Antibodies

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4 pages, 210 KiB

Open AccessEditorial

Design, Production, Characterization, and Use of Peptide Antibodies

by Nicole H. Trier and Gunnar Houen

Antibodies 2023, 12(1), 6; https://doi.org/10.3390/antib12010006 - 13 Jan 2023

Cited by 1 | Viewed by 3055

Abstract

Antibodies are key reagents in diagnostics, therapeutics, and experimental biology, capable of detecting numerous targets [...] Full article

(This article belongs to the Special Issue Design, Production and Characterization of Peptide Antibodies)

Research

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13 pages, 1364 KiB

Open AccessArticle

Epitope Mapping of Monoclonal Antibodies to Calreticulin Reveals That Charged Amino Acids Are Essential for Antibody Binding

by Ann Christina Bergmann, Cecilie Kyllesbech, Rimantas Slibinskas, Evaldas Ciplys, Peter Højrup, Nicole Hartwig Trier and Gunnar Houen

Antibodies 2021, 10(3), 31; https://doi.org/10.3390/antib10030031 - 4 Aug 2021

Cited by 6 | Viewed by 4357

Abstract

Calreticulin is a chaperone protein, which is associated with myeloproliferative diseases. In this study, we used resin-bound peptides to characterize two monoclonal antibodies (mAbs) directed to calreticulin, mAb FMC 75 and mAb 16, which both have significantly contributed to understanding the biological function [...] Read more.

Calreticulin is a chaperone protein, which is associated with myeloproliferative diseases. In this study, we used resin-bound peptides to characterize two monoclonal antibodies (mAbs) directed to calreticulin, mAb FMC 75 and mAb 16, which both have significantly contributed to understanding the biological function of calreticulin. The antigenicity of the resin-bound peptides was determined by modified enzyme-linked immunosorbent assay. Specific binding was determined to an 8-mer epitope located in the N-terminal (amino acids 34–41) and to a 12-mer peptide located in the C-terminal (amino acids 362–373). Using truncated peptides, the epitopes were identified as TSRWIESK and DEEQRLKEEED for mAb FMC 75 and mAb 16, respectively, where, especially the charged amino acids, were found to have a central role for a stable binding. Further studies indicated that the epitope of mAb FMC 75 is assessable in the oligomeric structure of calreticulin, making this epitope a potential therapeutic target. Full article

(This article belongs to the Special Issue Design, Production and Characterization of Peptide Antibodies)

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12 pages, 2222 KiB

Open AccessArticle

Specificity of Anti-Citrullinated Protein Antibodies to Citrullinated α-Enolase Peptides as a Function of Epitope Structure and Composition

by Ilaria Fanelli, Paolo Rovero, Paul Robert Hansen, Jette Frederiksen, Gunnar Houen and Nicole Hartwig Trier

Antibodies 2021, 10(3), 27; https://doi.org/10.3390/antib10030027 - 21 Jul 2021

Cited by 6 | Viewed by 4214

Abstract

Rheumatoid arthritis (RA) is an autoimmune disease affecting approximately 1–2% of the world population. In addition to the first discovered serologic markers for RA, the rheumatoid factors (RFs), anti-citrullinated protein antibodies (ACPAs) are even more specific for the disease compared to RFs and [...] Read more.

Rheumatoid arthritis (RA) is an autoimmune disease affecting approximately 1–2% of the world population. In addition to the first discovered serologic markers for RA, the rheumatoid factors (RFs), anti-citrullinated protein antibodies (ACPAs) are even more specific for the disease compared to RFs and are found in 70–80% of RA patient sera. RA etiopathogenesis still needs to be elucidated, as different factors are proposed to be involved, such as Epstein–Barr virus infection. Hence, understanding the interaction between ACPAs and their citrullinated peptide targets is relevant for a better knowledge of RA pathophysiology and for diagnostic purposes. In this study, a cohort of RA sera, healthy control sera and multiple sclerosis sera were screened for reactivity to a variety of citrullinated peptides originating from α-enolase, pro-filaggrin, proteoglycan and Epstein–Barr nuclear antigen-2 by enzyme-linked immunosorbent assay. ACPA reactivity to citrullinated α-enolase peptides was found to depend on peptide length and peptide conformation, favouring cyclic (disulfide bond) conformations for long peptides and linear peptides for truncated ones. Additional investigations about the optimal peptide conformation for ACPA detection, employing pro-filaggrin and EBNA-2 peptides, confirmed these findings, indicating a positive effect of cyclization of longer peptides of approximately 20 amino acids. Moreover, screening of the citrullinated peptides confirmed that ACPAs can be divided into two groups based on their reactivity. Approximately 90% of RA sera recognize several peptide targets, being defined as cross-reactive or overlapping reactivities, and whose reactivity to the citrullinated peptide is considered primarily to be backbone-dependent. In contrast, approximately 10% recognize a single target and are defined as nonoverlapping, primarily depending on the specific amino acid side-chains in the epitope for a stable interaction. Collectively, this study contributed to characterize epitope composition and structure for optimal ACPA reactivity and to obtain further knowledge about the cross-reactive nature of ACPAs. Full article

(This article belongs to the Special Issue Design, Production and Characterization of Peptide Antibodies)

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12 pages, 2490 KiB

Open AccessArticle

Recombinant Antibody Production Using a Dual-Promoter Single Plasmid System

by Stefania C. Carrara, David Fiebig, Jan P. Bogen, Julius Grzeschik, Björn Hock and Harald Kolmar

Antibodies 2021, 10(2), 18; https://doi.org/10.3390/antib10020018 - 13 May 2021

Cited by 8 | Viewed by 15941

Abstract

Monoclonal antibodies (mAbs) have demonstrated tremendous effects on the treatment of various disease indications and remain the fastest growing class of therapeutics. Production of recombinant antibodies is performed using mammalian expression systems to facilitate native antibody folding and post-translational modifications. Generally, mAb expression [...] Read more.

Monoclonal antibodies (mAbs) have demonstrated tremendous effects on the treatment of various disease indications and remain the fastest growing class of therapeutics. Production of recombinant antibodies is performed using mammalian expression systems to facilitate native antibody folding and post-translational modifications. Generally, mAb expression systems utilize co-transfection of heavy chain (hc) and light chain (lc) genes encoded on separate plasmids. In this study, we examine the production of two FDA-approved antibodies using a bidirectional (BiDi) vector encoding both hc and lc with mirrored promoter and enhancer elements on a single plasmid, by analysing the individual hc and lc mRNA expression levels and subsequent quantification of fully-folded IgGs on the protein level. From the assessment of different promoter combinations, we have developed a generic expression vector comprised of mirrored enhanced CMV (eCMV) promoters showing comparable mAb yields to a two-plasmid reference. This study paves the way to facilitate small-scale mAb production by transient cell transfection with a single vector in a cost- and time-efficient manner. Full article

(This article belongs to the Special Issue Design, Production and Characterization of Peptide Antibodies)

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20 pages, 3615 KiB

Open AccessArticle

Characterization and Modeling of Reversible Antibody Self-Association Provide Insights into Behavior, Prediction, and Correction

by Carl Mieczkowski, Alan Cheng, Thierry Fischmann, Mark Hsieh, Jeanne Baker, Makiko Uchida, Gopalan Raghunathan, Corey Strickland and Laurence Fayadat-Dilman

Antibodies 2021, 10(1), 8; https://doi.org/10.3390/antib10010008 - 15 Feb 2021

Cited by 11 | Viewed by 6807

Abstract

Reversible antibody self-association, while having major developability and therapeutic implications, is not fully understood or readily predictable and correctable. For a strongly self-associating humanized mAb variant, resulting in unacceptable viscosity, the monovalent affinity of self-interaction was measured in the low μM range, typical [...] Read more.

Reversible antibody self-association, while having major developability and therapeutic implications, is not fully understood or readily predictable and correctable. For a strongly self-associating humanized mAb variant, resulting in unacceptable viscosity, the monovalent affinity of self-interaction was measured in the low μM range, typical of many specific and biologically relevant protein–protein interactions. A face-to-face interaction model extending across both the heavy-chain (HC) and light-chain (LC) Complementary Determining Regions (CDRs) was apparent from biochemical and mutagenesis approaches as well as computational modeling. Light scattering experiments involving individual mAb, Fc, Fab, and Fab’2 domains revealed that Fabs self-interact to form dimers, while bivalent mAb/Fab’2 forms lead to significant oligomerization. Site-directed mutagenesis of aromatic residues identified by homology model patch analysis and self-docking dramatically affected self-association, demonstrating the utility of these predictive approaches, while revealing a highly specific and tunable nature of self-binding modulated by single point mutations. Mutagenesis at these same key HC/LC CDR positions that affect self-interaction also typically abolished target binding with notable exceptions, clearly demonstrating the difficulties yet possibility of correcting self-association through engineering. Clear correlations were also observed between different methods used to assess self-interaction, such as Dynamic Light Scattering (DLS) and Affinity-Capture Self-Interaction Nanoparticle Spectroscopy (AC-SINS). Our findings advance our understanding of therapeutic protein and antibody self-association and offer insights into its prediction, evaluation and corrective mitigation to aid therapeutic development. Full article

(This article belongs to the Special Issue Design, Production and Characterization of Peptide Antibodies)

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10 pages, 1773 KiB

Open AccessArticle

Selection and Characterization of Single-Domain Antibodies for Detection of Lassa Nucleoprotein

by George P. Anderson, Jinny L. Liu, Lisa C. Shriver-Lake and Ellen R. Goldman

Antibodies 2020, 9(4), 71; https://doi.org/10.3390/antib9040071 - 17 Dec 2020

Cited by 3 | Viewed by 4184

Abstract

Lassa virus is the etiologic agent of Lassa fever, an acute and often fatal illness endemic to West Africa. It is important to develop new reagents applicable either for the specific diagnosis or as improved therapeutics for the treatment of Lassa fever. Here, [...] Read more.

Lassa virus is the etiologic agent of Lassa fever, an acute and often fatal illness endemic to West Africa. It is important to develop new reagents applicable either for the specific diagnosis or as improved therapeutics for the treatment of Lassa fever. Here, we describe the development and initial testing of llama-derived single-domain antibodies that are specific for the Lassa virus nucleoprotein. Four sequence families based on complementarity-determining region (CDR) homology were identified by phage-based enzyme-linked immunosorbent assays, however, the highest affinity clones all belonged to the same sequence family which possess a second disulfide bond between Framework 2 and CDR3. The affinity and thermal stability were evaluated for each clone. A MagPlex-based homogeneous sandwich immunoassay for Lassa virus-like particles was also demonstrated to show their potential for further development as diagnostic reagents. Full article

(This article belongs to the Special Issue Design, Production and Characterization of Peptide Antibodies)

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24 pages, 3752 KiB

Open AccessArticle

Crystal Structure and Characterization of Human Heavy-Chain Only Antibodies Reveals a Novel, Stable Dimeric Structure Similar to Monoclonal Antibodies

by Carl Mieczkowski, Soheila Bahmanjah, Yao Yu, Jeanne Baker, Gopalan Raghunathan, Daniela Tomazela, Mark Hsieh, Mark McCoy, Corey Strickland and Laurence Fayadat-Dilman

Antibodies 2020, 9(4), 66; https://doi.org/10.3390/antib9040066 - 22 Nov 2020

Cited by 4 | Viewed by 5613

Abstract

We report the novel crystal structure and characterization of symmetrical, homodimeric humanized heavy-chain-only antibodies or dimers (HC2s). HC2s were found to be significantly coexpressed and secreted along with mAbs from transient CHO HC/LC cotransfection, resulting in an unacceptable mAb developability attribute. Expression of [...] Read more.

We report the novel crystal structure and characterization of symmetrical, homodimeric humanized heavy-chain-only antibodies or dimers (HC2s). HC2s were found to be significantly coexpressed and secreted along with mAbs from transient CHO HC/LC cotransfection, resulting in an unacceptable mAb developability attribute. Expression of full-length HC2s in the absence of LC followed by purification resulted in HC2s with high purity and thermal stability similar to conventional mAbs. The V_H and C_H1 portion of the heavy chain (or Fd) was also efficiently expressed and yielded a stable, covalent, and reducible dimer (Fd2). Mutagenesis of all heavy chain cysteines involved in disulfide bond formation revealed that Fd2 intermolecular disulfide formation was similar to Fabs and elucidated requirements for Fd2 folding and expression. For one HC2, we solved the crystal structure of the Fd2 domain to 2.9 Å, revealing a highly symmetrical homodimer that is structurally similar to Fabs and is mediated by conserved (C_H1) and variable (V_H) contacts with all CDRs positioned outward for target binding. Interfacial dimer contacts revealed by the crystal structure were mutated for two HC2s and were found to dramatically affect HC2 formation while maintaining mAb bioactivity, offering a potential means to modulate novel HC2 formation through engineering. These findings indicate that human heavy-chain dimers can be secreted efficiently in the absence of light chains, may show good physicochemical properties and stability, are structurally similar to Fabs, offer insights into their mechanism of formation, and may be amenable as a novel therapeutic modality. Full article

(This article belongs to the Special Issue Design, Production and Characterization of Peptide Antibodies)

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22 pages, 2151 KiB

Open AccessArticle

Bringing the Heavy Chain to Light: Creating a Symmetric, Bivalent IgG-Like Bispecific

by Anusuya Ramasubramanian, Rachel Tennyson, Maureen Magnay, Sagar Kathuria, Tara Travaline, Annu Jain, Dana M. Lord, Megan Salemi, Caitlin Sullivan, Tristan Magnay, Jiali Hu, Eva Bric-Furlong, Pierrick Rival, Yanfeng Zhou, Dietmar Hoffmann, William Brondyk, Katarina Radošević and Partha S. Chowdhury

Antibodies 2020, 9(4), 62; https://doi.org/10.3390/antib9040062 - 6 Nov 2020

Cited by 3 | Viewed by 7950

Abstract

Bispecific molecules are biologically significant, yet their complex structures pose important manufacturing and pharmacokinetic challenges. Nevertheless, owing to similarities with monoclonal antibodies (mAbs), IgG-like bispecifics conceptually align well with conventional expression and manufacturing platforms and often exhibit potentially favorable drug metabolism and pharmacokinetic [...] Read more.

Bispecific molecules are biologically significant, yet their complex structures pose important manufacturing and pharmacokinetic challenges. Nevertheless, owing to similarities with monoclonal antibodies (mAbs), IgG-like bispecifics conceptually align well with conventional expression and manufacturing platforms and often exhibit potentially favorable drug metabolism and pharmacokinetic (DMPK) properties. However, IgG-like bispecifics do not possess target bivalency and current designs often require tedious engineering and purification to ensure appropriate chain pairing. Here, we present a near-native IgG antibody format, the 2xVH, which can create bivalency for each target or epitope and requires no engineering for cognate chain pairing. In this modality, two different variable heavy (VH) domains with distinct binding specificities are grafted onto the first constant heavy (CH1) and constant light (CL) domains, conferring the molecule with dual specificity. To determine the versatility of this format, we characterized the expression, binding, and stability of several previously identified soluble human VH domains. By grafting these domains onto an IgG scaffold, we generated several prototype 2xVH IgG and Fab molecules that display similar properties to mAbs. These molecules avoided the post-expression purification necessary for engineered bispecifics while maintaining a capacity for simultaneous dual binding. Hence, the 2xVH format represents a bivalent, bispecific design that addresses limitations of manufacturing IgG-like bispecifics while promoting biologically-relevant dual target engagement. Full article

(This article belongs to the Special Issue Design, Production and Characterization of Peptide Antibodies)

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