Despite the advances in surface-display systems for directed evolution, variants with high affinity are not always enriched due to undesirable biases that increase target-unrelated variants during biopanning. Here, our goal was to design a library containing improved variants from the information of the "weakly enriched" library where functional variants were weakly enriched. Deep sequencing for the previous biopanning result, where no functional antibody mimetics were experimentally identified, revealed that weak enrichment was partly due to undesirable biases during phage infection and amplification steps. The clustering analysis of the deep sequencing data from appropriate steps revealed no distinct sequence patterns, but a Bayesian machine learning model trained with the selected deep sequencing data supplied nine clusters with distinct sequence patterns. Phage libraries were designed on the basis of the sequence patterns identified, and four improved variants with target-specific affinity (EC50 = 80-277 nM) were identified by biopanning. The selection and use of deep sequencing data without undesirable bias enabled us to extract the information on prospective variants. In summary, the use of appropriate deep sequencing data and machine learning with the sequence data has the possibility of finding sequence space where functional variants are enriched.
{"title":"Selection of target-binding proteins from the information of weakly enriched phage display libraries by deep sequencing and machine learning.","authors":"Tomoyuki Ito, Thuy Duong Nguyen, Yutaka Saito, Yoichi Kurumida, Hikaru Nakazawa, Sakiya Kawada, Hafumi Nishi, Koji Tsuda, Tomoshi Kameda, Mitsuo Umetsu","doi":"10.1080/19420862.2023.2168470","DOIUrl":"https://doi.org/10.1080/19420862.2023.2168470","url":null,"abstract":"<p><p>Despite the advances in surface-display systems for directed evolution, variants with high affinity are not always enriched due to undesirable biases that increase target-unrelated variants during biopanning. Here, our goal was to design a library containing improved variants from the information of the \"weakly enriched\" library where functional variants were weakly enriched. Deep sequencing for the previous biopanning result, where no functional antibody mimetics were experimentally identified, revealed that weak enrichment was partly due to undesirable biases during phage infection and amplification steps. The clustering analysis of the deep sequencing data from appropriate steps revealed no distinct sequence patterns, but a Bayesian machine learning model trained with the selected deep sequencing data supplied nine clusters with distinct sequence patterns. Phage libraries were designed on the basis of the sequence patterns identified, and four improved variants with target-specific affinity (EC<sub>50</sub> = 80-277 nM) were identified by biopanning. The selection and use of deep sequencing data without undesirable bias enabled us to extract the information on prospective variants. In summary, the use of appropriate deep sequencing data and machine learning with the sequence data has the possibility of finding sequence space where functional variants are enriched.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":null,"pages":null},"PeriodicalIF":5.3,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/51/e4/KMAB_15_2168470.PMC9872955.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10679887","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-01-01DOI: 10.1080/19420862.2023.2242548
Emma Kelsall, Claire Harris, Titash Sen, Diane Hatton, Sarah Dunn, Suzanne Gibson
Introns are included in genes encoding therapeutic proteins for their well-documented function of boosting expression. However, mis-splicing of introns in recombinant immunoglobulin (IgG) heavy chain (HC) transcripts can produce amino acid sequence product variants. These variants can affect product quality; therefore, purification process optimization may be needed to remove them, or if they cannot be removed, then in-depth characterization must be carried out to understand their effects on biological activity. In this study, HC transgene engineering approaches were investigated and were successful in significantly reducing the previously identified IgG HC splice variants to <0.5%. Subsequently, a comprehensive evaluation was conducted to understand the influence of the different introns in the HC genes on the expression of recombinant biotherapeutic antibodies. The data revealed an unexpected cooperation between specific introns for efficient splicing, where intron retention led to significant reductions in IgG expression of up to 75% for some intron combinations. Furthermore, it was shown that HC introns could be fully removed without significantly affecting productivity. This work paves the way for future biotherapeutic antibody transgene design with regard to inclusion of HC introns. By removing unnecessary introns, transgene mRNA transcript will no longer be mis-spliced, thereby eliminating HC splice variants and improving antibody product quality.
{"title":"Interplay of heavy chain introns influences efficient transcript splicing and affects product quality of recombinant biotherapeutic antibodies from CHO cells.","authors":"Emma Kelsall, Claire Harris, Titash Sen, Diane Hatton, Sarah Dunn, Suzanne Gibson","doi":"10.1080/19420862.2023.2242548","DOIUrl":"10.1080/19420862.2023.2242548","url":null,"abstract":"<p><p>Introns are included in genes encoding therapeutic proteins for their well-documented function of boosting expression. However, mis-splicing of introns in recombinant immunoglobulin (IgG) heavy chain (HC) transcripts can produce amino acid sequence product variants. These variants can affect product quality; therefore, purification process optimization may be needed to remove them, or if they cannot be removed, then in-depth characterization must be carried out to understand their effects on biological activity. In this study, HC transgene engineering approaches were investigated and were successful in significantly reducing the previously identified IgG HC splice variants to <0.5%. Subsequently, a comprehensive evaluation was conducted to understand the influence of the different introns in the HC genes on the expression of recombinant biotherapeutic antibodies. The data revealed an unexpected cooperation between specific introns for efficient splicing, where intron retention led to significant reductions in IgG expression of up to 75% for some intron combinations. Furthermore, it was shown that HC introns could be fully removed without significantly affecting productivity. This work paves the way for future biotherapeutic antibody transgene design with regard to inclusion of HC introns. By removing unnecessary introns, transgene mRNA transcript will no longer be mis-spliced, thereby eliminating HC splice variants and improving antibody product quality.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":null,"pages":null},"PeriodicalIF":5.3,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/f5/c6/KMAB_15_2242548.PMC10413919.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9980545","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-01-01DOI: 10.1080/19420862.2023.2181016
Kathryn Ball, Simon J Dovedi, Pavan Vajjah, Alex Phipps
Innovative approaches in the design of T cell-engaging (TCE) molecules are ushering in a new wave of promising immunotherapies for the treatment of cancer. Their mechanism of action, which generates an in trans interaction to create a synthetic immune synapse, leads to complex and interconnected relationships between the exposure, efficacy, and toxicity of these drugs. Challenges thus arise when designing optimal clinical dose regimens for TCEs with narrow therapeutic windows, with a variety of dosing strategies being evaluated to mitigate key side effects such as cytokine release syndrome, neurotoxicity, and on-target off-tumor toxicities. This review evaluates the current approaches to dose optimization throughout the preclinical and clinical development of TCEs, along with perspectives for improvement of these strategies. Quantitative approaches used to aid the understanding of dose-exposure-response relationships are highlighted, along with opportunities to guide the rational design of next-generation TCE molecules, and optimize their dose regimens in patients.
{"title":"Strategies for clinical dose optimization of T cell-engaging therapies in oncology.","authors":"Kathryn Ball, Simon J Dovedi, Pavan Vajjah, Alex Phipps","doi":"10.1080/19420862.2023.2181016","DOIUrl":"https://doi.org/10.1080/19420862.2023.2181016","url":null,"abstract":"<p><p>Innovative approaches in the design of T cell-engaging (TCE) molecules are ushering in a new wave of promising immunotherapies for the treatment of cancer. Their mechanism of action, which generates an in <i>trans</i> interaction to create a synthetic immune synapse, leads to complex and interconnected relationships between the exposure, efficacy, and toxicity of these drugs. Challenges thus arise when designing optimal clinical dose regimens for TCEs with narrow therapeutic windows, with a variety of dosing strategies being evaluated to mitigate key side effects such as cytokine release syndrome, neurotoxicity, and on-target off-tumor toxicities. This review evaluates the current approaches to dose optimization throughout the preclinical and clinical development of TCEs, along with perspectives for improvement of these strategies. Quantitative approaches used to aid the understanding of dose-exposure-response relationships are highlighted, along with opportunities to guide the rational design of next-generation TCE molecules, and optimize their dose regimens in patients.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":null,"pages":null},"PeriodicalIF":5.3,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/b5/8f/KMAB_15_2181016.PMC9980545.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10643374","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-01-01Epub Date: 2023-09-22DOI: 10.1080/19420862.2023.2256668
Inga Petersen, Muhammad Ilyas Ali, Alex Petrovic, Anders Jimmy Ytterberg, Karin Staxäng, Monika Hodik, Fadi Rofo, Sina Bondza, Greta Hultqvist
Soluble aggregates are reported to be the most neurotoxic species of α-Synuclein (αSyn) in Parkinson's disease (PD) and hence are a promising target for diagnosis and treatment of PD. However, the predominantly intracellular location of αSyn limits its accessibility, especially for antibody-based molecules and prompts the need for exceptionally strong soluble αSyn aggregate binders to enhance their sensitivity and efficacy for targeting the extracellular αSyn pool. In this study, we have created the multivalent antibodies TetraSynO2 and HexaSynO2, derived from the αSyn oligomer-specific antibody SynO2, to increase avidity binding to soluble αSyn aggregate species through more binding sites in close proximity. The multivalency was achieved through recombinant fusion of single-chain variable fragments of SynO2 to the antibodies' original N-termini. Our ELISA results indicated a 20-fold increased binding strength of the multivalent formats to αSyn aggregates, while binding to αSyn monomers and unspecific binding to amyloid β protofibrils remained low. Kinetic analysis using LigandTracer revealed that only 80% of SynO2 bound bivalently to soluble αSyn aggregates, whereas the proportion of TetraSynO2 and HexaSynO2 binding bi- or multivalently to soluble αSyn aggregates was increased to ~ 95% and 100%, respectively. The overall improved binding strength of TetraSynO2 and HexaSynO2 implies great potential for immunotherapeutic and diagnostic applications with targets of limited accessibility, like extracellular αSyn aggregates. The ability of the multivalent antibodies to bind a wider range of αSyn aggregate species, which are not targetable by conventional bivalent antibodies, thus could allow for an earlier and more effective intervention in the progression of PD.
{"title":"Multivalent design of the monoclonal SynO2 antibody improves binding strength to soluble α-Synuclein aggregates.","authors":"Inga Petersen, Muhammad Ilyas Ali, Alex Petrovic, Anders Jimmy Ytterberg, Karin Staxäng, Monika Hodik, Fadi Rofo, Sina Bondza, Greta Hultqvist","doi":"10.1080/19420862.2023.2256668","DOIUrl":"10.1080/19420862.2023.2256668","url":null,"abstract":"<p><p>Soluble aggregates are reported to be the most neurotoxic species of α-Synuclein (αSyn) in Parkinson's disease (PD) and hence are a promising target for diagnosis and treatment of PD. However, the predominantly intracellular location of αSyn limits its accessibility, especially for antibody-based molecules and prompts the need for exceptionally strong soluble αSyn aggregate binders to enhance their sensitivity and efficacy for targeting the extracellular αSyn pool. In this study, we have created the multivalent antibodies TetraSynO2 and HexaSynO2, derived from the αSyn oligomer-specific antibody SynO2, to increase avidity binding to soluble αSyn aggregate species through more binding sites in close proximity. The multivalency was achieved through recombinant fusion of single-chain variable fragments of SynO2 to the antibodies' original N-termini. Our ELISA results indicated a 20-fold increased binding strength of the multivalent formats to αSyn aggregates, while binding to αSyn monomers and unspecific binding to amyloid β protofibrils remained low. Kinetic analysis using LigandTracer revealed that only 80% of SynO2 bound bivalently to soluble αSyn aggregates, whereas the proportion of TetraSynO2 and HexaSynO2 binding bi- or multivalently to soluble αSyn aggregates was increased to ~ 95% and 100%, respectively. The overall improved binding strength of TetraSynO2 and HexaSynO2 implies great potential for immunotherapeutic and diagnostic applications with targets of limited accessibility, like extracellular αSyn aggregates. The ability of the multivalent antibodies to bind a wider range of αSyn aggregate species, which are not targetable by conventional bivalent antibodies, thus could allow for an earlier and more effective intervention in the progression of PD.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":null,"pages":null},"PeriodicalIF":5.3,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10519360/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41179245","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-01-01Epub Date: 2023-10-12DOI: 10.1080/19420862.2023.2261509
Hans Peter Grimm, Vanessa Schumacher, Martin Schäfer, Sabine Imhof-Jung, Per-Ola Freskgård, Kevin Brady, Carsten Hofmann, Petra Rüger, Tilman Schlothauer, Ulrich Göpfert, Maximilian Hartl, Sylvia Rottach, Adrian Zwick, Shanon Seger, Rachel Neff, Jens Niewoehner, Niels Janssen
There are few treatments that slow neurodegeneration in Alzheimer's disease (AD), and while therapeutic antibodies are being investigated in clinical trials for AD treatment, their access to the central nervous system is restricted by the blood-brain barrier. This study investigates a bispecific modular fusion protein composed of gantenerumab, a fully human monoclonal anti- amyloid-beta (Aβ) antibody under investigation for AD treatment, with a human transferrin receptor 1-directed Brainshuttle™ module (trontinemab; RG6102, INN trontinemab). In vitro, trontinemab showed a similar binding affinity to fibrillar Aβ40 and Aβ plaques in human AD brain sections to gantenerumab. A single intravenous administration of trontinemab (10 mg/kg) or gantenerumab (20 mg/kg) to non-human primates (NHPs, Macaca fascicularis), was well tolerated in both groups. Immunohistochemistry indicated increased trontinemab uptake into the brain endothelial cell layer and parenchyma, and more homogeneous distribution, compared with gantenerumab. Brain and plasma pharmacokinetic (PK) parameters for trontinemab were estimated by nonlinear mixed-effects modeling with correction for tissue residual blood, indicating a 4-18-fold increase in brain exposure. A previously developed clinical PK/pharmacodynamic model of gantenerumab was adapted to include a brain compartment as a driver of plaque removal and linked to the allometrically scaled above model from NHP. The new brain exposure-based model was used to predict trontinemab dosing regimens for effective amyloid reduction. Simulations from these models were used to inform dosing of trontinemab in the first-in-human clinical trial.
{"title":"Delivery of the Brainshuttle™ amyloid-beta antibody fusion trontinemab to non-human primate brain and projected efficacious dose regimens in humans.","authors":"Hans Peter Grimm, Vanessa Schumacher, Martin Schäfer, Sabine Imhof-Jung, Per-Ola Freskgård, Kevin Brady, Carsten Hofmann, Petra Rüger, Tilman Schlothauer, Ulrich Göpfert, Maximilian Hartl, Sylvia Rottach, Adrian Zwick, Shanon Seger, Rachel Neff, Jens Niewoehner, Niels Janssen","doi":"10.1080/19420862.2023.2261509","DOIUrl":"10.1080/19420862.2023.2261509","url":null,"abstract":"<p><p>There are few treatments that slow neurodegeneration in Alzheimer's disease (AD), and while therapeutic antibodies are being investigated in clinical trials for AD treatment, their access to the central nervous system is restricted by the blood-brain barrier. This study investigates a bispecific modular fusion protein composed of gantenerumab, a fully human monoclonal anti- amyloid-beta (Aβ) antibody under investigation for AD treatment, with a human transferrin receptor 1-directed Brainshuttle™ module (trontinemab; RG6102, INN trontinemab). <i>In vitro</i>, trontinemab showed a similar binding affinity to fibrillar Aβ<sub>40</sub> and Aβ plaques in human AD brain sections to gantenerumab. A single intravenous administration of trontinemab (10 mg/kg) or gantenerumab (20 mg/kg) to non-human primates (NHPs, <i>Macaca fascicularis</i>), was well tolerated in both groups. Immunohistochemistry indicated increased trontinemab uptake into the brain endothelial cell layer and parenchyma, and more homogeneous distribution, compared with gantenerumab. Brain and plasma pharmacokinetic (PK) parameters for trontinemab were estimated by nonlinear mixed-effects modeling with correction for tissue residual blood, indicating a 4-18-fold increase in brain exposure. A previously developed clinical PK/pharmacodynamic model of gantenerumab was adapted to include a brain compartment as a driver of plaque removal and linked to the allometrically scaled above model from NHP. The new brain exposure-based model was used to predict trontinemab dosing regimens for effective amyloid reduction. Simulations from these models were used to inform dosing of trontinemab in the first-in-human clinical trial.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":null,"pages":null},"PeriodicalIF":5.3,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/f7/9d/KMAB_15_2261509.PMC10572082.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41204580","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-01-01DOI: 10.1007/978-3-031-22669-4
R. Martínez-Guerra, Juan Pablo Flores-Flores
{"title":"An Approach to Multi-agent Systems as a Generalized Multi-synchronization Problem","authors":"R. Martínez-Guerra, Juan Pablo Flores-Flores","doi":"10.1007/978-3-031-22669-4","DOIUrl":"https://doi.org/10.1007/978-3-031-22669-4","url":null,"abstract":"","PeriodicalId":18206,"journal":{"name":"mAbs","volume":null,"pages":null},"PeriodicalIF":5.3,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84054751","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-01-01Epub Date: 2023-11-27DOI: 10.1080/19420862.2023.2285277
M Desai, A Kundu, M Hageman, H Lou, D Boisvert
Biologic drugs are used to treat a variety of cancers and chronic diseases. While most of these treatments are administered intravenously by trained healthcare professionals, a noticeable trend has emerged favoring subcutaneous (SC) administration. SC administration of biologics poses several challenges. Biologic drugs often require higher doses for optimal efficacy, surpassing the low volume capacity of traditional SC delivery methods like autoinjectors. Consequently, high concentrations of active ingredients are needed, creating time-consuming formulation obstacles. Alternatives to traditional SC delivery systems are therefore needed to support higher-volume biologic formulations and to reduce development time and other risks associated with high-concentration biologic formulations. Here, we outline key considerations for SC biologic drug formulations and delivery and explore a paradigm shift: the flexibility afforded by low-to-moderate-concentration drugs in high-volume formulations as an alternative to the traditionally difficult approach of high-concentration, low-volume SC formulation delivery.
{"title":"Monoclonal antibody and protein therapeutic formulations for subcutaneous delivery: high-concentration, low-volume vs. low-concentration, high-volume.","authors":"M Desai, A Kundu, M Hageman, H Lou, D Boisvert","doi":"10.1080/19420862.2023.2285277","DOIUrl":"10.1080/19420862.2023.2285277","url":null,"abstract":"<p><p>Biologic drugs are used to treat a variety of cancers and chronic diseases. While most of these treatments are administered intravenously by trained healthcare professionals, a noticeable trend has emerged favoring subcutaneous (SC) administration. SC administration of biologics poses several challenges. Biologic drugs often require higher doses for optimal efficacy, surpassing the low volume capacity of traditional SC delivery methods like autoinjectors. Consequently, high concentrations of active ingredients are needed, creating time-consuming formulation obstacles. Alternatives to traditional SC delivery systems are therefore needed to support higher-volume biologic formulations and to reduce development time and other risks associated with high-concentration biologic formulations. Here, we outline key considerations for SC biologic drug formulations and delivery and explore a paradigm shift: the flexibility afforded by low-to-moderate-concentration drugs in high-volume formulations as an alternative to the traditionally difficult approach of high-concentration, low-volume SC formulation delivery.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":null,"pages":null},"PeriodicalIF":5.3,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10793682/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138445259","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-01-01DOI: 10.1080/19420862.2023.2185924
Carl Mieczkowski, Xuejin Zhang, Dana Lee, Khanh Nguyen, Wei Lv, Yanling Wang, Yue Zhang, Jackie Way, Jean-Michel Gries
Large-molecule antibody biologics have revolutionized medicine owing to their superior target specificity, pharmacokinetic and pharmacodynamic properties, safety and toxicity profiles, and amenability to versatile engineering. In this review, we focus on preclinical antibody developability, including its definition, scope, and key activities from hit to lead optimization and selection. This includes generation, computational and in silico approaches, molecular engineering, production, analytical and biophysical characterization, stability and forced degradation studies, and process and formulation assessments. More recently, it is apparent these activities not only affect lead selection and manufacturability, but ultimately correlate with clinical progression and success. Emerging developability workflows and strategies are explored as part of a blueprint for developability success that includes an overview of the four major molecular properties that affect all developability outcomes: 1) conformational, 2) chemical, 3) colloidal, and 4) other interactions. We also examine risk assessment and mitigation strategies that increase the likelihood of success for moving the right candidate into the clinic.
{"title":"Blueprint for antibody biologics developability.","authors":"Carl Mieczkowski, Xuejin Zhang, Dana Lee, Khanh Nguyen, Wei Lv, Yanling Wang, Yue Zhang, Jackie Way, Jean-Michel Gries","doi":"10.1080/19420862.2023.2185924","DOIUrl":"10.1080/19420862.2023.2185924","url":null,"abstract":"<p><p>Large-molecule antibody biologics have revolutionized medicine owing to their superior target specificity, pharmacokinetic and pharmacodynamic properties, safety and toxicity profiles, and amenability to versatile engineering. In this review, we focus on preclinical antibody developability, including its definition, scope, and key activities from hit to lead optimization and selection. This includes generation, computational and <i>in silico</i> approaches, molecular engineering, production, analytical and biophysical characterization, stability and forced degradation studies, and process and formulation assessments. More recently, it is apparent these activities not only affect lead selection and manufacturability, but ultimately correlate with clinical progression and success. Emerging developability workflows and strategies are explored as part of a blueprint for developability success that includes an overview of the four major molecular properties that affect all developability outcomes: 1) conformational, 2) chemical, 3) colloidal, and 4) other interactions. We also examine risk assessment and mitigation strategies that increase the likelihood of success for moving the right candidate into the clinic.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":null,"pages":null},"PeriodicalIF":5.6,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/ec/f7/KMAB_15_2185924.PMC10012935.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9483654","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-01-01DOI: 10.1080/19420862.2023.2175311
Tiphanie Pruvost, Magali Mathieu, Steven Dubois, Bernard Maillère, Emmanuelle Vigne, Hervé Nozach
Delineating the precise regions on an antigen that are targeted by antibodies has become a key step for the development of antibody therapeutics. X-ray crystallography and cryogenic electron microscopy are considered the gold standard for providing precise information about these binding sites at atomic resolution. However, they are labor-intensive and a successful outcome is not guaranteed. We used deep mutational scanning (DMS) of the human LAMP-1 antigen displayed on yeast surface and leveraged next-generation sequencing to observe the effect of individual mutants on the binding of two LAMP-1 antibodies and to determine their functional epitopes on LAMP-1. Fine-tuned epitope mapping by DMS approaches is augmented by knowledge of experimental antigen structure. As human LAMP-1 structure has not yet been solved, we used the AlphaFold predicted structure of the full-length protein to combine with DMS data and ultimately finely map antibody epitopes. The accuracy of this method was confirmed by comparing the results to the co-crystal structure of one of the two antibodies with a LAMP-1 luminal domain. Finally, we used AlphaFold models of non-human LAMP-1 to understand the lack of mAb cross-reactivity. While both epitopes in the murine form exhibit multiple mutations in comparison to human LAMP-1, only one and two mutations in the Macaca form suffice to hinder the recognition by mAb B and A, respectively. Altogether, this study promotes a new application of AlphaFold to speed up precision mapping of antibody-antigen interactions and consequently accelerate antibody engineering for optimization.
{"title":"Deciphering cross-species reactivity of LAMP-1 antibodies using deep mutational epitope mapping and AlphaFold.","authors":"Tiphanie Pruvost, Magali Mathieu, Steven Dubois, Bernard Maillère, Emmanuelle Vigne, Hervé Nozach","doi":"10.1080/19420862.2023.2175311","DOIUrl":"10.1080/19420862.2023.2175311","url":null,"abstract":"<p><p>Delineating the precise regions on an antigen that are targeted by antibodies has become a key step for the development of antibody therapeutics. X-ray crystallography and cryogenic electron microscopy are considered the gold standard for providing precise information about these binding sites at atomic resolution. However, they are labor-intensive and a successful outcome is not guaranteed. We used deep mutational scanning (DMS) of the human LAMP-1 antigen displayed on yeast surface and leveraged next-generation sequencing to observe the effect of individual mutants on the binding of two LAMP-1 antibodies and to determine their functional epitopes on LAMP-1. Fine-tuned epitope mapping by DMS approaches is augmented by knowledge of experimental antigen structure. As human LAMP-1 structure has not yet been solved, we used the AlphaFold predicted structure of the full-length protein to combine with DMS data and ultimately finely map antibody epitopes. The accuracy of this method was confirmed by comparing the results to the co-crystal structure of one of the two antibodies with a LAMP-1 luminal domain. Finally, we used AlphaFold models of non-human LAMP-1 to understand the lack of mAb cross-reactivity. While both epitopes in the murine form exhibit multiple mutations in comparison to human LAMP-1, only one and two mutations in the Macaca form suffice to hinder the recognition by mAb B and A, respectively. Altogether, this study promotes a new application of AlphaFold to speed up precision mapping of antibody-antigen interactions and consequently accelerate antibody engineering for optimization.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":null,"pages":null},"PeriodicalIF":5.6,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/b7/ba/KMAB_15_2175311.PMC9980635.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9130205","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-01-01DOI: 10.1080/19420862.2022.2149057
Maria L Jaramillo, Traian Sulea, Yves Durocher, Mauro Acchione, Melissa J Schur, Anna Robotham, John F Kelly, Marie-France Goneau, Alma Robert, Yuneivy Cepero-Donates, Michel Gilbert
Effective processes for synthesizing antibody-drug conjugates (ADCs) require: 1) site-specific incorporation of the payload to avoid interference with binding to the target epitope, 2) optimal drug/antibody ratio to achieve sufficient potency while avoiding aggregation or solubility problems, and 3) a homogeneous product to facilitate approval by regulatory agencies. In conventional ADCs, the drug molecules are chemically attached randomly to antibody surface residues (typically Lys or Cys), which can interfere with epitope binding and targeting, and lead to overall product heterogeneity, long-term colloidal instability and unfavorable pharmacokinetics. Here, we present a more controlled process for generating ADCs where drug is specifically conjugated to only Fab N-linked glycans in a narrow ratio range through functionalized sialic acids. Using a bacterial sialytransferase, we incorporated N-azidoacetylneuraminic acid (Neu5NAz) into the Fab glycan of cetuximab. Since only about 20% of human IgG1 have a Fab glycan, we extended the application of this approach by using molecular modeling to introduce N-glycosylation sites in the Fab constant region of other therapeutic monoclonal antibodies. We used trastuzumab as a model for the incorporation of Neu5NAz in the novel Fab glycans that we designed. ADCs were generated by clicking the incorporated Neu5NAz with monomethyl auristatin E (MMAE) attached to a self-immolative linker terminated with dibenzocyclooctyne (DBCO). Through this process, we obtained cetuximab-MMAE and trastuzumab-MMAE with drug/antibody ratios in the range of 1.3 to 2.5. We confirmed that these ADCs still bind their targets efficiently and are as potent in cytotoxicity assays as control ADCs obtained by standard conjugation protocols. The site-directed conjugation to Fab glycans has the additional benefit of avoiding potential interference with effector functions that depend on Fc glycan structure.
有效的合成抗体-药物偶联物(adc)的工艺需要:1)有效载荷的位点特异性结合,以避免与目标表位的结合受到干扰;2)最佳的药物/抗体比例,以获得足够的效力,同时避免聚集或溶解度问题;3)均质产品,以方便监管机构的批准。在传统adc中,药物分子随机附着在抗体表面残基(通常为赖氨酸或胱氨酸)上,这可能干扰表位结合和靶向,导致整体产物异质性、长期胶体不稳定和不利的药代动力学。在这里,我们提出了一种更可控的生成adc的过程,其中药物通过功能化唾液酸在一个狭窄的比例范围内特异性地偶联到Fab n -链聚糖。利用细菌唾液转移酶,我们将n -叠氮多乙酰神经氨酸(Neu5NAz)掺入西妥昔单抗的Fab聚糖中。由于只有约20%的人IgG1具有Fab聚糖,我们通过使用分子模型在其他治疗性单克隆抗体的Fab恒定区引入n -糖基化位点,扩展了该方法的应用。我们使用曲妥珠单抗作为模型,将Neu5NAz纳入我们设计的新型Fab聚糖中。adc是通过点击掺入的Neu5NAz生成的,该Neu5NAz与单甲基aurisatin E (MMAE)连接在以二苯并环辛(DBCO)为末端的自溶连接体上。通过这一过程,我们获得了西妥昔单抗- mmae和曲妥珠单抗- mmae,药抗比在1.3 - 2.5之间。我们证实这些adc仍然有效地结合它们的靶标,并且在细胞毒性试验中与通过标准偶联方案获得的对照adc一样有效。与Fab聚糖的定点偶联具有避免对依赖于Fc聚糖结构的效应函数的潜在干扰的额外好处。
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