Advertisement

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

Abstract

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.

Keywords

Myasthenia gravis Antibody engineering Antibody humanization scFv Biacore Expression optimization 

Notes

Acknowledgements

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).

References

  1. Avidan N, Le Panse R, Berrih-Aknin S, Miller A (2014) Genetic basis of myasthenia gravis—a comprehensive review. J Autoimmun 52:146–153CrossRefPubMedGoogle Scholar
  2. Ayyar BV, Hearty S, O’Kennedy R (2010) Highly sensitive recombinant antibodies capable of reliably differentiating heart-type fatty acid binding protein from noncardiac isoforms. Anal Biochem 407:165–171CrossRefPubMedGoogle Scholar
  3. Ayyar BV, Aoki KR, Atassi MZ (2015a) The C-terminal heavy-chain domain of botulinum neurotoxin A is not the only site that binds neurons, as the N-terminal heavy-chain domain also plays a very active role in toxin-cell binding and interactions. Infect Immun 83:1465–1476CrossRefPubMedPubMedCentralGoogle Scholar
  4. Ayyar BV, Hearty S, O’Kennedy R (2015b) Facile domain rearrangement abrogates expression recalcitrance in a rabbit scFv. Appl Microbiol Biotechnol 99:2693–2703CrossRefPubMedGoogle Scholar
  5. Ayyar BV, Tajhya RB, Beeton C, Zouhair Atassi M (2015c) Antigenic sites on the HN domain of botulinum neurotoxin A stimulate protective antibody responses against active toxin. Sci Rep 5:15776CrossRefPubMedGoogle Scholar
  6. Ayyar BV, Arora S, Ravi SS (2017) Optimizing antibody expression: the nuts and bolts. Methods 116:51–62CrossRefPubMedGoogle Scholar
  7. Berrih-Aknin S, Morel E, Raimond F, Safar D, Gaud C, Binet JP, Levasseur P, Bach JF (1987) The role of the thymus in myasthenia gravis: immunohistological and immunological studies in 115 cases. Ann N Y Acad Sci 505:50–70CrossRefPubMedGoogle Scholar
  8. Brochet X, Lefranc M-P, Giudicelli V (2008) IMGT/V-QUEST: the highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis. Nucleic Acids Res 36:W503–W508CrossRefPubMedPubMedCentralGoogle Scholar
  9. Bujotzek A, Lipsmeier F, Harris SF, Benz J, Kuglstatter A, Georges G (2016) VH-VL orientation prediction for antibody humanization candidate selection: a case study. MAbs 8:288–305CrossRefPubMedGoogle Scholar
  10. Chamberlain N, Massad C, Oe T, Cantaert T, Herold KC, Meffre E (2016) Rituximab does not reset defective early B cell tolerance checkpoints. J Clin Invest 126:282–287CrossRefPubMedGoogle Scholar
  11. Conti-Fine BM, Diethelm-Okita B, Ostlie N, Wang W, Milani M (2009) Immunopathogenesis of myasthenia gravis. In: Kaminski H J (ed) Myasthenia gravis and related disorders. Humana Press, Totowa, pp 43–70Google Scholar
  12. Deitiker PR, Oshima M, Smith RG, Mosier DR, Atassi MZ (2006) Subtle differences in HLA DQ haplotype-associated presentation of AChR α-chain peptides may suffice to mediate myasthenia gravis. Autoimmunity 39:277–288CrossRefPubMedGoogle Scholar
  13. Deitiker PR, Oshima M, Smith RG, Mosier D, Atassi MZ (2011) Association with HLA DQ of early onset myasthenia gravis in Southeast Texas region of the United States. Int J Immunogenet 38:55–62CrossRefPubMedGoogle Scholar
  14. Drachman DB (1994) Myasthenia gravis. N Engl J Med 330:1797–1810CrossRefPubMedGoogle Scholar
  15. Evans L, Hughes M, Waters J, Cameron J, Dodsworth N, Tooth D, Greenfield A, Sleep D (2010) The production, characterisation and enhanced pharmacokinetics of scFv–albumin fusions expressed in Saccharomyces cerevisiae. Protein Expr Purif 73:113–124CrossRefPubMedGoogle Scholar
  16. Fernando MMA, Stevens CR, Walsh EC, De Jager PL, Goyette P, Plenge RM, Vyse TJ, Rioux JD (2008) Defining the role of the MHC in autoimmunity: a review and pooled analysis. PLoS Genet 4:e1000024CrossRefPubMedPubMedCentralGoogle Scholar
  17. Foote J, Winter G (1992) Antibody framework residues affecting the conformation of the hypervariable loops. J Mol Biol 224:487–499CrossRefPubMedGoogle Scholar
  18. Furrer E, Berdugo M, Stella C, Behar-Cohen F, Gurny R, Feige U, Lichtlen P, Urech DM (2009) Pharmacokinetics and posterior segment biodistribution of ESBA105, an anti–TNF-α single-chain antibody, upon topical administration to the rabbit eye. Invest Ophthalmol Vis Sci 50:771–778CrossRefPubMedGoogle Scholar
  19. Gil D, Schrum AG (2013) Strategies to stabilize compact folding and minimize aggregation of antibody-based fragments. Adv Biosci Biotechnol 4:73–84CrossRefPubMedPubMedCentralGoogle Scholar
  20. Giraud M, Vandiedonck C, Garchon H-J (2008) Genetic factors in autoimmune myasthenia gravis. Ann N Y Acad Sci 1132:180–192CrossRefPubMedGoogle Scholar
  21. Giudicelli V, Lefranc M-P (2011) IMGT/JunctionAnalysis: IMGT standardized analysis of the V-J and V-D-J junctions of the rearranged immunoglobulins (Ig) and T cell receptors (TR). Cold Spring Harb Protoc 2011:716–725PubMedGoogle Scholar
  22. Giudicelli V, Brochet X, Lefranc M-P (2011) IMGT/V-QUEST: IMGT standardized analysis of the immunoglobulin (Ig) and T cell receptor (TR) nucleotide sequences. Cold Spring Harb Protoc 2011:695–715PubMedGoogle Scholar
  23. Guo J-Q, Li Q-M, Zhou J-Y, Zhang G-P, Yang Y-Y, Xing G-X, Zhao D, You S-Y, Zhang C-Y (2006) Efficient recovery of the functional IP10-scFv fusion protein from inclusion bodies with an on-column refolding system. Protein Expr Purif 45:168–174CrossRefPubMedGoogle Scholar
  24. Hohlfeld R, Kalies I, Kohleisen B, Heininger K, Conti-Tronconi B, Toyka KV (1986) Myasthenia gravis: stimulation of antireceptor autoantibodies by autoreactive T cell lines. Neurology 36:618–621CrossRefPubMedGoogle Scholar
  25. Kabat E, Wu TT, Perry HM, Gottesman KS, Foeller C (1991) Sequence of proteins of immunological interest. Publication No 91-3242. US Public Health Services, NIH, BethesdaGoogle Scholar
  26. Kay J, Matteson EL, Dasgupta B, Nash P, Durez P, Hall S, Hsia EC, Han J, Wagner C, Xu Z, Visvanathan S, Rahman MU (2008) Golimumab in patients with active rheumatoid arthritis despite treatment with methotrexate: a randomized, double-blind, placebo-controlled, dose-ranging study. Arthritis Rheum 58:964–975CrossRefPubMedGoogle Scholar
  27. Kim Y-J, Neelamegam R, Heo M-A, Edwardraja S, Paik H-J, Lee S-G (2008) Improving the productivity of single-chain Fv antibody against c-Met by rearranging the order of its variable domains. J Microbiol Biotechnol 18:1186–1190PubMedGoogle Scholar
  28. Koerber JT, Hornsby MJ, Wells JA (2015) An improved single-chain Fab platform for efficient display and recombinant expression. J Mol Biol 427:576–586CrossRefPubMedGoogle Scholar
  29. Kovalenko OV, Olland A, Piché-Nicholas N, Godbole A, King D, Svenson K, Calabro V, Müller MR, Barelle CJ, Somers W, Gill DS, Mosyak L, Tchistiakova L (2013) Atypical antigen recognition mode of a shark immunoglobulin new antigen receptor (IgNAR) variable domain characterized by humanization and structural analysis. J Biol Chem 288(24):17408–17419CrossRefPubMedPubMedCentralGoogle Scholar
  30. Le Panse R, Bismuth J, Cizeron-Clairac G, Weiss JM, Cufi P, Dartevelle P, De Rosbo NK, Berrih-Aknin S (2010) Thymic remodeling associated with hyperplasia in myasthenia gravis. Autoimmunity 43:401–412CrossRefPubMedGoogle Scholar
  31. Leite MI, Jones M, Ströbel P, Marx A, Gold R, Niks E, Verschuuren JJGM, Berrih-Aknin S, Scaravilli F, Canelhas A, Morgan BP, Vincent A, Willcox N (2007) Myasthenia gravis thymus: complement vulnerability of epithelial and myoid cells, complement attack on them, and correlations with autoantibody status. Am J Pathol 171:893–905CrossRefPubMedPubMedCentralGoogle Scholar
  32. Lu D, Jimenez X, Witte L, Zhu Z (2004) The effect of variable domain orientation and arrangement on the antigen-binding activity of a recombinant human bispecific diabody. Biochem Biophys Res Commun 318:507–513CrossRefPubMedGoogle Scholar
  33. Mamalaki A, Trakas N, Tzartos SJ (1993) Bacterial expression of a single-chain Fv fragment which efficiently protects the acetylcholine receptor against antigenic modulation caused by myasthenic antibodies. Eur J Immunol 23:1839–1845CrossRefPubMedGoogle Scholar
  34. Maniaol AH, Elsais A, Lorentzen ÅR, Owe JF, Viken MK, Sæther H, Flåm ST, Bråthen G, Kampman MT, Midgard R, Christensen M, Rognerud A, Kerty E, Gilhus NE, Tallaksen CME, Lie BA, Harbo HF (2012) Late onset myasthenia gravis is associated with HLA DRB1*15:01 in the Norwegian population. PLoS One 7:e36603CrossRefPubMedPubMedCentralGoogle Scholar
  35. Marino M, Maiuri MT, Di Sante G, Scuderi F, La Carpia F, Trakas N, Provenzano C, Zisimopoulou P, Ria F, Tzartos SJ, Evoli A, Bartoccioni E (2014) T cell repertoire in DQ5-positive MuSK-positive myasthenia gravis patients. J Autoimmun 52:113–121CrossRefPubMedGoogle Scholar
  36. McDevitt HO (2000) Discovering the role of the major histocompatibility complex in the immune response. Annu Rev Immunol 18:1–17CrossRefPubMedGoogle Scholar
  37. Monod MY, Giudicelli V, Chaume D, Lefranc M-P (2004) IMGT/JunctionAnalysis: the first tool for the analysis of the immunoglobulin and T cell receptor complex V–J and V–D–J JUNCTIONs. Bioinformatics 20:i379–i385CrossRefGoogle Scholar
  38. Mulac-Jericevic B, Manshouri T, Yokoi T, Atassi MZ (1988) The regions of α-neurotoxin binding on the extracellular part of the α-subunit of human acetylcholine receptor. J Protein Chem 7:173–177CrossRefPubMedGoogle Scholar
  39. Nakayashiki N, Oshima M, Deitiker PR, Ashizawa T, Atassi MZ (2000) Suppression of experimental myasthenia gravis by monoclonal antibodies against MHC peptide region involved in presentation of a pathogenic T-cell epitope. J Neuroimmunol 105:131–144CrossRefPubMedGoogle Scholar
  40. Nancy P, Berrih-Aknin S (2005) Differential estrogen receptor expression in autoimmune myasthenia gravis. Endocrinology 146:2345–2353CrossRefPubMedPubMedCentralGoogle Scholar
  41. Ofosu-Appiah W, Mokhtarian F, Shirazian D, Grob D (1994) Production of anti-acetylcholine receptor-a antibody in vitro by peripheral blood lymphocytes of patients with myasthenia gravis: role of immunoregulatory T cells and monocytes. J Lab Clin Med 124:231–241PubMedGoogle Scholar
  42. Ojima-Kato T, Fukui K, Yamamoto H, Hashimura D, Miyake S, Hirakawa Y, Yamasaki T, Kojima T, Nakano H (2016) ‘Zipbody’ leucine zipper-fused Fab in E. coli in vitro and in vivo expression systems. Protein Eng Des Sel 29:149–157CrossRefPubMedGoogle Scholar
  43. Oshima M, Atassi MZ (1995) Effect of amino acid substitutions within the region 62-76 of I-A beta b on binding with and antigen presentation of Torpedo acetylcholine receptor alpha-chain peptide 146-162. J Immunol 154:5245–5254PubMedGoogle Scholar
  44. Oshima M, Yokoi T, Deitiker P, Atassi MZ (1998) T cell responses in EAMG-susceptible and non-susceptible mouse strains after immunization with overlapping peptides encompassing the extracellular part of Torpedo californica acetylcholine receptor alpha chain. Implication to role in myasthenia gravis of autoimmune T-cell responses against receptor degradation products. Autoimmunity 27(2):79–90CrossRefPubMedGoogle Scholar
  45. Oshima M, Deitiker PR, Mosier DR, Smith RG, Atassi MZ (2005a) Responses in vitro of peripheral blood lymphocytes from patients with myasthenia gravis to stimulation with human acetylcholine receptor α-chain peptides: analysis in relation to age, thymic abnormality, and ethnicity. Hum Immunol 66:32–42CrossRefPubMedGoogle Scholar
  46. Oshima M, Ohtani M, Deitiker PR, Glenn Smith R, Mosier DR, Zouhair Atassi M (2005b) Suppression by mAbs against DQB1 peptides of in vitro proliferation of AChR-specific T cells from myasthenia gravis patients. Autoimmunity 38:161–169CrossRefPubMedGoogle Scholar
  47. Papanastasiou D, Mamalaki A, Eliopoulos E, Poulas K, Liolitsas C, Tzartos SJ (1999) Construction and characterization of a humanized single chain Fv antibody fragment against the main immunogenic region of the acetylcholine receptor. J Neuroimmunol 94:182–195CrossRefPubMedGoogle Scholar
  48. Protopapadakis E, Kokla A, Tzartos SJ, Mamalaki A (2005) Isolation and characterization of human anti-acetylcholine receptor monoclonal antibodies from transgenic mice expressing human immunoglobulin loci. Eur J Immunol 35:1960–1968CrossRefPubMedGoogle Scholar
  49. Robeson KR, Kumar A, Keung B, DiCapua DB, Grodinsky E, Patwa HS, Stathopoulos PA, Goldstein JM, O’Connor KC, Nowak RJ (2017) Durability of the rituximab response in acetylcholine receptor autoantibody-positive myasthenia gravis. JAMA Neurol 74:60–66CrossRefPubMedGoogle Scholar
  50. Rosenberg JS, Oshima M, Atassi MZ (1996) B-cell activation in vitro by helper T cells specific to region alpha 146-162 of Torpedo californica nicotinic acetylcholine receptor. J Immunol 157:3192–3199PubMedGoogle Scholar
  51. Saruhan-Direskeneli G, Kiliç A, Parman Y, Serdaroğlu P, Deymeer F (2006) HLA-DQ polymorphism in Turkish patients with myasthenia gravis. Hum Immunol 67:352–358CrossRefPubMedGoogle Scholar
  52. Saruhan-Direskeneli G, Hughes T, Yilmaz V, Durmus H, Adler A, Alahgholi-Hajibehzad M, Aysal F, Yentür SP, Akalin MA, Dogan O, Marx A, Gülsen-Parman Y, Oflazer P, Deymeer F, Sawalha AH (2016) Genetic heterogeneity within the HLA region in three distinct clinical subgroups of myasthenia gravis. Clin Immunol 166–167:81–88CrossRefPubMedGoogle Scholar
  53. Shenoy M, Oshima M, Atassi MZ, Christadoss P (1993) Suppression of experimental autoimmune myasthenia gravis by epitope-specific neonatal tolerance to synthetic region alpha 146-162 of acetylcholine receptor. Clin Immunol Immunopathol 66:230–238CrossRefPubMedGoogle Scholar
  54. Sun H, Wu G, Chen Y, Tian Y, Yue Y, Zhang G (2014) Expression, production, and renaturation of a functional single-chain variable antibody fragment (scFv) against human ICAM-1. Braz J Med Biol Res 47:540–547CrossRefPubMedPubMedCentralGoogle Scholar
  55. Teplyakov A, Obmolova G, Malia T, Gilliland G (2011) Antigen recognition by antibody C836 through adjustment of VL/VH packing. Acta Crystallogr Sect F Struct Biol Cryst Commun 67:1165–1167CrossRefPubMedPubMedCentralGoogle Scholar
  56. Testi M, Terracciano C, Guagnano A, Testa G, Marfia GA, Pompeo E, Andreani M, Massa R (2012) Association of HLA-DQB1∗05:02 and DRB1∗16 alleles with late-onset, nonthymomatous, AChR-Ab-positive myasthenia gravis. Autoimmune Dis 2012:541760PubMedPubMedCentralGoogle Scholar
  57. Tiftikcioglu BI, Uludag IF, Zorlu Y, Pirim İ, Sener U, Tokucoglu F, Korucuk M (2017) Human leucocyte antigen B50 is associated with conversion to generalized myasthenia gravis in patients with pure ocular onset. Med Princ Pract 26:71–77CrossRefPubMedGoogle Scholar
  58. Tzartos SJ, Seybold ME, Lindstrom JM (1982) Specificities of antibodies to acetylcholine receptors in sera from myasthenia gravis patients measured by monoclonal antibodies. Proc Natl Acad Sci U S A 79:188–192CrossRefPubMedPubMedCentralGoogle Scholar
  59. Tzartos S, Hochschwender S, Vasquez P, Lindstrom J (1987) Passive transfer of experimental autoimmune myasthenia gravis by monoclonal antibodies to the main immunogenic region of the acetylcholine receptor. J Neuroimmunol 15:185–194CrossRefPubMedGoogle Scholar
  60. Vander Heiden JA, Stathopoulos P, Zhou JQ, Chen L, Gilbert TJ, Bolen CR, Barohn RJ, Dimachkie MM, Ciafaloni E, Broering TJ, Vigneault F, Nowak RJ, Kleinstein SH, O’Connor KC (2017) Dysregulation of B cell repertoire formation in myasthenia gravis patients revealed through deep sequencing. J Immunol 198:1460–1473CrossRefPubMedGoogle Scholar
  61. Vandiedonck C, Beaurain G, Giraud M, Hue-Beauvais C, Eymard B, Tranchant C, Gajdos P, Dausset J, Garchon H-J (2004) Pleiotropic effects of the 8.1 HLA haplotype in patients with autoimmune myasthenia gravis and thymus hyperplasia. Proc Natl Acad Sci U S A 101:15464–15469CrossRefPubMedPubMedCentralGoogle Scholar
  62. Vijayan N, Vijayan VK, Dreyfus PM (1977) Acetylcholinesterase activity and menstrual remissions in myasthenia gravis. J Neurol Neurosurg Psychiatry 40:1060–1065CrossRefPubMedPubMedCentralGoogle Scholar
  63. Vrolix K, Fraussen J, Losen M, Stevens J, Lazaridis K, Molenaar PC, Somers V, Bracho MA, Le Panse R, Stinissen P, Berrih-Aknin S, Maessen JG, Van Garsse L, Buurman WA, Tzartos SJ, De Baets MH, Martinez-Martinez P (2014) Clonal heterogeneity of thymic B cells from early-onset myasthenia gravis patients with antibodies against the acetylcholine receptor. J Autoimmun 52:101–112CrossRefPubMedGoogle Scholar
  64. Wang Z, Raifu M, Howard M, Smith L, Hansen D, Goldsby R, Ratner D (2000) Universal PCR amplification of mouse immunoglobulin gene variable regions: the design of degenerate primers and an assessment of the effect of DNA polymerase 3′ to 5′ exonuclease activity. J Immunol Methods 233:167–177CrossRefPubMedGoogle Scholar
  65. Willcox N, Leite MI, Kadota Y, Jones M, Meager A, Subrahmanyam P, Dasgupta B, Morgan BP, Vincent A (2008) Autoimmunizing mechanisms in thymoma and thymus. Ann N Y Acad Sci 1132:163–173CrossRefPubMedGoogle Scholar
  66. Wolfe GI, Kaminski HJ, Aban IB, Minisman G, Kuo H-C, Marx A, Ströbel P, Mazia C, Oger J, Cea JG, Heckmann JM, Evoli A, Nix W, Ciafaloni E, Antonini G, Witoonpanich R, King JO, Beydoun SR, Chalk CH, Barboi AC, Amato AA, Shaibani AI, Katirji B, Lecky BRF, Buckley C, Vincent A, Dias-Tosta E, Yoshikawa H, Waddington-Cruz M, Pulley MT, Rivner MH, Kostera-Pruszczyk A, Pascuzzi RM, Jackson CE, Garcia Ramos GS, Verschuuren JJGM, Massey JM, Kissel JT, Werneck LC, Benatar M, Barohn RJ, Tandan R, Mozaffar T, Conwit R, Odenkirchen J, Sonett JR, Jaretzki AI, Newsom-Davis J, Cutter GR (2016) Randomized trial of thymectomy in myasthenia gravis. N Engl J Med 375:511–522CrossRefPubMedPubMedCentralGoogle Scholar
  67. Wörn A, Plückthun A (2001) Stability engineering of antibody single-chain Fv fragments. J Mol Biol 305(5):989–1010CrossRefPubMedGoogle Scholar
  68. Yamanouchi J, Rainbow D, Serra P, Howlett S, Hunter K, Garner VES, Gonzalez-Munoz A, Clark J, Veijola R, Cubbon R, Chen S-L, Rosa R, Cumiskey AM, Serreze DV, Gregory S, Rogers J, Lyons PA, Healy B, Smink LJ, Todd JA, Peterson LB, Wicker LS, Santamaria P (2007) Interleukin-2 gene variation impairs regulatory T cell function and causes autoimmunity. Nat Genet 39:329–337CrossRefPubMedPubMedCentralGoogle Scholar
  69. Yousefipour G-A, Salami Z, Farjadian S (2009) Association of HLA-DQA1* 0101/2 and DQB1* 0502 with myasthenia gravis in southern Iranian patients. Iran J Immunol 6:99–102PubMedGoogle Scholar
  70. Yuan X, Gubbins MJ, Berry JD (2004) A simple and rapid protocol for the sequence determination of functional kappa light chain cDNAs from aberrant-chain-positive murine hybridomas. J Immunol Methods 294:199–207CrossRefPubMedGoogle Scholar
  71. Zagoriti Z, Kambouris ME, Patrinos GP, Tzartos SJ, Poulas K (2013) Recent advances in genetic predisposition of myasthenia gravis. Biomed Res Int 2013:404053CrossRefPubMedPubMedCentralGoogle Scholar
  72. Zhang K, Geddie ML, Kohli N, Kornaga T, Kirpotin DB, Jiao Y, Rennard R, Drummond DC, Nielsen UB, Xu L, Lugovskoy AA (2015) Comprehensive optimization of a single-chain variable domain antibody fragment as a targeting ligand for a cytotoxic nanoparticle. MAbs 7:42–52CrossRefPubMedGoogle Scholar

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

Personalised recommendations