RESUMO
In a biologic therapeutic landscape that requires versatility in targeting specificity, valency and half-life modulation, the monomeric Fc fusion platform holds exciting potential for the creation of a class of monovalent protein therapeutics that includes fusion proteins and bispecific targeting molecules. Here we report a structure-guided approach to engineer monomeric Fc molecules to adapt multiple versions of half-life extension modifications. Co-crystal structures of these monomeric Fc variants with Fc neonatal receptor (FcRn) shed light into the binding interactions that could serve as a guide for engineering the half-life of antibody Fc fragments. These engineered monomeric Fc molecules also enabled the generation of a novel monovalent bispecific molecular design, which translated the FcRn binding enhancement to improvement of in vivo serum half-life.
Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Fragmentos Fc das Imunoglobulinas/metabolismo , Receptores Fc/metabolismo , Animais , Meia-Vida , Antígenos de Histocompatibilidade Classe I/farmacologia , Humanos , Fragmentos Fc das Imunoglobulinas/farmacologia , Camundongos , Camundongos Transgênicos , Engenharia de ProteínasRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0234268.].
RESUMO
Neuregulin protein 1 (NRG1) is a large (> 60-amino-acid) natural peptide ligand for the ErbB protein family members HER3 and HER4. We developed an agonistic antibody modality, termed antibody ligand mimetics (ALM), by incorporating complex ligand agonists such as NRG1 into an antibody scaffold. We optimized the linker and ligand length to achieve native ligand activity in HEK293 cells and cardiomyocytes derived from induced pluripotent stem cells (iPSCs) and used a monomeric Fc-ligand fusion platform to steer the ligand specificity toward HER4-dominant agonism. With the help of selectivity engineering, these enhanced ALM molecules can provide an antibody scaffold with increased receptor specificity and the potential to greatly improve the pharmacokinetics, stability, and downstream developability profiles from the natural ligand approach. This ligand mimetic design and optimization approach can be expanded to apply to other cardiovascular disease targets and emerging therapeutic areas, providing differentiated drug molecules with increased specificity and extended half-life.
Assuntos
Anticorpos Monoclonais/química , Neuregulina-1/química , Receptor ErbB-4/agonistas , Anticorpos Monoclonais/metabolismo , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/química , Células-Tronco Pluripotentes Induzidas/metabolismo , Cinética , Ligantes , Miócitos Cardíacos/química , Miócitos Cardíacos/metabolismo , Neuregulina-1/metabolismo , Ligação Proteica , Receptor ErbB-4/metabolismo , Transdução de SinaisRESUMO
Monoclonal antibodies from human sources are being increasingly recognized as valuable options in many therapeutic areas. These antibodies can show exquisite specificity and high potency while maintaining a desirable safety profile, having been matured and tolerized within human patients. However, the discovery of these antibodies presents important challenges, since the B cells encoding therapeutic antibodies can be rare in a typical blood draw and are short-lived ex vivo. Furthermore, the unique pairing of VH and VL domains in each B cell contributes to specificity and function; therefore, maintaining antibody chain pairing presents a throughput limitation. This work will review the various approaches aimed at addressing these challenges with an eye to next-generation methods for high-throughput discovery from the human B-cell repertoire.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Linfócitos B/imunologia , Descoberta de Drogas , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , HumanosRESUMO
Annexin A1 (anxA1) is an immunomodulatory protein that has been proposed as a tumor vascular target for antitumor biologic agents, yet to date the vascular expression of anxA1 in specific tumor indications has not been systematically assessed. Attempts to evaluate vascular anxA1 expression by immunohistochemistry are complicated by a lack of available antibodies that are both specific for anxA1 and bind the N-terminal-truncated form of anxA1 that has previously been identified in tumor vasculature. To study the vascular expression pattern of anxA1 in non-small-cell lung carcinoma (NSCLC), we isolated an antibody capable of binding N-terminal-truncated anxA127-346 and employed it in immunohistochemical studies of human lung specimens. Lung tumor specimens evaluated with this antibody revealed vascular (endothelial) anxA1 expression in five of eight tumor samples studied, but no vascular anxA1 expression was observed in normal lung tissue. Tumor microarray analysis further demonstrated positive vascular staining for anxA1 in 30 of 80 NSCLC samples, and positive staining of neoplastic cells was observed in 54 of 80 samples. No correlation was observed between vascular and parenchymal anxA1 expression. Two rodent tumor models, B16-F10 and Py230, were determined to have upregulated anxA1 expression in the intratumoral vasculature. These data validate anxA1 as a potential vascular anti-tumor target in a subset of human lung tumors and identify rodent models which demonstrate anxA1 expression in tumor vasculature.
Assuntos
Anexina A1/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Regulação para Cima , Animais , Carcinoma Pulmonar de Células não Pequenas/irrigação sanguínea , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Neoplasias Pulmonares/irrigação sanguínea , CamundongosRESUMO
Efficacious use of therapeutic gene delivery via nanoparticles is hampered by the challenges associated with targeted delivery to tissues of interest. Systemic administration of lipid nanoparticle (LNP)-encapsulated mRNA leads to a protein expressed predominantly in the liver and spleen. Here, LNP encapsulating mRNA was covalently conjugated to an antibody, specifically binding plasmalemma vesicle-associated protein (PV1) as a means to target lung tissue. Systemic administration of PV1-targeted LNPs demonstrated significantly increased delivery of mRNA to the lungs and a 40-fold improvement in protein expression in the lungs, compared with control LNPs. We also investigated the effect of LNP size to determine optimal tissue distribution and transfection. Larger-size PV1-targeted LNPs not only have the elasticity to target the PV1 expressed in the caveolae but also enable robust mRNA expression in the lungs. Targeted delivery of mRNA to the lungs is a promising approach in the treatment of lung diseases.
Assuntos
Portadores de Fármacos/química , Lipídeos/química , Pulmão/metabolismo , Nanopartículas/química , RNA Mensageiro/farmacologia , Animais , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Cavéolas/imunologia , Feminino , Técnicas de Transferência de Genes , Proteínas de Membrana/imunologia , Camundongos Endogâmicos BALB CRESUMO
Targeted strategies to deliver and retain drugs to kidneys are needed to improve drug accumulation and efficacy in a myriad of kidney diseases. These drug delivery systems show potential for improving the therapeutic windows of drugs acting in the kidney. Biodistribution of antibody-based therapeutics in vivo is governed by several factors including binding affinity, size, and valency. Investigations of how the biophysical and biochemical properties of biologics enable them to overcome biological barriers and reach kidneys are therefore of interest. Although renal accumulation of antibody fragments in cancer diagnostics and treatment has been observed, reports on effective delivery of antibody fragments to the kidneys remain scarce. Previously, we demonstrated that targeting plasmalemma vesicle-associated protein (PV1), a caveolae-associated protein, can promote accumulation of antibodies in both the lungs and the kidneys. Here, by fine-tuning the binding affinity of an antibody toward PV1, we observe that the anti-PV1 antibody with reduced binding affinity lost the capability for kidney targeting while retaining the lung targeting activity, suggesting that binding affinity is a critical factor for kidney targeting of the anti-PV1 antibody. We next use the antibody fragment F(ab')2 targeting PV1 to assess the dual effects of rapid kidney filtration and PV1 targeting on kidney-selective targeting. Ex vivo fluorescence imaging results demonstrated that after rapidly accumulating in kidneys at 4 h, PV1-targeted F(ab')2 was continually retained in the kidney at 24 h, whereas the isotype control F(ab')2 underwent urinary elimination with significantly reduced signaling in the kidney. Confocal imaging studies confirmed the localization of PV1-targeted F(ab')2 in the kidney. In addition, the monovalent antibody fragment (Fab-C4) lost the capability for kidney homing, indicating that the binding avidity of anti-PV1 F(ab')2 is important for kidney targeting. Our findings suggest that PV1-targeted F(ab')2 might be useful as a drug carrier for renal targeting and highlight the importance of affinity optimization for tissue targeting antibodies.
Assuntos
Anticorpos Monoclonais/imunologia , Cavéolas/metabolismo , Portadores de Fármacos/farmacocinética , Fragmentos Fab das Imunoglobulinas/imunologia , Rim/efeitos dos fármacos , Proteínas de Membrana/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacocinética , Afinidade de Anticorpos , Portadores de Fármacos/administração & dosagem , Feminino , Células HEK293 , Humanos , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Rim/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Distribuição TecidualRESUMO
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
G-protein coupled receptors (GPCRs) constitute major drug targets due to their involvement in critical biological functions and pathophysiological disorders. The leading challenge in their structural and functional characterization has been the need for a lipid environment to accommodate their hydrophobic cores. Here, we report an antibody scaffold mimetic (ASM) platform where we have recapitulated the extracellular functional domains of the GPCR, C-X-C chemokine receptor 4 (CXCR4) on a soluble antibody framework. The engineered ASM molecule can accommodate the N-terminal loop and all three extracellular loops of CXCR4. These extracellular features are important players in ligand recruitment and interaction for allostery and signal transduction. Our study shows that ASMCXCR4 can be recognized by the anti-CXCR4 antibodies, MEDI3185, 2B11, and 12G5, and that ASMCXCR4 can bind the HIV-1 glycoprotein ligand gp120, and the natural chemokine ligand SDF-1α. Further, we show that ASMCXCR4 can competitively inhibit the SDF-1α signaling pathway, and be used as an immunogen to generate CXCR4-specific antibodies. This platform will be useful in the study of GPCR biology in a soluble receptor context for evaluating its extracellular ligand interactions.
Assuntos
Biomimética/métodos , Receptores CXCR4/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Anticorpos/genética , Quimiocina CXCL12/metabolismo , Células HEK293 , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Ligantes , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas , Receptores CXCR4/genética , Receptores Acoplados a Proteínas G/genética , Transdução de SinaisRESUMO
Systemic administration of bio-therapeutics can result in only a fraction of drug reaching targeted tissues, with the majority of drug being distributed to tissues irrelevant to the drug's site of action. Targeted delivery to specific organs may allow for greater accumulation, better efficacy, and improved safety. We investigated how targeting plasmalemma vesicle-associated protein (PV1), a protein found in the endothelial caveolae of lungs and kidneys, can promote accumulation in these organs. Using ex vivo fluorescence imaging, we show that intravenously administered αPV1 antibodies localize to mouse lungs and kidneys. In a bleomycin-induced idiopathic pulmonary fibrosis (IPF) mouse model, αPV1 conjugated to Prostaglandin E2 (PGE2), a known anti-fibrotic agent, significantly reduced collagen content and fibrosis whereas a non-targeted PGE2 antibody conjugate failed to slow fibrosis progression. Our results demonstrate that PV1 targeting can be utilized to deliver therapeutics to lungs and this approach is potentially applicable for various lung diseases.
Assuntos
Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Proteínas de Membrana/metabolismo , Animais , Biomarcadores , Bleomicina/efeitos adversos , Dinoprostona/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Humanos , Fibrose Pulmonar Idiopática/etiologia , Fibrose Pulmonar Idiopática/patologia , Imuno-Histoquímica , Rim/metabolismo , Rim/patologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Proteínas de Membrana/química , Proteínas de Membrana/genética , CamundongosRESUMO
Interleukin-21 (IL-21), a member of the common cytokine receptor γ chain (γc) family, is secreted by CD4+ T cells and natural killer T cells and induces effector function through interactions with the IL-21 receptor (IL-21R)/γc complex expressed on both immune and non-immune cells. Numerous studies suggest that IL-21 plays a significant role in autoimmune disorders. Therapeutic intervention to disrupt the IL-21/IL-21R/γc interaction and inhibit subsequent downstream signal transduction could offer a treatment paradigm for these diseases. Potent neutralizing antibodies reported in the literature were generated after extensive immunizations with human IL-21 alone and in combination with various adjuvants. To circumvent the laborious method of antibody generation while targeting a conserved functional epitope, we designed a novel alternating-antigen immunization strategy utilizing both human and cynomolgus monkey (cyno) IL-21. Despite the high degree of homology between human and cyno IL-21, our alternating-immunization strategy elicited higher antibody titers and more potent neutralizing hybridomas in mice than did the immunization with human IL-21 antigen alone. The lead hybridoma clone was humanized by grafting the murine complementarity-determining regions onto human germline framework templates, using a unique rational design. The final humanized and engineered antibody, MEDI7169, encodes only one murine residue at the variable heavy/light-chain interface, retains the sub-picomolar affinity for IL-21, specifically inhibits IL-21/IL-21R-mediated signaling events and is currently under clinical development as a potential therapeutic agent for autoimmune diseases. This study provides experimental evidence of the immune system's potential to recognize and respond to shared epitopes of antigens from distinct species, and presents a generally applicable, novel method for the rapid generation of exceptional therapeutic antibodies using the hybridoma platform.
Assuntos
Anticorpos Monoclonais Humanizados/metabolismo , Anticorpos Neutralizantes/metabolismo , Interleucinas/imunologia , Macaca fascicularis/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Modelos Animais de Doenças , Humanos , Hibridomas/imunologia , Imunização , CamundongosRESUMO
Cell line development (CLD) for biotherapeutics is a time- and resource-intensive process requiring the isolation and screening of large numbers of clones to identify high producers. Novel methods aimed at enhancing cell line screening efficiency using markers predictive of productivity early in the CLD process are needed to reliably generate high-yielding cell lines. To enable efficient and selective isolation of antibody expressing Chinese hamster ovary cells by fluorescence-activated cell sorting, we developed a strategy for the expression of antibodies containing a switchable membrane-associated domain to anchor an antibody to the membrane of the expressing cell. The switchable nature of the membrane domain is governed by the function of an orthogonal aminoacyl transfer RNA synthetase/tRNApyl pair, which directs a nonnatural amino acid (nnAA) to an amber codon encoded between the antibody and the membrane anchor. The process is "switchable" in response to nnAA in the medium, enabling a rapid transition between the surface display and secretion. We demonstrate that the level of cell surface display correlates with productivity and provides a method for enriching phenotypically stable high-producer cells. The strategy provides a means for selecting high-producing cells with potential applications to multiple biotherapeutic protein formats.
Assuntos
Códon de Terminação , Vetores Genéticos/genética , Imunoglobulina G/genética , Proteínas Recombinantes/genética , Animais , Técnicas de Cultura Celular por Lotes/métodos , Células CHO , Cricetulus , Humanos , Transfecção/métodosRESUMO
The human antibody repertoire is increasingly being recognized as a valuable source of therapeutic grade antibodies. However, methods for mining primary antibody-expressing B cells are limited in their ability to rapidly isolate rare and antigen-specific binders. Here we show the encapsulation of two million primary B cells into picoliter-sized droplets, where their cognate V genes are fused in-frame to form a library of scFv cassettes. We used this approach to construct natively paired phage-display libraries from healthy donors and drove selection towards cross-reactive antibodies targeting influenza hemagglutinin. Within 4 weeks we progressed from B cell isolation to a panel of unique monoclonal antibodies, including seven that displayed broad reactivity to different clinically relevant influenza hemagglutinin subtypes. Most isolated antibody sequences were not detected by next-generation sequencing of the paired repertoire, illustrating how this method can isolate extremely rare leads not likely found by existing technologies.
RESUMO
IgG antibodies are abundantly present in the vasculature but to a much lesser extent in mucosal tissues. This contrasts with antibodies of the IgA and IgM isotype that are present at high concentration in mucosal secretions due to active delivery by the polymeric Ig receptor (pIgR). IgG is the preferred isotype for therapeutic mAb development due to its long serum half-life and robust Fc-mediated effector function, and it is utilized to treat a diverse array of diseases with antigen targets located in the vasculature, serosa, and mucosa. As therapeutic IgG antibodies targeting the luminal side of mucosal tissue lack an active transport delivery mechanism, we sought to generate IgG antibodies that could be transported via pIgR, similarly to dimeric IgA and pentameric IgM. We show that an anti-Pseudomonas aeruginosa IgG fused with pIgR-binding peptides gained the ability to transcytose and be secreted via pIgR. Consistent with these results, pIgR-binding IgG antibodies exhibit enhanced localization to the bronchoalveolar space when compared with the parental IgG antibody. Furthermore, pIgR-binding mAbs maintained Fc-mediated functional activity and promoted enhanced survival compared with the parental mAb in a P. aeruginosa acute pneumonia model. Our results suggest that increasing IgG accumulation at mucosal surfaces by pIgR-mediated active transport can improve the efficacy of therapeutic mAbs that act at these sites.
Assuntos
Imunoglobulina G/imunologia , Imunoglobulina G/farmacologia , Mucosa/imunologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/efeitos dos fármacos , Animais , Transporte Biológico/imunologia , Células CHO , Cricetulus , Cães , Imunoglobulina A/imunologia , Imunoglobulina M/imunologia , Células Madin Darby de Rim Canino , Camundongos Endogâmicos BALB C , Mucosa/microbiologia , Receptores de Imunoglobulina Polimérica , Componente Secretório , Transcitose/imunologiaRESUMO
The accumulation, dissemination and clearance of monoclonal antibody-based therapeutics or imaging reagents targeting tumor associated antigens is governed by several factors including affinity, size, charge, and valency. Tumor targeting antibody fragments have distinct advantages over intact monoclonal antibodies such as enhanced penetration within the tumor and rapid accumulation but are subject to rapid clearance. Polyethylene glycol (PEG)-modified antibody fragments can provide a way to balance tumor penetration and accumulation with improved serum persistence. In this study, we use a diabody, the dimeric antibody fragment, targeting the 5T4 antigen to assess the impact of PEGs of distinct size and shape on tumor accumulation and pharmacokinetics (PK). We show that PEG-modified diabodies improved the PK of the parental diabody from a half-life of 40â¯min to over 40â¯h for the higher molecular weight PEG conjugated diabodies. This improvement correlates with the increasing hydrodynamic size of pegylated diabodies, and can serve as a better predictor of the PK behavior of pegylated molecules than molecular weight alone. Tumor uptake profiles determined by quantitative PET imaging differed significantly based on PEG size and shape with diabody-PEG5K showing peak accumulation early on, but with the larger diabody-PEG20K showing better sustained tumor uptake at later time points. In addition, we demonstrate that a diabody-PEG20K-B with a hydrodynamic radius (Rh) of 6â¯nm had superior tumor uptake than the larger diabody-PEG40K-B with Rh of 12â¯nm, indicating that beyond 6â¯nm, larger pegylated diabodies have a slower tumor uptake rate while having comparable clearance kinetics. Our data demonstrate that pegylated diabodies with Rh of ~6â¯nm have an optimal size and PK profile for tumor uptake. Understanding the impact of pegylation on PK and tumor uptake could facilitate the development of pegylated diabodies as therapeutics.
Assuntos
Sistemas de Liberação de Medicamentos , Fragmentos de Imunoglobulinas/administração & dosagem , Neoplasias/metabolismo , Polietilenoglicóis/química , Animais , Transporte Biológico , Linhagem Celular Tumoral , Feminino , Meia-Vida , Humanos , Hidrodinâmica , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/metabolismo , Camundongos , Camundongos Nus , Peso Molecular , Tomografia por Emissão de Pósitrons , Distribuição TecidualRESUMO
Membrane proteins play key roles in the evolution of numerous diseases and as a result have become the most dominant class of targets for therapeutic intervention. However, their poor expression and detection oftentimes prohibit drug discovery and screening efforts. Herein, we have developed an approach, named 'Tag-on-Demand' that exploits amber suppression to control the expression of 'tagged' membrane proteins for detection and selections, yet can be turned off for expression of the protein in its native form. Utilizing an engineered Chinese hamster ovary cell line capable of efficient amber suppression, we evaluated the expression of a diverse panel of model membrane proteins and demonstrated the enrichment of cells with improved expression profiles, where ~200-800% improvement in total protein expression levels were observed over pre-sorted populations after a single round of fluorescence-activated cell sorting. Furthermore, results were most striking for the typically difficult-to-express G protein-coupled receptor, CXCR2, where ~2.5-fold improvement in surface expression was observed. We anticipate that the Tag-on-Demand approach will be suitable not only for membrane protein cell line development but also for the development of intracellular and secreted protein cell lines in expression systems for which amber suppression technology exists, including bacterial, yeast, insect and cell-free expression systems.
Assuntos
Códon de Terminação/genética , Engenharia Genética/métodos , Proteínas de Membrana/genética , Animais , Células CHO , Cricetulus , Avaliação Pré-Clínica de Medicamentos , Expressão Gênica , Células HEK293 , HumanosRESUMO
Phage display antibody libraries are a rich resource for discovery of potential therapeutic antibodies. Single-chain variable fragment (scFv) libraries are the most common format due to the efficient display of scFv by phage particles and the ease by which soluble scFv antibodies can be expressed for high-throughput screening. Typically, a cascade of screening and triaging activities are performed, beginning with the assessment of large numbers of E. coli-expressed scFv, and progressing through additional assays with individual reformatting of the most promising scFv to full-length IgG. However, use of high-throughput screening of scFv for the discovery of full-length IgG is not ideal because of the differences between these molecules. Furthermore, the reformatting step represents a bottle neck in the process because each antibody has to be handled individually to preserve the unique VH and VL pairing. These problems could be resolved if populations of scFv could be reformatted to full-length IgG before screening without disrupting the variable region pairing. Here, we describe a novel strategy that allows the reformatting of diverse populations of scFv from phage selections to full-length IgG in a batch format. The reformatting process maintains the diversity and variable region pairing with high fidelity, and the resulted IgG pool enables high-throughput expression of IgG in mammalian cells and cell-based functional screening. The improved process led to the discovery of potent candidates that are comparable or better than those obtained by traditional methods. This strategy should also be readily applicable to Fab-based phage libraries. Our approach, Screening in Product Format (SiPF), represents a substantial improvement in the field of antibody discovery using phage display.
RESUMO
Bispecific antibodies are considered attractive bio-therapeutic agents owing to their ability to target two distinct disease mediators. Cross-arm avidity targeting of antigen double-positive cancer cells over single-positive normal tissue is believed to enhance the therapeutic efficacy, restrict major escape mechanisms and increase tumor-targeting selectivity, leading to reduced systemic toxicity and improved therapeutic index. However, the interplay of factors regulating target selectivity is not well understood and often overlooked when developing clinically relevant bispecific therapeutics. We show in vivo that dual targeting alone is not sufficient to endow selective tumor-targeting, and report the pivotal roles played by the affinity of the individual arms, overall avidity and format valence. Specifically, a series of monovalent and bivalent bispecific IgGs composed of the anti-HER2 trastuzumab moiety paired with affinity-modulated VH and VL regions of the anti-EGFR GA201 mAb were tested for selective targeting and eradication of double-positive human NCI-H358 non-small cell lung cancer target tumors over single-positive, non-target NCI-H358-HER2 CRISPR knock out tumors in nude mice bearing dual-flank tumor xenografts. Affinity-reduced monovalent bispecific variants, but not their bivalent bispecific counterparts, mediated a greater degree of tumor targeting selectivity, while the overall efficacy against the targeted tumor was not substantially affected.
Assuntos
Anticorpos Biespecíficos/imunologia , Afinidade de Anticorpos , Especificidade de Anticorpos , Carcinoma Pulmonar de Células não Pequenas/imunologia , Trastuzumab/imunologia , Animais , Anticorpos Biespecíficos/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Receptores ErbB/imunologia , Humanos , Camundongos Nus , Receptor ErbB-2/imunologia , Trastuzumab/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Multiple mutation combinations in the IgG Fc have been characterized to tailor immune effector function or IgG serum persistence to fit desired biological outcomes for monoclonal antibody therapeutics. An unintended consequence of introducing mutations in the Fc (particularly the CH2 domain) can be a reduction in biophysical stability which can correlate with increased aggregation propensity, poor manufacturability, and lower solubility. Herein, we characterize the changes in IgG conformational and colloidal stability when 2 sets of CH2 mutations "TM" (L234F/L235E/P331S) and "YTE" (M252Y/S254T/T256E) are combined to generate an antibody format lacking immune receptor binding and exhibiting extended half-life. In addition to significantly lowered thermostability, we observe greater conformational flexibility for TM-YTE in CH2, increased self-association, and poorer solubility and aggregation profiles. To improve these properties, we dissected the contributions of individual mutations within TM-YTE on thermostability and substituted destabilizing mutations with new mutations that raise thermostability. One novel combination, FQQ-YTE (L234F/L235Q/K322Q/M252Y/S254T/T256E), had significantly improved conformational and colloidal stability, and was found to retain the same biological activities as TM-YTE (extended half-life and lack of antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity activity). Our engineering approach offers a way to improve the developability of antibodies containing Fc mutations while retaining tailored biological activity.
Assuntos
Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/química , Imunoglobulina G/genética , Animais , Inativação Gênica , Células HEK293 , Meia-Vida , Humanos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de IgG/química , Receptores de IgG/genéticaRESUMO
Antibody therapy against antibiotics resistant Klebsiella pneumoniae infections represents a promising strategy, the success of which depends critically on the ability to identify appropriate antibody targets. Using a target-agnostic strategy, we recently discovered MrkA as a potential antibody target and vaccine antigen. Interestingly, the anti-MrkA monoclonal antibodies isolated through phage display and hybridoma platforms all recognize an overlapping epitope, which opens up important questions including whether monoclonal antibodies targeting different MrkA epitopes can be generated and if they possess different protective profiles. In this study we generated four anti-MrkA antibodies targeting different epitopes through phage library panning against recombinant MrkA protein. These anti-MrkA antibodies elicited strong in vitro and in vivo protections against a multi-drug resistant Klebsiella pneumoniae strain. Furthermore, mutational and epitope analysis suggest that the two cysteine residues may play essential roles in maintaining a MrkA structure that is highly compacted and exposes limited antibody binding/neutralizing epitopes. These results suggest the need for further in-depth understandings of the structure of MrkA, the role of MrkA in the pathogenesis of Klebsiella pneumoniae and the protective mechanism adopted by anti-MrkA antibodies to fully explore the potential of MrkA as an efficient therapeutic target and vaccine antigen.