Crystal Structure and Characterization of Human Heavy-Chain Only Antibodies Reveals a Novel, Stable Dimeric Structure Similar to Monoclonal Antibodies
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 develo...
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doaj-1e33758d71ef4c78a8ebb00f371910352020-11-25T04:11:29ZengMDPI AGAntibodies2073-44682020-11-019666610.3390/antib9040066Crystal Structure and Characterization of Human Heavy-Chain Only Antibodies Reveals a Novel, Stable Dimeric Structure Similar to Monoclonal AntibodiesCarl Mieczkowski0Soheila Bahmanjah1Yao Yu2Jeanne Baker3Gopalan Raghunathan4Daniela Tomazela5Mark Hsieh6Mark McCoy7Corey Strickland8Laurence Fayadat-Dilman9Discovery Biologics, Protein Sciences, Merck & Co., Inc., South San Francisco, CA 94080, USADepartment of Chemistry, Modeling and Informatics, Merck & Co., Inc., Kenilworth, NJ 07033, USADiscovery Biologics, Protein Sciences, Merck & Co., Inc., South San Francisco, CA 94080, USADiscovery Biologics, Protein Sciences, Merck & Co., Inc., South San Francisco, CA 94080, USADiscovery Biologics, Protein Sciences, Merck & Co., Inc., South San Francisco, CA 94080, USADiscovery Biologics, Protein Sciences, Merck & Co., Inc., South San Francisco, CA 94080, USADiscovery Biologics, Protein Sciences, Merck & Co., Inc., South San Francisco, CA 94080, USADepartment of Pharmacology, Mass Spectrometry & Biophysics, Merck & Co., Inc., Kenilworth, NJ 07033, USADepartment of Chemistry, Modeling and Informatics, Merck & Co., Inc., Kenilworth, NJ 07033, USADiscovery Biologics, Protein Sciences, Merck & Co., Inc., South San Francisco, CA 94080, USAWe 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<sub>H</sub> and C<sub>H</sub>1 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<sub>H</sub>1) and variable (V<sub>H</sub>) 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.https://www.mdpi.com/2073-4468/9/4/66antibodyheavy-chain dimerheavy-chain antibodycrystal structure |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Carl Mieczkowski Soheila Bahmanjah Yao Yu Jeanne Baker Gopalan Raghunathan Daniela Tomazela Mark Hsieh Mark McCoy Corey Strickland Laurence Fayadat-Dilman |
spellingShingle |
Carl Mieczkowski Soheila Bahmanjah Yao Yu Jeanne Baker Gopalan Raghunathan Daniela Tomazela Mark Hsieh Mark McCoy Corey Strickland Laurence Fayadat-Dilman Crystal Structure and Characterization of Human Heavy-Chain Only Antibodies Reveals a Novel, Stable Dimeric Structure Similar to Monoclonal Antibodies Antibodies antibody heavy-chain dimer heavy-chain antibody crystal structure |
author_facet |
Carl Mieczkowski Soheila Bahmanjah Yao Yu Jeanne Baker Gopalan Raghunathan Daniela Tomazela Mark Hsieh Mark McCoy Corey Strickland Laurence Fayadat-Dilman |
author_sort |
Carl Mieczkowski |
title |
Crystal Structure and Characterization of Human Heavy-Chain Only Antibodies Reveals a Novel, Stable Dimeric Structure Similar to Monoclonal Antibodies |
title_short |
Crystal Structure and Characterization of Human Heavy-Chain Only Antibodies Reveals a Novel, Stable Dimeric Structure Similar to Monoclonal Antibodies |
title_full |
Crystal Structure and Characterization of Human Heavy-Chain Only Antibodies Reveals a Novel, Stable Dimeric Structure Similar to Monoclonal Antibodies |
title_fullStr |
Crystal Structure and Characterization of Human Heavy-Chain Only Antibodies Reveals a Novel, Stable Dimeric Structure Similar to Monoclonal Antibodies |
title_full_unstemmed |
Crystal Structure and Characterization of Human Heavy-Chain Only Antibodies Reveals a Novel, Stable Dimeric Structure Similar to Monoclonal Antibodies |
title_sort |
crystal structure and characterization of human heavy-chain only antibodies reveals a novel, stable dimeric structure similar to monoclonal antibodies |
publisher |
MDPI AG |
series |
Antibodies |
issn |
2073-4468 |
publishDate |
2020-11-01 |
description |
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<sub>H</sub> and C<sub>H</sub>1 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<sub>H</sub>1) and variable (V<sub>H</sub>) 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. |
topic |
antibody heavy-chain dimer heavy-chain antibody crystal structure |
url |
https://www.mdpi.com/2073-4468/9/4/66 |
work_keys_str_mv |
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