one-step process. The uncatalyzed DA reaction between SCpHK and maleimide in aqueous buffer was rapid (1.8-5.4 M-1 s-1), and the antibody-drug adduct was stable in rat serum for at least 1 week at 37 °C. Anti-EphA2 DA-ADCs containing AZ1508 or SG3249 maleimide drug-linkers were potent inhibitors of tumor growth in PC3 tumor models in vivo. The DA bioconjugation strategy described here represents a simple method to produce site-specific and stable ADCs with maleimide drug-linkers.
Assuntos
Imunoconjugados/química , Maleimidas/química , Animais , Células CHO , Sobrevivência Celular/efeitos dos fármacos , Cricetulus , Reação de Cicloadição , Humanos , Imunoconjugados/farmacologia , Modelos Moleculares , Células PC-3 , Conformação Proteica , Compostos de Espiro/químicaRESUMO
Despite some clinical success with antibody-drug conjugates (ADCs) in patients with solid tumors and hematological malignancies, improvements in ADC design are still desirable due to the narrow therapeutic window of these compounds. Tumor-targeting antibody fragments have distinct advantages over monoclonal antibodies, including more rapid tumor accumulation and enhanced penetration, but are subject to rapid clearance. Half-life extension technologies such as PEGylation and albumin-binding domains (ABDs) have been widely used to improve the pharmacokinetics of many different types of biologics. PEGylation improves pharmacokinetics by increasing hydrodynamic size to reduce renal clearance, whereas ABDs extend half-life via FcRn-mediated recycling. In this study, we used an anti-oncofetal antigen 5T4 diabody conjugated with a highly potent cytotoxic pyrrolobenzodiazepine (PBD) warhead to assess and compare the effects of PEGylation and albumin binding on the in vivo efficacy of antibody fragment drug conjugates. Conjugation of 2× PEG20K to a diabody improved half-life from 40 min to 33 h, and an ABD-diabody fusion protein exhibited a half-life of 45 h in mice. In a xenograft model of breast cancer MDA-MB-436, the ABD-diabody-PBD showed greater tumor growth suppression and better tolerability than either PEG-diabody-PBD or diabody-PBD. These results suggest that the mechanism of half-life extension is an important consideration for designing cytotoxic antitumor agents.
Assuntos
Antineoplásicos/uso terapêutico , Imunoconjugados/uso terapêutico , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Ligação Competitiva , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Ensaio de Imunoadsorção Enzimática , Feminino , Meia-Vida , Humanos , Imunoconjugados/química , Imunoconjugados/farmacocinética , Camundongos , Camundongos Nus , Polietilenoglicóis/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Here, we describe a diene-containing noncanonical amino acid (ncAA) capable of undergoing fast and selective normal electron-demand Diels-Alder (DA) reactions following its incorporation into antibodies. A cyclopentadiene derivative of lysine (CpHK) served as the reactive handle for DA transformations and the substrate for genetic incorporation. CpHK incorporated into antibodies with high efficiency and was available for maleimide conjugation or self-reaction depending on position in the amino acid sequence. CpHK at position K274 reacted with the maleimide drug-linker AZ1508 at a rate of ≈79 m-1 s-1 to produce functional antibody-drug conjugates (ADCs) in a one-step process. Incorporation of CpHK at position S239 resulted in dimerization, which covalently linked antibody heavy chains together. The diene ncAA described here is capable of producing therapeutic protein conjugates with clinically validated and widely available maleimide compounds, while also enabling proximity-based stapling through a DA dimerization reaction.
Assuntos
Alcadienos/química , Aminoácidos/química , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Maleimidas/química , Reação de Cicloadição , Dimerização , Humanos , Modelos Moleculares , Estrutura MolecularRESUMO
Antibody therapeutics offer effective treatment options for a broad range of diseases. One of the greatest benefits of antibody therapeutics is their extraordinarily long serum half-life, allowing infrequent dosing with long-lasting effects. A characteristic of antibodies that drives long half-life is the ability to interact with the recycling receptor, FcRn, in a pH-dependent manner. The benefit of long half-life, however, carries with it liabilities. Although the positive effects of antibody therapeutics are long-lasting, any acute adverse events or chronic negative impacts, such as immunosuppression in the face of an infection, are also long-lasting. Therefore, we sought to develop antibodies with a chemical handle that alone would enjoy the long half-life of normal antibodies but, upon addition of a small-molecule antidote, would interact with the chemical handle and inhibit the antibody recycling mechanism, thus leading to rapid degradation and shortened half-life in vivo Here we present a proof of concept study where we identify sites to incorporate a non-natural amino acid that can be chemically modified to modulate FcRn interaction in vitro and antibody half-life in vivo This is an important first step in developing safer therapeutics, and the next step will be development of technology that can perform the modifying chemistry in vivo.
Assuntos
Anticorpos/química , Antídotos/química , Antígenos de Histocompatibilidade Classe I/química , Receptores Fc/química , Anticorpos/uso terapêutico , Antídotos/uso terapêutico , Antígenos de Histocompatibilidade Classe I/uso terapêutico , Humanos , Receptores Fc/uso terapêuticoRESUMO
Soluble ligands have commonly been targeted by antibody therapeutics for cancers and other diseases. Although monoclonal antibodies targeting such ligands can block their interactions with their cognate receptors, they can also significantly increase the half-life of their ligands by FcRn-mediated antibody recycling, thereby evading ligand renal clearance and requiring increasingly high antibody doses to neutralize the increasing pool of target. To overcome this issue, we generated a bispecific/biparatopic antibody (BiSAb) that targets two different epitopes on IL-6 to block IL-6-mediated signaling. The BiSAb formed large immune complexes with IL-6 that can bind Fcγ receptors on phagocytic cells and are rapidly internalized. In addition, rapid clearance of the BiSAb·IL-6 complex was observed in mice while the parental antibodies prolonged the serum half-life of IL-6. Intravital imaging of the liver in mice confirmed that the rapid clearance of these large immune complexes was associated with Fcγ receptor-dependent binding to Kupffer cells in the liver. The approach described here provides a general strategy for therapeutic antibodies with the ability to not only neutralize but also actively drive clearance of their soluble antigens.
Assuntos
Anticorpos Biespecíficos/metabolismo , Anticorpos Monoclonais/metabolismo , Complexo Antígeno-Anticorpo/imunologia , Interleucina-6/antagonistas & inibidores , Receptores de IgG/metabolismo , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais/imunologia , Células HEK293 , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Interleucina-6/imunologia , Células de Kupffer/citologia , Células de Kupffer/metabolismo , Fígado/citologia , Fígado/metabolismo , Camundongos , Ligação Proteica , Receptores de IgG/imunologiaRESUMO
The thiol-maleimide linkage is widely used for antibody-drug conjugate (ADC) production; however, conjugation of maleimide-drugs could be improved by simplified procedures and reliable conjugate stability. Here, we report the evaluation of electron-rich and cyclic dienes that can be appended to antibodies and reacted with maleimide-containing drugs through the Diels-Alder (DA) reaction. Drug conjugation is fast and quantitative due to reaction acceleration in water, and the linkage is more stable in serum than in the corresponding thiol-maleimide adduct with the same drug. ADCs produced using the DA reaction (DAADCs) are effective in vitro and in vivo, demonstrating the utility of this reaction in producing effective biotherapeutics. Given the large number of commercially available maleimide compounds, this conjugation approach could be readily applied to the production of a wide range of antibody (or protein) conjugates.
Assuntos
Reação de Cicloadição/métodos , Imunoconjugados/química , Maleimidas/química , Alcenos , Anticorpos/química , Reagentes de Ligações Cruzadas/química , Estabilidade de Medicamentos , Maleimidas/uso terapêutico , Preparações Farmacêuticas/químicaRESUMO
Codelivery of multiple therapeutic agents with different anticancer mechanisms can overcome drug resistance as well as generate additive or synergistic anticancer effects that may enhance the antitumor efficacy. Antibody-drug conjugates (ADCs) can be used for highly specific delivery of multiple therapeutic agents with different anticancer mechanisms, though more research is required towards designing flexible platforms on which dual drug ADCs could be prepared. Herein, we describe the synthesis of a heterotrifunctional linker that could be used to construct flexible platforms for preparing dual-cytotoxic drug conjugates in a site-specific manner. As a proof of concept, we synthesized dual drug ADCs carrying monomethyl auristain E (MMAE, tubulin polymerization inhibitor) and pyrrolobenzodiazepine dimer (PBD, DNA minor groove alkylator). We then evaluated the dual drug ADCs for in vitro efficacy and confirmed the dual mechanism of action.
Assuntos
Imunoconjugados/química , Moduladores de Tubulina/química , Aminobenzoatos/química , Anticorpos Monoclonais/química , Antineoplásicos Alquilantes/química , Benzodiazepinas/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Química Click , Humanos , Imunoconjugados/farmacologia , Oligopeptídeos/química , Pirróis/químicaRESUMO
Antibody-drug conjugates (ADCs) are a class of biopharmaceuticals that combine the specificity of antibodies with the high-potency of cytotoxic drugs. Engineering cysteine residues in the antibodies using mutagenesis is a common method to prepare site-specific ADCs. With this approach, solvent accessible amino acids in the antibody have been selected for substitution with cysteine for conjugating maleimide-bearing cytotoxic drugs, resulting in homogeneous and stable site-specific ADCs. Here we describe a cysteine engineering approach based on the insertion of cysteines before and after selected sites in the antibody, which can be used for site-specific preparation of ADCs. Cysteine-inserted antibodies have expression level and monomeric content similar to the native antibodies. Conjugation to a pyrrolobenzodiazepine dimer (SG3249) resulted in comparable efficiency of site-specific conjugation between cysteine-inserted and cysteine-substituted antibodies. Cysteine-inserted ADCs were shown to have biophysical properties, FcRn, and antigen binding affinity similar to the cysteine-substituted ADCs. These ADCs were comparable for serum stability to the ADCs prepared using cysteine-mutagenesis and had selective and potent cytotoxicity against human prostate cancer cells. Two of the cysteine-inserted variants abolish binding of the resulting ADCs to FcγRs in vitro, thereby potentially preventing non-target mediated uptake of the ADCs by cells of the innate immune system that express FcγRs, which may result in mitigating off-target toxicities. A selected cysteine-inserted ADC demonstrated potent dose-dependent anti-tumor activity in a xenograph tumor mouse model of human breast adenocarcinoma expressing the oncofetal antigen 5T4.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Cisteína/química , Animais , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/uso terapêutico , Linhagem Celular Tumoral , Feminino , Humanos , Imunoconjugados/química , Imunoconjugados/uso terapêutico , Neoplasias Mamárias Experimentais/tratamento farmacológico , Camundongos , Camundongos Nus , Trastuzumab/química , Trastuzumab/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Targeted nanomedicines are a promising technology for treatment of disease; however, preparation and characterization of well-defined protein-nanoparticle systems remain challenging. Here, we describe a platform technology to prepare antibody binding fragment (Fab)-bearing nanoparticles and an accompanying real-time cell-based assay to determine their cellular uptake compared to monoclonal antibodies (mAbs) and Fabs. The nanoparticle platform was composed of core-cross-linked polyion complex (PIC) micelles prepared from azide-functionalized PEG-b-poly(amino acids), that is, azido-PEG-b-poly(l-lysine) [N3-PEG-b-PLL] and azido-PEG-b-poly(aspartic acid) [N3-PEG-b-PAsp]. These PIC micelles were 30 nm in size and contained approximately 10 polymers per construct. Fabs were derived from an antibody binding the EphA2 receptor expressed on cancer cells and further engineered to contain a reactive cysteine for site-specific attachment and a cleavable His tag for purification from cell culture expression systems. Azide-functionalized micelles and thiol-containing Fab were linked using a heterobifunctional cross-linker (FPM-PEG4-DBCO) that contained a fluorophenyl-maleimide for stable conjugation to Fabs thiols and a strained alkyne (DBCO) group for coupling to micelle azide groups. Analysis of Fab-PIC micelle conjugates by fluorescence correlation spectroscopy, size exclusion chromatography, and UV-vis absorbance determined that each nanoparticle contained 2-3 Fabs. Evaluation of cellular uptake in receptor positive cancer cells by real-time fluorescence microscopy revealed that targeted Fab-PIC micelles achieved higher cell uptake than mAbs and Fabs, demonstrating the utility of this approach to identify targeted nanoparticle constructs with unique cellular internalization properties.
Assuntos
Anticorpos Monoclonais/química , Reagentes de Ligações Cruzadas/química , Fragmentos Fab das Imunoglobulinas/química , Nanopartículas/química , Polímeros/química , Neoplasias da Próstata/metabolismo , Receptor EphA2/metabolismo , Anticorpos Monoclonais/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Masculino , Micelas , Polímeros/metabolismo , Células Tumorais CultivadasRESUMO
B cell activation is regulated by a variety of signals. CD19 positively regulates B cell activation, augmenting signals delivered through the BCR complex. In contrast, CD32b contains an ITIM and negatively regulates BCR signaling. Importantly, there are drugs currently in clinical trials and preclinical development that cross-link CD32b to molecules within the BCR complex. We wanted to address how single engagement versus cotargeting these molecules affects human B cell function. When B cells from healthy individuals were activated by signals that mimic a T cell response (IL-21 costimulation), ligation of CD32b, but not CD19, inhibited B cell expansion and plasma cell (PC) differentiation. In contrast, when B cells were activated through TLR, anti-CD19, but not anti-CD32b, blunted the response. However, when both CD19 and CD32b were coengaged by a bispecific anti-CD19×CD32b Ab, both types of stimuli were potently inhibited. Cross-linking CD19 with CD32b also inhibited Ab-independent functions of B cells, such as HLA upregulation, cytokine production, and the ability of B cells to prime CD4(+) T cells. Finally, although cross-linking CD19 and CD32b inhibited PC differentiation of primary B cells, it did not alter Ig production from pre-established PCs. These data elucidate the mechanism by which a complex set of signals determines the fate of B cell responsiveness. Although signals through CD19 influence TLR-driven activation, CD32b impacts the magnitude of the response following IL-21 costimulation. Therefore, simultaneous targeting of multiple surface molecules may be a necessary approach to comprehensively modulate B cell activation in vivo.
Assuntos
Antígenos CD19/metabolismo , Linfócitos B/imunologia , Ativação Linfocitária/imunologia , Plasmócitos/metabolismo , Receptores de IgG/metabolismo , Anticorpos/imunologia , Antígenos CD19/biossíntese , Antígenos CD19/imunologia , Antígenos de Diferenciação de Linfócitos B/imunologia , Doenças Autoimunes/imunologia , Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/imunologia , Morte Celular/imunologia , Diferenciação Celular , Células Cultivadas , Reagentes de Ligações Cruzadas , Humanos , Memória Imunológica/imunologia , Interleucinas/metabolismo , Ligação Proteica/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de IgG/biossíntese , Receptores de IgG/imunologia , Transdução de Sinais/imunologia , Receptores Toll-Like/metabolismoRESUMO
We report the three-dimensional structure of human neonatal Fc receptor (FcRn) bound concurrently to its two known ligands. More particularly, we solved the crystal structure of the complex between human FcRn, wild-type human serum albumin (HSA), and a human Fc engineered for improved pharmacokinetics properties (Fc-YTE). The crystal structure of human FcRn bound to wild-type HSA alone is also presented. HSA domain III exhibits an extensive interface of contact with FcRn, whereas domain I plays a lesser role. A molecular explanation for the HSA recycling mechanism is provided with the identification of FcRn His(161) as the only potential direct contributor to the corresponding pH-dependent process. At last, this study also allows an accurate structural definition of residues considered for decades as important to the human IgG/FcRn interaction and reveals Fc His(310) as a significant contributor to pH-dependent binding. Finally, we explain various structural mechanisms by which several Fc mutations (including YTE) result in increased human IgG binding to FcRn. Our study provides an unprecedented relevant understanding of the molecular basis of human Fc interaction with human FcRn.
Assuntos
Antígenos de Histocompatibilidade Classe I/química , Imunoglobulina G/química , Receptores Fc/química , Albumina Sérica/química , Cristalização , Cristalografia por Raios X , Células HEK293 , Humanos , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Fragmentos Fc das Imunoglobulinas/química , Ligantes , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/químicaRESUMO
Antibody-drug conjugates (ADCs) have emerged as an important class of therapeutics for cancer treatment that combine the target specificity of antibodies with the killing activity of anticancer chemotherapeutics. Early conjugation technologies relied upon random conjugation to either lysine or cysteine residues, resulting in heterogeneous ADCs. Recent technology advancements have resulted in the preparation of homogeneous ADCs through the site-specific conjugation at engineered cysteines, glycosylated amino acids, and bioorthogonal unnatural amino acids. Here we describe for the first time the conjugation of an anti-mitotic drug to an antibody following the mild and selective oxidation of a serine residue engineered at the N-terminus of the light chain. Using an alkoxyamine-derivatized monomethyl auristatine E payload, we have prepared a hydrolytically stable ADC that retains binding to its antigen and displays potent in vitro cytotoxicity and in vivo tumor growth inhibition.
Assuntos
Anticorpos/química , Anticorpos/farmacologia , Engenharia de Proteínas/métodos , Animais , Anticorpos/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Humanos , Hidrólise , Camundongos Nus , Oximas/química , Estabilidade Proteica , Ratos , Receptor EphA2/imunologia , Receptor EphA2/metabolismo , Serina/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Targeting Eph (erythropoietin producing hepatoma) receptors with monoclonal antibodies is being explored as therapy for several types of cancer. To test whether simultaneous targeting of EphA2, EphA4, and EphB4 would be an effective approach to cancer therapy, we generated a recombinant trispecific antibody using the variable domain genes of anti-EphA2, anti-EphA4, and anti-EphB4 monoclonal antibodies. A multidisciplinary approach combining biochemical, biophysical, and cellular-based assays was used to characterize the trispecific antibody in vitro and in vivo. Here we demonstrate that the trispecific antibody is expressed at high levels by mammalian cells, monodispersed in solution, thermostable, capable of simultaneously binding the three receptors, and able to activate the three targets effectively as evidenced by receptor internalization and degradation both in vitro and in vivo. Furthermore, pharmacokinetic analysis using tumor-bearing nude mice showed that the trispecific antibody remains in the circulation similarly to its respective parental antibodies. These results indicate that simultaneous blockade of EphA2, EphA4, and EphB4 could be an attractive approach to cancer therapy.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos/imunologia , Desenho de Fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias da Próstata/tratamento farmacológico , Receptor EphA2/antagonistas & inibidores , Receptor EphA4/antagonistas & inibidores , Receptor EphB4/antagonistas & inibidores , Animais , Varredura Diferencial de Calorimetria , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Masculino , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ligação Proteica , Receptor EphA2/imunologia , Receptor EphA4/imunologia , Receptor EphB4/imunologia , Ressonância de Plasmônio de Superfície , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Assessment of drug activation and subsequent interaction with targets in living tissues could guide nanomedicine design, but technologies enabling insight into how a drug reaches and binds its target are limited. We show that a Hoechst-based reporter system can monitor drug release and engagement from a nanoparticle delivery system in vitro and in vivo, elucidating differences in target-bound drug distribution related to drug-linker and nanoparticle properties. Drug engagement is defined as chemical detachment of drug or reporter from a nanoparticle and subsequent binding to a subcellular target, which in the case of Hoechst results in a fluorescence signal. Hoechst-based nanoreporters for drug activation contain prodrug elements such as dipeptide linkers, conjugation handles, and nanoparticle modifications such as targeting ligands to determine how nanomedicine design affects distribution of drug engaged with a subcellular target, which is tracked via cellular nuclear fluorescence in situ. Furthermore, the nanoplatform is amenable toward common maleimide-based linkers found in many prodrug-based delivery systems including polymer-, peptide-, and antibody-drug conjugates. Findings from the Hoechst reporter system were applied to develop highly potent, targeted, anticancer micelle nanoparticles delivering a monomethyl auristatin E (MMAE) prodrug comprising the same linkers employed in Hoechst studies. MMAE nanomedicine with the optimal drug-linker resulted in effective tumor growth inhibition in mice without associated acute toxicity, whereas the nonoptimal linker that showed broader drug activation in Hoechst reporter studies resulted in severe toxicity. Our results demonstrate the potential to synergize direct visualization of drug engagement with nanomedicine drug-linker design to optimize safety and efficacy.
Assuntos
Antineoplásicos , Imunoconjugados , Nanopartículas , Pró-Fármacos , Camundongos , Animais , Pró-Fármacos/química , Ensaios Antitumorais Modelo de Xenoenxerto , Imunoconjugados/química , Micelas , Nanopartículas/uso terapêutico , Nanopartículas/química , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/química , Sistemas de Liberação de MedicamentosRESUMO
Many SARS-CoV-2 neutralizing antibodies (nAbs) lose potency against variants of concern. In this study, we developed 2 strategies to produce mutation-resistant antibodies. First, a yeast library expressing mutant receptor binding domains (RBDs) of the spike protein was utilized to screen for potent nAbs that are least susceptible to viral escape. Among the candidate antibodies, P5-22 displayed ultrahigh potency for virus neutralization as well as an outstanding mutation resistance profile. Additionally, P14-44 and P15-16 were recognized as mutation-resistant antibodies with broad betacoronavirus neutralization properties. P15-16 has only 1 binding hotspot, which is K378 in the RBD of SARS-CoV-2. The crystal structure of the P5-22, P14-44, and RBD ternary complex clarified the unique mechanisms that underlie the excellent mutation resistance profiles of these antibodies. Secondly, polymeric IgG enhanced antibody avidity by eliminating P5-22's only hotspot, residue F486 in the RBD, thereby potently blocking cell entry by mutant viruses. Structural and functional analyses of antibodies screened using both potency assays and the yeast RBD library revealed rare, ultrapotent, mutation-resistant nAbs against SARS-CoV-2.