RESUMO
The spread of cancer from organ to organ (metastasis) is responsible for the vast majority of cancer deaths; however, most current anti-cancer drugs are designed to arrest or reverse tumor growth without directly addressing disease spread. It was recently discovered that tumor cell-secreted interleukin-6 (IL-6) and interleukin-8 (IL-8) synergize to enhance cancer metastasis in a cell-density dependent manner, and blockade of the IL-6 and IL-8 receptors (IL-6R and IL-8R) with a novel bispecific antibody, BS1, significantly reduced metastatic burden in multiple preclinical mouse models of cancer. Bispecific antibodies (BsAbs), which combine two different antigen-binding sites into one molecule, are a promising modality for drug development due to their enhanced avidity and dual targeting effects. However, while BsAbs have tremendous therapeutic potential, elucidating the mechanisms underlying their binding and inhibition will be critical for maximizing the efficacy of new BsAb treatments. Here, we describe a quantitative, computational model of the BS1 BsAb, exhibiting how modeling multivalent binding provides key insights into antibody affinity and avidity effects and can guide therapeutic design. We present detailed simulations of the monovalent and bivalent binding interactions between different antibody constructs and the IL-6 and IL-8 receptors to establish how antibody properties and system conditions impact the formation of binary (antibody-receptor) and ternary (receptor-antibody-receptor) complexes. Model results demonstrate how the balance of these complex types drives receptor inhibition, providing important and generalizable predictions for effective therapeutic design.
Assuntos
Anticorpos Biespecíficos , Receptores de Interleucina-6 , Receptores de Interleucina-8 , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/química , Receptores de Interleucina-6/antagonistas & inibidores , Receptores de Interleucina-6/imunologia , Receptores de Interleucina-6/metabolismo , Humanos , Receptores de Interleucina-8/metabolismo , Receptores de Interleucina-8/antagonistas & inibidores , Animais , Biologia Computacional , Simulação por Computador , Interleucina-6/metabolismo , Interleucina-6/imunologia , Camundongos , Interleucina-8/metabolismo , Interleucina-8/imunologia , Interleucina-8/antagonistas & inibidores , Neoplasias/imunologia , Neoplasias/tratamento farmacológicoRESUMO
The use of bispecific antibodies as T cell engagers can bypass the normal T cell receptor-major histocompatibility class interaction, redirect the cytotoxic activity of T cells, and lead to highly efficient tumor cell killing. However, this immunotherapy also causes significant on-target off-tumor toxicologic effects, especially when it is used to treat solid tumors. To avoid these adverse events, it is necessary to understand the fundamental mechanisms involved in the physical process of T cell engagement. We developed a multiscale computational framework to reach this goal. The framework combines simulations on the intercellular and multicellular levels. On the intercellular level, we simulated the spatial-temporal dynamics of three-body interactions among bispecific antibodies, CD3 and tumor-associated antigens (TAAs). The derived number of intercellular bonds formed between CD3 and TAAs was further transferred to the multicellular simulations as the input parameter of adhesive density between cells. Through the simulations under various molecular and cellular conditions, we were able to gain new insights into how to adopt the most appropriate strategy to maximize the drug efficacy and avoid the off-target effect. For instance, we discovered that the low antibody-binding affinity resulted in the formation of large clusters at the cell-cell interface, which could be important to control the downstream signaling pathways. We also tested different molecular architectures of the bispecific antibody and suggested the existence of an optimal length in regulating the T cell engagement. Overall, the current multiscale simulations serve as a proof-of-concept study to help in the future design of new biological therapeutics.
Assuntos
Anticorpos Biespecíficos , Neoplasias , Humanos , Linfócitos T , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/uso terapêutico , Complexo CD3/farmacologia , Neoplasias/tratamento farmacológico , Imunoterapia/métodosRESUMO
Bispecific antibodies (BsAbs) are undergoing continued development for applications in oncology and autoimmune diseases. While increasing activity by having more than one targeting arm, most BsAb engineering employs single Fc engagement as monoclonal antibodies. Here, we designed a novel immunoglobulin gamma-1 (IgG1)-derived dual-Fc BsAb containing two Fc regions and two distinct asymmetric antigen binding arms comprising a Fab arm and another VHH domain. In conjunction with the knob-into-hole technology, dual-Fc BsAbs could be produced with a high yield and good stability. We explore how Fc engineering effects on dual-Fc constructs could boost the desired therapeutic efficacy. This new format enabled simultaneous bispecific binding to corresponding antigens. Furthermore, compared to the one-Fc control molecules, dual-Fc BsAbs were shown to increase the avidity-based binding to FcγRs to result in higher ADCC and ADCP activities by potent avidity via binding to two antigens and Fc receptors. Overall, this novel BsAb format with enhanced effector functionalities provides a new option for antibody-based immunotherapy.
Assuntos
Anticorpos Biespecíficos , Anticorpos Biespecíficos/química , Fragmentos Fc das Imunoglobulinas/genética , Anticorpos MonoclonaisRESUMO
Bispecific antibodies possess exceptional potential as therapeutic agents due to their capacity to bind to two different antigens simultaneously. However, challenges pertain to unsatisfactory stability, manufacturing complexity, and limited tumor penetration hinder their broad applicability. In this study, a versatile technology is presented for the rapid generation of bispecific nanobody-aptamer conjugates with efficient tumor penetration. The approach utilizes microbial transglutaminase (MTGase) and click chemistry to achieve site-specific conjugation of nanobodies and aptamers, which are termed nanotamers. The nanotamers recognize and bind to two types of molecular targets expressed on cancer cells. As a prototype, a bispecific nanotamer is developed that binds both clusters of differentiation 47 (CD47) and mesenchymal epithelial transition receptor (Met) expressed on the tumor cell membrane. This CD47-Met nanotamer demonstrates high affinity and specificity toward tumor cells expressing both targets, exhibits improved receptor functional inhibition through a strong steric hindrance effect. Moreover, its capacity for deep tumor penetration greatly enhances the impact of conventional chemotherapy on antitumor efficacy. The as-developed nanotamer synthesis approach shows promise to customize bispecific molecular probes targeting different cancer types and different therapeutic goals.
Assuntos
Anticorpos Biespecíficos , Aptâmeros de Nucleotídeos , Neoplasias , Anticorpos de Domínio Único , Humanos , Aptâmeros de Nucleotídeos/química , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/farmacologia , Neoplasias/tratamento farmacológico , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Linhagem Celular Tumoral , AnimaisRESUMO
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
For a certain number of mAbs, bispecific antibodies (bsAbs) and Fc-fusion proteins that we worked on, the Protein A capture step experienced low yield (i.e., â¼80%). A previous case study suggested that non-binding aggregate formed in cell culture was the root cause of low Protein A step yield. In the current work, we selected five projects with the low Protein A yield issue to further illustrate this phenomenon. In all cases, existence of non-binding aggregates was confirmed by size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) analysis of Protein A load and flow-through. In addition, we demonstrated that aggregates failed to bind to Protein A resin mainly due to their large sizes, which prevented them from entering the resin beads. As the data suggested, SEC-HPLC analysis of Protein A load and flow-through, although not a standard procedure, can provide information that is critical for understanding the unexpected performance of Protein A chromatography in cases like those being presented here. Thus, SEC-HPLC analysis of Protein A load and flow-through is highly recommended for antibodies/Fc-fusions suffering from low Protein A yield.
Assuntos
Anticorpos Biespecíficos , Técnicas de Cultura de Células , Cromatografia Líquida de Alta Pressão , Cromatografia em Gel , Anticorpos Monoclonais/química , Anticorpos Biespecíficos/química , Proteína Estafilocócica A/químicaRESUMO
Size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) is an analytical method routinely used for assessing aggregation content in protein samples. As SEC-HPLC separates analytes based on their hydrodynamic radius, it generally lacks the capability of differentiating species that are similar in size. Recently while purifying a bispecific antibody (bsAb), we noticed that SEC-HPLC can provide certain degree of resolution between the target bsAb and a disulfide scrambled form, although these two species were identical in molecular weight. In seeing the unexpected potential of SEC-HPLC at resolving species with similar size, we further tested Zenix SEC-300, a mixed-mode SEC-HPLC column from Sepax, which was reported to be capable of separating protein analytes based on other factors besides size. The Zenix column indeed provided resolution much better than the regular SEC-HPLC column. Upon further optimization, the Zenix column allowed close to baseline separation of the correctly folded and the disulfide scrambled species. The current study, as a complement to the previous reports, further demonstrates that mix-mode SEC-HPLC is capable of separating protein analytes that are close in size but are different in conformation and/or surface characteristics.
Assuntos
Anticorpos Biespecíficos , Cromatografia em Gel , Dissulfetos , Anticorpos Biespecíficos/isolamento & purificação , Anticorpos Biespecíficos/química , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia em Gel/métodos , Dissulfetos/química , Humanos , AnimaisRESUMO
Protein A affinity chromatography has been widely used for initial product capture in recombinant antibody/Fc-fusion purification. However, in general Protein A lacks the capability of separating aggregates (unless the aggregates are too large to enter the pores of resin beads or have their Protein A binding sites buried, in which case the aggregates do not bind). In the current work, we demonstrated that CaptureSelect FcXP affinity medium exhibited strong aggregate separation capability and effectively removed aggregates under pH or conductivity gradient elution in two bispecific antibody (bsAb) cases. For these two cases, aggregate contents were reduced from >16% and >22% (in the feed) to <1% and <5% (in the eluate) for the first and second bsAbs, respectively. While more case studies are required to further demonstrate FcXP's superiority in aggregate removal, findings from the current study suggest that FcXP can potentially be a better alternative than Protein A for product capture in cases where aggregate content is high.
Assuntos
Anticorpos Biespecíficos , Cromatografia de Afinidade , Proteína Estafilocócica A , Cromatografia de Afinidade/métodos , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/isolamento & purificação , Proteína Estafilocócica A/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/genética , Agregados Proteicos , Humanos , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/isolamento & purificaçãoRESUMO
Introduction: The therapeutic potential of bispecific antibodies is becoming widely recognised, with over a hundred formats already described. For many applications, enhanced tissue penetration is sought, so bispecifics with low molecular weight may offer a route to enhanced potency. Here we report the design of bi- and tri-specific antibody-based constructs with molecular weights as low as 14.5 and 22 kDa respectively. Methods: Autonomous bovine ultra-long CDR H3 (knob domain peptide) modules have been engineered with artificial coiled-coil stalks derived from Sin Nombre orthohantavirus nucleocapsid protein and human Beclin-1, and joined in series to produce bi- and tri-specific antibody-based constructs with exceptionally low molecular weights. Results: Knob domain peptides with coiled-coil stalks retain high, independent antigen binding affinity, exhibit exceptional levels of thermal stability, and can be readily joined head-to-tail yielding the smallest described multi-specific antibody format. The resulting constructs are able to bind simultaneously to all their targets with no interference. Discussion: Compared to existing bispecific formats, the reduced molecular weight of the knob domain fusions may enable enhanced tissue penetration and facilitate binding to cryptic epitopes that are inaccessible to conventional antibodies. Furthermore, they can be easily produced at high yield as recombinant products and are free from the heavy-light chain mispairing issue. Taken together, our approach offers an efficient route to modular construction of minimalistic bi- and multi-specifics, thereby further broadening the therapeutic scope for knob domain peptides.
Assuntos
Anticorpos Biespecíficos , Animais , Bovinos , Humanos , Anticorpos Biespecíficos/química , Peptídeos , Proteínas do NucleocapsídeoRESUMO
Developing a knob-into-hole asymmetric bispecific IgG1 monoclonal antibody (mAb) poses manufacturing challenges due to the expression of chain pairing variants, also called mispaired species, in the desired product. The incorrect pairing of light and heavy chains could result in heterogeneous mispaired species of homodimers, heterodimers, light chain swapping, and low molecular weight species (LMWS). Standard chromatography, capillary electrophoretic, or spectroscopic methods poorly resolve these from the main variants. Here, we report a highly sensitive reverse-phase polyphenyl ultra-high-performance liquid chromatography (RP-UHPLC) method to accurately measure mispaired species of Duet mAb format, an asymmetric IgG1 bispecific mAb, for both process development and quality control analytical tests. Coupled with electrospray ionization mass spectrometry (ESI-MS), it enabled direct online characterization of mispaired species. This single direct assay detected diverse mispaired IgG-like species and LMWS. The method resolved eight disulfide bonds dissociated LMWS and three mispaired LMWS. It also resolved three different types of IgG-like mispaired species, including two homodimers and one heterodimer. The characterization and quantification simultaneously enabled the cell line selection that produces a lesser heterogeneity and lower levels of mispaired species with the desired correctly paired product. The biological activity assessment of samples with increased levels of these species quantified by the method exhibited a linear decline in potency with increasing levels of mispaired species in the desired product. We also demonstrated the utility of the technique for testing in-process intermediate materials to determine and assess downstream purification process capability in removing diverse mispaired IgG-like species and LMWS to a certain level during the downstream purification process. Our investigation demonstrates that adopting this method was vital in developing asymmetric bispecific mAb from the initial stage of cell line development to manufacturing process development. Therefore, this tool could be used in the control strategy to monitor and control mispaired species during manufacturing, thus improving the quality control of the final product.
Assuntos
Anticorpos Biespecíficos , Espectrometria de Massas por Ionização por Electrospray , Imunoglobulina G/química , Cromatografia de Fase Reversa , Domínios Proteicos , Anticorpos Biespecíficos/química , Anticorpos Monoclonais/químicaRESUMO
Bispecific antibodies (BsAbs) represent an emerging class of biologics that can recognize two different antigens or epitopes. T-cell engagers (TcEs) bind two targets in trans on the cell surface of the effector and target cell to induce proximal immune effects, opening exciting windows for immunotherapies. To date, the engineering of BsAbs has been mainly focused on tuning the molecular weight and valency. However, the effects of spatial factors on the biological functions of BsAbs have been less explored due to the lack of biochemical methods to precisely manipulate protein geometry. Here, we studied the geometric effects of the TcEs. First, by genetically inserting rigidly designed ankyrin repeat proteins into TcEs, we revealed that the efficacy progressively decreased as the spacer distance of the two binding domains increased. Then, we constructed 26 pairs of TcEs with the same size but varying orientations using click chemistry-mediated conjugation at different mutation sites. We found that linear ligation sites play a minor role in modulating cell-killing efficacy. Next, we rendered the TcEs' advanced topology by cyclization chemistry using the SpyTag/SpyCatcher pair or sortase ligation approaches. Cyclized TcEs were generally more potent than their linear counterparts. Particularly, sortase A cyclized TcEs, bearing a minimal tagging motif, exhibited better cell-killing efficacy in vitro and improved stability both in vitro and in vivo compared to the linear TcE. This work combines modern bioconjugation chemistry and protein engineering tools for antibody engineering, shedding light on the elusive spatial factors of BsAbs functionality.
Assuntos
Anticorpos Biespecíficos , Linfócitos T , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Biespecíficos/química , Química Click , Engenharia de Proteínas/métodos , Proteínas , Linfócitos T/imunologia , HumanosRESUMO
Biotherapeutics hold great promise for the treatment of several diseases and offer innovative possibilities for new treatments that target previously unaddressed medical needs. Despite successful transitions from preclinical to clinical stages and regulatory approval, there are instances where adverse reactions arise, resulting in product withdrawals. As a result, it is essential to conduct thorough evaluations of safety and effectiveness on an individual basis. This article explores current practices, challenges, and future approaches in conducting comprehensive preclinical assessments to ensure the safety and efficacy of biotherapeutics including monoclonal antibodies, toxin-conjugates, bispecific antibodies, single-chain antibodies, Fc-engineered antibodies, antibody mimetics, and siRNA-antibody/peptide conjugates.
Assuntos
Anticorpos Monoclonais , Avaliação Pré-Clínica de Medicamentos , Humanos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/imunologia , Imunoconjugados/químicaRESUMO
Bispecific antibodies expressed and assembled from a single upstream culture require the correct balance and pairing of four different heavy and light chains (HC and LC). The increased potential for chain-mispaired species challenges the downstream purification of this new format. While clearance of HC-mispaired species, including homodimers and half-antibodies, has been assessed, removal of LC mispairs requires a more stringent approach. Here, we report two case studies in which separation is achieved, as well as the structural basis of these separations: (A) In the first case, a main species with a positively charged patch in the correctly formed variable fragment (Fv) is disrupted when paired with the wrong LC. This LC-mispaired variant binds more weakly to a cation exchange resin and can be washed off in a chromatography step. (B) A second molecule whose LC mispair introduces a negative-charge patch and hydrophobic patch in close proximity, presenting increased binding to a multimodal anion exchange resin. This LC-mispaired variant can be retained on the column under conditions in which the bispecific is recovered. In both case studies, the molecular structural analysis by protein surface properties models correlated well with the chromatography experiments. The comprehensive interpretation of experimental and computational results has provided a better understanding of strategies and potential applications for predicting the downstream purification of complex molecules.
Assuntos
Anticorpos Biespecíficos , Cadeias Leves de Imunoglobulina , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/isolamento & purificação , Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/isolamento & purificação , Cromatografia por Troca Iônica/métodos , Animais , Células CHO , Cricetulus , Modelos MolecularesRESUMO
The growing clinical success of bispecific antibodies (bsAbs) has led to rapid interest in leveraging dual targeting in order to generate novel modes of therapeutic action beyond mono-targeting approaches. While bsAbs that bind targets on two different cells (trans-targeting) are showing promise in the clinic, the co-targeting of two proteins on the same cell surface through cis-targeting bsAbs (cis-bsAbs) is an emerging strategy to elicit new functionalities. This includes the ability to induce proximity, enhance binding to a target, increase target/cell selectivity, and/or co-modulate function on the cell surface with the goal of altering, reversing, or eradicating abnormal cellular activity that contributes to disease. In this review, we focus on the impact of cis-bsAbs in the clinic, their emerging applications, and untangle the intricacies of improving bsAb discovery and development.
Assuntos
Anticorpos Biespecíficos , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/imunologia , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , AnimaisRESUMO
The development of bispecific antibodies that bind at least two different targets relies on bringing together multiple binding domains with different binding properties and biophysical characteristics to produce a drug-like therapeutic. These building blocks play an important role in the overall quality of the molecule and can influence many important aspects from potency and specificity to stability and half-life. Single-domain antibodies, particularly camelid-derived variable heavy domain of heavy chain (VHH) antibodies, are becoming an increasingly popular choice for bispecific construction due to their single-domain modularity, favorable biophysical properties, and potential to work in multiple antibody formats. Here, we review the use of VHH domains as building blocks in the construction of multispecific antibodies and the challenges in creating optimized molecules. In addition to exploring traditional approaches to VHH development, we review the integration of machine learning techniques at various stages of the process. Specifically, the utilization of machine learning for structural prediction, lead identification, lead optimization, and humanization of VHH antibodies.
Assuntos
Anticorpos Biespecíficos , Aprendizado de Máquina , Anticorpos de Domínio Único , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/química , Humanos , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/química , Animais , Engenharia de Proteínas/métodos , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/químicaRESUMO
Bispecific agents are a rapidly growing class of cancer therapeutics, and immune targeted bispecific agents have the potential to expand functionality well beyond monoclonal antibody agents. Humabodiesâ are fully human single domain antibodies that can be linked in a modular fashion to form multispecific therapeutics. However, the effect of heterogeneous delivery on the efficacy of crosslinking bispecific agents is currently unclear. In this work, we utilize a PSMA-CD137 Humabody with an albumin binding half-life extension (HLE) domain to determine the impact of tissue penetration on T cell activating bispecific agents. Using heterotypic spheroids, we demonstrate that increased tissue penetration results in higher T cell activation at sub-saturating concentrations. Next, we tested the effect of two different albumin binding moieties on tissue distribution using albumin-specific HLE domains with varying affinities for albumin and a non-specific lipophilic dye. The results show that a specific binding mechanism to albumin does not influence tissue penetration, but a non-specific mechanism reduced both spheroid uptake and distribution in the presence of albumin. These results highlight the potential importance of tissue penetration on bispecific agent efficacy and describe how the design parameters including albumin-binding domains can be selected to maximize the efficacy of bispecific agents.
Assuntos
Anticorpos Biespecíficos , Antineoplásicos , Neoplasias , Humanos , Linfócitos T , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/química , Albuminas/uso terapêutico , Neoplasias/tratamento farmacológico , Antineoplásicos/uso terapêuticoRESUMO
Appended bispecific antibody (aBsAb) with two single chain variable fragments (scFv) linked at the c-terminus of its heavy chains is one of the promising formats in bispecific therapeutics. The presence of hydrophobic and flexible scFv fragments render aBsAb molecules higher molecule hydrophobicity and structural flexibility compared to monoclonal antibody (mAb), thus making its purification more challenging. We set out to investigate how the unique molecular properties of aBsAb affect its performance on Protein A chromatography. We showed that aBsAb has a high propensity for chromatography-induced aggregation due to its high molecule hydrophobicity, and this couldn't be improved by the addition of common chaotropic salts. Moreover, the presence of chaotropic salts, such as arginine hydrochloride (Arg-HCl), retarded aBsAb elution during Protein A chromatography rather than facilitating which was widely observed in mAb Protein A elution. Nevertheless, we were able to overcome the aggregation issue by optimizing elution condition and improved aBsAb purity from 29 % to 93 % in Protein A eluate with a high molecular weight (HMW) species of less than 5 %. We also showed that the high molecular flexibility of aBsAb leads to different hydrodynamic sizes of the aBsAb molecule post Protein A elution, neutralization, and re-acidification, which are pH dependent. This is different from mAbs where their sizes do not change post neutralization even with re-exposure to acid. The above unique observations of aBsAb in Protein A chromatography were clearly explained from the perspectives of its high molecular hydrophobicity and structural flexibility.
Assuntos
Anticorpos Biespecíficos , Interações Hidrofóbicas e Hidrofílicas , Proteína Estafilocócica A , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/isolamento & purificação , Proteína Estafilocócica A/química , Cromatografia de Afinidade/métodos , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/isolamento & purificação , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Concentração de Íons de Hidrogênio , HumanosRESUMO
The emergence of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) has posed a significant global health concern due to its severe respiratory illness and high fatality rate. Currently, despite the potential for resurgence, there are no specific treatments for MERS-CoV, and only supportive care is available. Our study aimed to address this therapeutic gap by developing a potent neutralizing bispecific antibody (bsAb) against MERS-CoV. Initially, we isolated four human monoclonal antibodies (mAbs) that specifically target the MERS-CoV receptor-binding domain (RBD) using phage display technology and an established human antibody library. Among these four selected mAbs, our intensive in vitro functional analyses showed that the MERS-CoV RBD-specific mAb K111.3 exhibited the most potent neutralizing activity against MERS-CoV pseudoviral infection and the molecular interaction between MERS-CoV RBD and human dipeptidyl peptidase 4. Consequently, we engineered a novel bsAb, K207.C, by utilizing K111.3 as the IgG base and fusing it with the single-chain variable fragment of its non-competing pair, K111.1. This engineered bsAb showed significantly enhanced neutralization potential against MERS-CoV compared to its parental mAb. These findings suggest that K207.C may serve as a potential candidate for effective MERS-CoV neutralization, further highlighting the promise of the bsAb dual-targeting approach in MERS-CoV neutralization.
Assuntos
Anticorpos Biespecíficos , Anticorpos Neutralizantes , Anticorpos Antivirais , Coronavírus da Síndrome Respiratória do Oriente Médio , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Humanos , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Animais , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/química , Anticorpos Monoclonais/imunologia , Ligação Proteica , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Dipeptidil Peptidase 4/imunologia , Camundongos , Testes de NeutralizaçãoRESUMO
The precise regulation of protein function is essential in biological systems and a key goal in chemical biology and protein engineering. Here, we describe a straightforward method to engineer functional control into the isopeptide bond-forming SpyTag/SpyCatcher protein ligation system. First, we perform a cysteine scan of the structured region of SpyCatcher. Except for two known reactive and catalytic residues, none of these mutations abolish reactivity. In a second screening step, we modify the cysteines with disulfide bond-forming small molecules. Here we identify 8 positions at which modifications strongly inhibit reactivity. This inhibition can be reversed by reducing agents. We call such a reversibly inhibitable SpyCatcher "SpyLock". Using "BiLockCatcher", a genetic fusion of wild-type SpyCatcher and SpyLock, and SpyTagged antibody fragments, we generate bispecific antibodies in a single, scalable format, facilitating the screening of a large number of antibody combinations. We demonstrate this approach by screening anti-PD-1/anti-PD-L1 bispecific antibodies using a cellular reporter assay.
Assuntos
Anticorpos Biespecíficos , Cisteína , Engenharia de Proteínas , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/química , Humanos , Engenharia de Proteínas/métodos , Cisteína/química , Cisteína/metabolismo , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Antígeno B7-H1/metabolismo , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Células HEK293 , Dissulfetos/química , AnimaisRESUMO
Cancer immunotherapy by immune checkpoint inhibitors (ICIs) acts on antitumor responses by stimulating the immune system to attack cancer cells. However, this powerful therapy is hampered by its high treatment cost and limited efficacy. Here, it is shown that the development of an antibody-conjugated nanogel (ANGel), consisting of N-isopropylacrylamide-co-acrylic acid and antibody-binding protein (protein A), potentiates the efficacy of two ICI monoclonal antibodies (mAbs) (cytotoxic-T-lymphocyte-associated antigen 4 and programmed death ligand-1 mAbs). Compared with mAb treatment alone, treatment with a bispecific ANGel surface-conjugated with the mAbs significantly decreases both the survival of Michigan Cancer Foundation-7 (MCF-7) and M D Anderson-Metastatic Breast-231 (MDA-MB-231) breast cancer cells in vitro and the burden of 4T1-luciferase-2-derived orthotopic syngeneic tumors in vivo. The bispecific ANGel is also more potent than the conventional treatment at prolonging survival in animals with triple-negative breast cancer. The advantage of the bispecific ANGel over other engineered bispecific antibodies arises not only from the adaptability to link multiple antibodies quickly and easily, but also from the capability to maintain the anticancer effect steadily at subcutaneously delivered tumor site. This finding has an important implication for cancer immunotherapy, opening a new paradigm to treat solid tumors.