Applied Microbiology and Biotechnology

, Volume 101, Issue 22, pp 8165–8179 | Cite as

Development of humanized scFv antibody fragment(s) that targets and blocks specific HLA alleles linked to myasthenia gravis

  • B. Vijayalakshmi AyyarEmail author
  • M. Zouhair AtassiEmail author
Applied genetics and molecular biotechnology


Myasthenia gravis (MG) is an autoimmune disease caused by sensitization of the immune system to self-antigens. We have previously shown that targeting MG-susceptible alleles can significantly inhibit proliferation of disease-specific T cells. In this work, we humanized a murine monoclonal antibody (mAb) LG11, capable of blocking MG-associated DQ beta 1 (DQB1) allele and reformatted it into single-chain fragment variable (scFv). A fully functional humanized scFv was obtained by optimizing variable domain orientations and linker lengths, along with the optimization of expression conditions and codons to suit Escherichia coli expression machinery. Characterization of humanized scFv (FL8) revealed that the reformatted scFv, despite recognizing the same epitope as the parent murine LG11 mAb, exhibited superior binding affinity (0.97 nM) compared to the LG11 mAb, towards the immunizing antigen (DQB1*0601/70-90) and was able to block the proliferation of T cells cultured from PBLs of MG-patients typed DQB1*0601. The scFv was also capable of binding a variant MG-associated allele (DQB1*0502/70-90) with moderate affinity (18.7 nM), a feature that was absent in the LG11. To our knowledge, this is the first report of humanizing a MG-associated human leukocyte antigen (HLA) scFv for preclinical studies.


Myasthenia gravis Antibody engineering Antibody humanization scFv Biacore Expression optimization 



The authors wish to acknowledge Prof. Carlos F. Barbas III (The Scripps Research Institute, La Jolla, CA) for providing pComb3XSS vector; Dr. Minako Oshima for providing anti-DQB1*0601 mAb, synthetic peptides, and reagents for T cell assay; and Dr. Philip Deitiker for technical help with beta counter. Additionally, the authors would like to thank Dr. Timothy Palzkill and Dr. Zhizeng Sun for allowing us to use their Biacore instrument and providing useful suggestions during analysis. The authors appreciate the assistance of Dr. Sushrut Arora with the proof-reading and scientific editing.

Compliance with ethical standards

Ethical statement

The authors accept the rules of good scientific practice and agree with the COPE guidelines followed by the Applied Microbiology and Biotechnology Journal.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

Procedures performed in studies involving human participants were in accordance with the ethical standards of the Institutional Review Board of Baylor College of Medicine. This article does not contain any studies with animals performed by any of the authors.

Informed consent

Blood samples were obtained from patients with the clinical diagnosis of generalized myasthenia. A written informed consent was obtained from the patients, which was approved by the Institutional Review Board of Baylor College of Medicine.

Supplementary material

253_2017_8557_MOESM1_ESM.pdf (203 kb)
ESM 1 (PDF 202 kb).


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Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular BiologyBaylor College of MedicineHoustonUSA
  2. 2.Department of Molecular Virology and MicrobiologyBaylor College of MedicineHoustonUSA
  3. 3.Department of Pathology and ImmunologyBaylor College of MedicineHoustonUSA

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