Single B cell technologies for monoclonal antibody discovery

Trends in Immunology, Journal Year: 2021, Volume and Issue: 42(12), P. 1143 - 1158

Published: Nov. 4, 2021

Monoclonal antibodies (mAbs) are among the most important type of biologic drugs on pharmaceutical market, as well for diagnostic purposes. Presently, more than 100 mAbs have been approved by US FDA against a variety diseases such cancer, infectious diseases, autoimmune and neurological disorders.Primary antigen-specific B cells main source obtaining mAb sequences, particularly using human specimens peripheral blood mononuclear cells. Also, humanization derived from other species (e.g. mice, rats, rabbits) has become easier efficient.Currently, single cell screening systems bear multiple advantages over systems, display technologies. Particularly, in vivo development favors safety profile overall developability. It also reduced off-target binding to proteome.Single technologies significantly evolved, becoming faster higher throughput before. Nonetheless, hybridoma technology, first technique this field, still represents an methodology is known within scientific community.At present no gold standard exists relying broad different discovery – each with its disadvantages. often selected hosts, which notably short-lived ex culture conditions hence, arduous interrogate. The several new techniques protocols facilitated isolation retrieval antibody-coding sequences leveraging miniaturization reaction volumes. Alternatively, can be generated independently cells, comprising and, recently, artificial intelligence-driven algorithms. Consequently, considerable used, raising demand better consolidation. In review, we discuss major available interrogate isolate mAbs, including their Highly specific functionally surpass polyclonal (pAbs) (see Glossary), heterogeneous undefined mixture [1.Kohler G. Milstein C. Continuous cultures fused secreting antibody predefined specificity.Nature. 1975; 256: 495-4927Crossref PubMed Google Scholar,2.Lipman N.S. et al.Monoclonal versus antibodies: distinguishing characteristics, applications, information resources.ILAR J. 2005; 46: 258-268Crossref Scholar]. broadly used highly tools, therapeutic modalities context viral infections, tumors, metabolic transplantation [3.Wilson P.C. Andrews S.F. Tools therapeutically harness response.Nat. Rev. Immunol. 2012; 12: 709-719Crossref Scopus (92) Scholar,4.Weiner G.J. Building monoclonal antibody-based therapeutics.Nat. Cancer. 2015; 15: 361-370Crossref (358) Particularly field immuno-oncology, variants thereof, namely chain variable fragments (scFvs), antibody–drug conjugates (ADCs), bispecific antibodies, etc. gaining importance leading [4.Weiner Scholar,5.Walsh Biopharmaceutical benchmarks 2018.Nat. Biotechnol. 2018; 36: 1136-1145Crossref (337) Since USA approval Orthoclone OKT3 (anti-CD3 mAb) 1986 kidney rejection (the mAb), [6.Mullard A. approves 100th product.Nat. Drug Discov. 2021; 20: 491-495Crossref (17) Furthermore, current COVID-19 pandemic highlights how class biologics crucial setting rapid efficient [7.Taylor al.Neutralizing treatment COVID-19.Nat. 21: 382-393Crossref (53) Moreover, potentially play key roles prophylactic settings (passive immunization), especially when considering pathogens respiratory syncytial virus, effective vaccines unavailable [8.Slifka M.K. Amanna I.J. Passive immunization.Plotkin's Vaccines. : 84-95.e10Crossref Following classical technology developed mAbs), many methodologies refined improve or even radically change (e.g., phage technology) (Box 1) Scholar,9.Mondon P. al.Human libraries: race engineer explore larger diversity.Front. Biosci. 2008; 13: 1117-1129Crossref (45) Scholar,10.Walker L.M. Burton D.R. immunotherapy infections: 'super-antibodies' enter fray.Nat. 18: 297-308Crossref (139) Table 1 (Key table) lists some published that use primary identification features, time expenditure, costs, ability target difficult antigens, affinity retrieved rare binders, throughput, ease use.Box 1The advantage selectionFrom clinical point view, there cell-derived display. Because maturation, they show rather high specificity towards targets, usually proteins, allowing tailored response low host proteins [15.Carter P.J. Potent therapeutics design.Nat. 2006; 6: 343-357Crossref (855) Related characteristic, immunogenicity upon administration [115.Le Basle Y. al.Physicochemical stability review.J. Pharm. Sci. 2020; 109: 169-190Abstract Full Text PDF Thus, hybridoma-derived had, average, general developability (a aspect, considered 'Achilles heel' biologics) compared from, example, libraries [116.Spencer S. al.Solubility evaluation murine antibodies.MAbs. 4: 319-325Crossref (36) Scholar, 117.Nixon A.E. al.Drugs display: candidate practice.MAbs. 2014; 73-85Crossref (0) 118.Kaleli N.E. al.Phage enriched aliphatic content: Insights issues.Proteins. 2019; 87: 607-618Crossref (10) 119.Sun Benet L.Z. Late-stage failures drugs: retrospective case study analysis.Pharmacology. 105: 145-163Crossref (9) 120.Garripelli V.K. al.Developability assessment drug candidates: study.Pharm. Dev. Technol. 26: 11-20Crossref (1) This due native post-translational modification profile, decreases possibility aggregation vitro importantly, [121.Jefferis R. Posttranslational modifications biotherapeutics.J Immunol Res. 2016; 20165358272Crossref (63) Scholar,122.Jenkins N. al.Post-translational recombinant proteins: significance biopharmaceuticals.Mol. 39: 113-118Crossref Scholar].The theoretical size repertoire between 1012 1018 clones (defined mathematical V(D)J recombinations) [123.Elhanati al.Inferring processes underlying B-cell diversity.Philos. Trans. Soc. Lond. Ser. Biol. 37020140243Crossref (84) However, reality number individual lower (around 1×107–2×107 circulating clonotypes) [124.Soto al.High frequency shared clonotypes receptor repertoires.Nature. 566: 398-402Crossref (90) might factors, sample donor estimate clonal repertoire, out-of-frame mutations inserted during recombination, formation self-reactive BCRs eventually undergo deletion 124.Soto 125.Rees A.R. Understanding repertoire.MAbs. 121729683Crossref (11) Yet, one must acknowledge technologies, display, naïve above 109 possible combinations, outpacing de facto natural given [126.Lim C.C. al.Development panning strategy utilizing crude antigens: MERS-CoV nucleoprotein antibodies.Sci. Rep. 9: 6088Crossref Scholar,127.Sheets M.D. al.Efficient construction large nonimmune library: production high-affinity single-chain protein antigens.Proc. Natl. Acad. U. 1998; 95: 6157-6162Crossref aspect partially compensated PBMC-derived healthy donors patients.An retrieving (single) reliable PCR-based amplification VH VL genes, pairing, sequencing. A degenerate primers/primer sets (usually nested PCR) designed commonly analyzed species, humans mice. our experience, primer von Boehmer al. allow consistently amplify Ig genes [51.von L. al.Sequencing cloning mouse memory cells.Nat. Protoc. 11: 1908-1923Crossref (60) For Friedensohn demonstrated good results [128.Friedensohn al.Synthetic standards combined error bias correction accuracy quantitative resolution sequencing naive cells.Front. 1401Crossref Scholar].Table 1Key table. Platforms discover cellsbThe discussed review were relevant parameters requirement, cost, antigens 3), chances identifying screening., aColor legend: green: performance, yellow: medium red: performancea Color performanceb screening. From patients. An Review, focus these frequently based identify mAbs. includes conventional laboratory equipment, surface staining techniques, miniaturized replica genetic analysis (Figure 1, Figure 2). With aim providing sound overview supports selection suitable embarking isolation, explained at technical level, followed discussion disadvantages (Table 1). section, presented cultures, while second section comprises microfluidics-based technologies.Figure 2Single equipment.Show full caption(A) Single methodologies. Usually, ASCs all rely secreted antibodies. activated used. We divided into open closed assays. Among assays Beacon (Berkeley Lights) microfluidic chamber, microengraved microcapillary system. Closed comprise encapsulation water-in-oil droplets. Both mainly detection via either colocalization beads displaying antigen surface, soluble bound anti-IgG-captured fluorophores generation Förster resonance energy transfer (FRET) signal. (B) replica. Primary encapsulated droplet paired VH–VL amplified sequence oligos containing complementary constant region sequences. order do so, lysed both cell-mRNAs anneal oligos. Upon emulsion breaking, library then prepared through RT-PCR cloned expression vectors. transformed yeast, phage, mammalian obtained population further screenings fluorescently labeled ELISA engineered (C) (VH–VL paired). together bearing barcoded-oligos subsequently lysed, single-cell total mRNAs. next-generation After data clonotype expansion (particular pairing), but characteristics text). Antibody expanded (or taking account additional features) synthesized, plasmids, generated; tested ELISA. figure was created BioRender (https://biorender.com). Abbreviation: mAb, antibody.View Large Image ViewerDownload Hi-res image Download (PPT) (A) antibody. survival generally sustain, poses challenge expressing (immunoglobulin). To increase lifespan, immortalization procedures developed, fusion immortalized myeloma (hybridoma technology), Epstein–Barr virus (EBV) infection. to: (i) maintain antibody-secreting long enough screen function; (ii) times minimized enrichment describe provide graphical illustration 1. probably best-known since development, revolutionized discovery. principle immortalize (ASCs) generating immortal whose supernatant screened presence [11.Parray H.A. al.Hybridoma versatile method applicability across limitations, advancement future perspectives.Int. Immunopharmacol. 85106639Crossref (21) Most commonly, immunized mice rechallenged interest few days later, spleen ASCs, procedure organs compartments, bone marrow, lymph nodes, (PBMCs) [12.Basalp Yucel F. Development hybridomas lymphocytes spleen, node, marrow.Hybrid Hybridomics. 2003; 22: 329-331Crossref original protocol relied PEG-mediated splenic naturally enzyme hypoxanthine–guanine phosphoribosyltransferase (HGPRT+), (HGPRT-) Scholar,13.de Almeida al.Enhanced immunization obtain 10: 46-54Crossref (6) accommodate Scholar].Box 2Immunization protocolThis step immunizing common heavily species. volunteers, individuals, animals seropositive interest. Different improvements strategy, adjuvants along optimized schedules, elicit strong humoral responses, those membrane GPCRs, ion channels, carbohydrates [131.Dodd R.B. al.Therapeutic complex targets: strategies.BioDrugs. 32: 339-355Crossref (8) 132.Chiarella Fazio V.M. Mouse biological research: strategies high-throughput production.Biotechnol. Lett. 30: 1303-1310Crossref 133.Aucouturier al.Adjuvants veterinary vaccines.Vaccine. 2001; 19: 2666-2672Crossref researchers prime–boost boosting schedules respect boosts intervals them. carefully because remain conformation

Language: Английский

---

Hybridoma technology; advancements, clinical significance, and future aspects

Journal of Genetic Engineering and Biotechnology, Journal Year: 2021, Volume and Issue: 19(1), P. 159 - 159

Published: Oct. 18, 2021

Hybridoma technology is one of the most common methods used to produce monoclonal antibodies. In this process, antibody-producing B lymphocytes are isolated from mice after immunizing with specific antigen and fused immortal myeloma cell lines form hybrid cells, called hybridoma lines. These cells cultured in a lab antibodies, against antigen. This can be achieved by an vivo or vitro method. It preferred above all available antibodies because thus produced high purity highly sensitive specific. Monoclonal useful diagnostic, imaging, therapeutic purposes have very clinical significance. Once become stable, these offer limitless production homogenized method also cost-effective. The targeted important tool various fields research such as toxicology, animal biotechnology, medicine, pharmacology, cell, molecular biology. extensively diagnosis applications. Radiolabeled probes detect tumor antigens living system; radioisotope coupled for target action on oncogenic cells.

Language: Английский

---

An overview on display systems (phage, bacterial, and yeast display) for production of anticancer antibodies; advantages and disadvantages

International Journal of Biological Macromolecules, Journal Year: 2022, Volume and Issue: 208, P. 421 - 442

Published: March 24, 2022

Language: Английский

---

Cancer therapy with antibodies

Nature reviews. Cancer, Journal Year: 2024, Volume and Issue: 24(6), P. 399 - 426

Published: May 13, 2024

Language: Английский

---

High-throughput spatiotemporal monitoring of single-cell secretions via plasmonic microwell arrays

Nature Biomedical Engineering, Journal Year: 2023, Volume and Issue: 7(7), P. 943 - 958

Published: April 3, 2023

Methods for the analysis of cell secretions at single-cell level only provide semiquantitative endpoint readouts. Here we describe a microwell array real-time spatiotemporal monitoring extracellular from hundreds single cells in parallel. The incorporates gold substrate with arrays nanometric holes functionalized receptors specific analyte, and is illuminated light spectrally overlapping device's spectrum extraordinary optical transmission. Spectral shifts surface plasmon resonance resulting analyte-receptor bindings around secreting are recorded by camera as variations intensity transmitted while machine-learning-assisted tracking eliminates influence movements. We used to characterize antibody-secretion profiles hybridoma rare subset antibody-secreting sorted human donor peripheral blood mononuclear cells. High-throughput measurements secretory will aid study physiological mechanisms governing protein secretion.

Language: Английский

---

Principles and Current Clinical Landscape of Multispecific Antibodies against Cancer

International Journal of Molecular Sciences, Journal Year: 2021, Volume and Issue: 22(11), P. 5632 - 5632

Published: May 26, 2021

Building upon the resounding therapeutic success of monoclonal antibodies, and supported by accelerating progress in engineering methods, field multispecific antibodies is growing rapidly. Over 140 different molecules are currently clinical testing, with excellent results recent phase 1–3 trials for several them. Multivalent bispecific IgG-modified formats predominate today, a clear tendency more target antigens further increased valency newer constructs. The strategies to augment anticancer efficacy equally divided between disruption multiple surface antigens, additional redirection cytotoxic T or NK lymphocytes against tumor. Both effects complement other modern modalities, such as tyrosine kinase inhibitors adoptive cell therapies, which multispecifics increasingly applied combination merged, example, form antibody producing CAR-T cells oncolytics. While mainly focused on B-cell malignancies early on, contemporary sector accommodates twice many solid compared hematologic cancers. An exciting emerging prospect targeting intracellular neoantigens using T-cell receptor (TCR) fusion proteins TCR-mimic fragments. Considering fact that introduction PD-(L)1 only few years ago has already facilitated 5-year survival rates 30–50% per se highly lethal neoplasms, metastatic melanoma non-small-cell lung carcinoma, upcoming enforcement current treatments “next-generation” immunotherapeutics, offers justified hope cure some advanced cancers near future.

Language: Английский

---

Inhalation monoclonal antibody therapy: a new way to treat and manage respiratory infections

Applied Microbiology and Biotechnology, Journal Year: 2021, Volume and Issue: 105(16-17), P. 6315 - 6332

Published: Aug. 1, 2021

Language: Английский

---

Advanced green analytical chemistry for environmental pesticide detection

Current Opinion in Green and Sustainable Chemistry, Journal Year: 2021, Volume and Issue: 30, P. 100488 - 100488

Published: March 27, 2021

Language: Английский

---

Monoclonal antibodies against rabies: current uses in prophylaxis and in therapy

Current Opinion in Virology, Journal Year: 2022, Volume and Issue: 53, P. 101204 - 101204

Published: Feb. 10, 2022

Rabies is a severe viral infection that causes an acute encephalomyelitis, which presents case fatality of nearly 100% after the manifestation neurological clinical signs. can be efficiently prevented with post-exposure prophylaxis (PEP), composed vaccines and anti-rabies immunoglobulins (RIGs); however, no treatment exists for symptomatic rabies. The PEP protocol faces access implementation obstacles in resource-limited settings, could partially overcome by substituting RIGs monoclonal antibodies (mAbs). mAbs offer lower production costs, consistent supply availability, long-term storage/stability, improved safety profile. Here we summarize key features different available against rabies, focusing on their application highlighting potential novel therapeutic approach.

Language: Английский

---

Immune- and Non-Immune-Mediated Adverse Effects of Monoclonal Antibody Therapy: A Survey of 110 Approved Antibodies

Antibodies, Journal Year: 2022, Volume and Issue: 11(1), P. 17 - 17

Published: Feb. 25, 2022

Identification of new disease-associated biomarkers; specific targeting such markers by monoclonal antibodies (mAbs); and application advances in recombinant technology, including the production humanized fully human antibodies, has enabled many improved treatment outcomes successful biological treatments some diseases previously neglected or with poor prognoses. Of 110 mAbs preparations currently approved FDA and/or EMA, 46 (including 13 antibody–drug conjugates) recognizing 29 different targets are indicated for cancers, 66, 48 targets, non-cancer disorders. Despite their expected accompanying reduced collateral damage normal healthy non-involved cells, mAbs, may cause types I (anaphylaxis, urticaria), II (e.g., hemolytic anemia, possibly early-onset neutropenia), III (serum sickness, pneumonitis), IV (Stevens–Johnson syndrome, toxic epidermal necrolysis) hypersensitivities as well other cutaneous, pulmonary, cardiac, liver adverse events. MAbs can provoke severe infusion reactions that resemble anaphylaxis induce a number systemic, potentially life-threatening syndromes low frequency. A common feature most these is release cascade cytokines associated inflammatory immunological processes. Epidermal growth factor receptor-targeted papulopustular mucocutaneous eruptions not immune-mediated.

Language: Английский

---