FREE SHIPPING over $40
OVER 9000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Home / Equine Research Bank / Front Immunol / Centaur antibodies: Engineered chimeric equine-human recombi...
Frontiers in immunology2022; 13; 942317; doi: 10.3389/fimmu.2022.942317

Centaur antibodies: Engineered chimeric equine-human recombinant antibodies.

Abstract: Hyper-immune antisera from large mammals, in particular horses, are routinely used for life-saving anti-intoxication intervention. While highly efficient, the use of these immunotherapeutics is complicated by possible recipient reactogenicity and limited availability. Accordingly, there is an urgent need for alternative improved next-generation immunotherapies to respond to this issue of high public health priority. Here, we document the development of previously unavailable tools for equine antibody engineering. A novel primer set, EquPD v2020, based on equine V-gene data, was designed for efficient and accurate amplification of rearranged horse antibody V-segments. The primer set served for generation of immune phage display libraries, representing highly diverse V-gene repertoires of horses immunized against botulinum A or B neurotoxins. Highly specific scFv clones were selected and expressed as full-length antibodies, carrying equine V-genes and human Gamma1/Lambda constant genes, to be referred as "Centaur antibodies". Preliminary assessment in a murine model of botulism established their therapeutic potential. The experimental approach detailed in the current report, represents a valuable tool for isolation and engineering of therapeutic equine antibodies.
Copyright © 2022 Rosenfeld, Alcalay, Zvi, Ben-David, Noy-Porat, Chitlaru, Epstein, Israeli, Lazar, Caspi, Barnea, Dor, Chomsky, Pitel, Makdasi, Zichel and Mazor.
Get Full Text
Publication Date: 2022-08-19 PubMed ID: 36059507PubMed Central: PMC9437483DOI: 10.3389/fimmu.2022.942317Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research article is about the development of new tools for engineering equine antibodies through a method they called “Centaur antibodies.” The antibodies generated showed therapeutic potential in a murine model of botulism, offering an alternative to current immunotherapies made from the anti-sera of large mammals.

Introduction and Motivation

  • This study arises from the need to develop alternative immunotherapies due to the limitations of current therapies created from large mammals’ hyper-immune antisera.
  • The use of these immunotherapeutics, while effective, can lead to recipient reactogenicity and are also constrained by limited availability.
  • Therefore, the research addresses the development of improved, next-generation immunotherapies to mitigate these challenges that are of high public health concern.

Research Methodology

  • The researchers devised a new primer set, named EquPD v2020, based on equine V-gene data.
  • This primer set was designed to enable efficient and accurate amplification of the rearranged horse antibody V-segments.
  • The amplified V-segments were used to create immune phage display libraries representing highly diverse V-gene repertoires of horses immunized against botulinum A or B neurotoxins.
  • From the libraries, the researchers selected and expressed scFv clones as full-length antibodies. These contained equine V-genes and human Gamma1/Lambda constant genes, making them chimeric, or “Centaur,” antibodies.

Findings

  • Tests on a murine model of botulism showed that these Centaur antibodies have therapeutic potential, making them a valuable tool in the field of immunotherapy.
  • This approach opens up new possibilities for producing therapeutic equine antibodies, providing an alternative to current immunotherapies derived from large mammals’ antisera.

Conclusion

  • The approach detailed in this study represents a significant development in the field of antibody engineering.
  • These engineered antibodies could serve as a more readily available and less reactogenic intervention for intoxications that current immunotherapies treat.

Cite This Article

APA
Rosenfeld R, Alcalay R, Zvi A, Ben-David A, Noy-Porat T, Chitlaru T, Epstein E, Israeli O, Lazar S, Caspi N, Barnea A, Dor E, Chomsky I, Pitel S, Makdasi E, Zichel R, Mazor O. (2022). Centaur antibodies: Engineered chimeric equine-human recombinant antibodies. Front Immunol, 13, 942317. https://doi.org/10.3389/fimmu.2022.942317

Publication

Frontiers in immunology
ISSN: 1664-3224
NlmUniqueID: 101560960
Country: Switzerland
Language: English
Volume: 13
Pages: 942317
PII: 942317

Researcher Affiliations

Rosenfeld, Ronit
  • Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness Ziona, Israel.
Alcalay, Ron
  • Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness Ziona, Israel.
Zvi, Anat
  • Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness Ziona, Israel.
Ben-David, Alon
  • Department of Biotechnology, Israel Institute for Biological Research, Ness Ziona, Israel.
Noy-Porat, Tal
  • Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness Ziona, Israel.
Chitlaru, Theodor
  • Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness Ziona, Israel.
Epstein, Eyal
  • Department of Biotechnology, Israel Institute for Biological Research, Ness Ziona, Israel.
Israeli, Ofir
  • Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness Ziona, Israel.
Lazar, Shirley
  • Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness Ziona, Israel.
Caspi, Noa
  • Veterinary Center for Preclinical Research, Israel Institute for Biological Research, Ness Ziona, Israel.
Barnea, Ada
  • Department of Biotechnology, Israel Institute for Biological Research, Ness Ziona, Israel.
Dor, Eyal
  • Department of Biotechnology, Israel Institute for Biological Research, Ness Ziona, Israel.
Chomsky, Inbar
  • Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness Ziona, Israel.
Pitel, Shani
  • Department of Biotechnology, Israel Institute for Biological Research, Ness Ziona, Israel.
Makdasi, Efi
  • Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness Ziona, Israel.
Zichel, Ran
  • Department of Biotechnology, Israel Institute for Biological Research, Ness Ziona, Israel.
Mazor, Ohad
  • Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness Ziona, Israel.

MeSH Terms

  • Animals
  • Antibodies / genetics
  • Cell Surface Display Techniques
  • Horses
  • Humans
  • Immunoglobulin Variable Region / genetics
  • Mammals
  • Mice
  • Neurotoxins
  • Recombinant Proteins / genetics

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

This article includes 48 references
  1. Theakston RD, Warrell DA, Griffiths E. Report of a WHO workshop on the standardization and control of antivenoms.. Toxicon 2003 Apr;41(5):541-57.
    doi: 10.1016/S0041-0101(02)00393-8pubmed: 12676433google scholar: lookup
  2. Chippaux JP. Snakebite envenomation turns again into a neglected tropical disease!. J Venom Anim Toxins Incl Trop Dis 2017;23:38.
    doi: 10.1186/s40409-017-0127-6pmc: PMC5549382pubmed: 28804495google scholar: lookup
  3. Laustsen AH, Dorrestijn N. Integrating Engineering, Manufacturing, and Regulatory Considerations in the Development of Novel Antivenoms.. Toxins (Basel) 2018 Jul 31;10(8).
    doi: 10.3390/toxins10080309pmc: PMC6115708pubmed: 30065185google scholar: lookup
  4. Adekar SP, Takahashi T, Jones RM, Al-Saleem FH, Ancharski DM, Root MJ, Kapadnis BP, Simpson LL, Dessain SK. Neutralization of botulinum neurotoxin by a human monoclonal antibody specific for the catalytic light chain.. PLoS One 2008 Aug 20;3(8):e3023.
    doi: 10.1371/journal.pone.0003023pmc: PMC2515629pubmed: 18714390google scholar: lookup
  5. Nowakowski A, Wang C, Powers DB, Amersdorfer P, Smith TJ, Montgomery VA, Sheridan R, Blake R, Smith LA, Marks JD. Potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody.. Proc Natl Acad Sci U S A 2002 Aug 20;99(17):11346-50.
    doi: 10.1073/pnas.172229899pmc: PMC123259pubmed: 12177434google scholar: lookup
  6. Razai A, Garcia-Rodriguez C, Lou J, Geren IN, Forsyth CM, Robles Y, Tsai R, Smith TJ, Smith LA, Siegel RW, Feldhaus M, Marks JD. Molecular evolution of antibody affinity for sensitive detection of botulinum neurotoxin type A.. J Mol Biol 2005 Aug 5;351(1):158-69.
    doi: 10.1016/j.jmb.2005.06.003pubmed: 16002090google scholar: lookup
  7. Grilo AL, Mantalaris A. The Increasingly Human and Profitable Monoclonal Antibody Market.. Trends Biotechnol 2019 Jan;37(1):9-16.
    doi: 10.1016/j.tibtech.2018.05.014pubmed: 29945725google scholar: lookup
  8. Kaplon H, Reichert JM. Antibodies to watch in 2021.. MAbs 2021 Jan-Dec;13(1):1860476.
    doi: 10.1080/19420862.2020.1860476pmc: PMC7833761pubmed: 33459118google scholar: lookup
  9. Lu RM, Hwang YC, Liu IJ, Lee CC, Tsai HZ, Li HJ, Wu HC. Development of therapeutic antibodies for the treatment of diseases.. J Biomed Sci 2020 Jan 2;27(1):1.
    doi: 10.1186/s12929-019-0592-zpmc: PMC6939334pubmed: 31894001google scholar: lookup
  10. Almagro JC, Martinez L, Smith SL, Alagon A, Estevez J, Paniagua J. Analysis of the horse V(H) repertoire and comparison with the human IGHV germline genes, and sheep, cattle and pig V(H) sequences.. Mol Immunol 2006 Apr;43(11):1836-45.
    doi: 10.1016/j.molimm.2005.10.017pubmed: 16337682google scholar: lookup
  11. Benson DA, Cavanaugh M, Clark K, Karsch-Mizrachi I, Lipman DJ, Ostell J, Sayers EW. GenBank.. Nucleic Acids Res 2013 Jan;41(Database issue):D36-42.
    doi: 10.1093/nar/gks1195pmc: PMC3531190pubmed: 23193287google scholar: lookup
  12. Ford JE, Home WA, Gibson DM. Light chain isotype regulation in the horse. Characterization of Ig kappa genes.. J Immunol 1994 Aug 1;153(3):1099-111.
    pubmed: 8027543
  13. Giudicelli V, Duroux P, Ginestoux C, Folch G, Jabado-Michaloud J, Chaume D, Lefranc MP. IMGT/LIGM-DB, the IMGT comprehensive database of immunoglobulin and T cell receptor nucleotide sequences.. Nucleic Acids Res 2006 Jan 1;34(Database issue):D781-4.
    doi: 10.1093/nar/gkj088pmc: PMC1347451pubmed: 16381979google scholar: lookup
  14. Sun Y, Wang C, Wang Y, Zhang T, Ren L, Hu X, Zhang R, Meng Q, Guo Y, Fei J, Li N, Zhao Y. A comprehensive analysis of germline and expressed immunoglobulin repertoire in the horse.. Dev Comp Immunol 2010 Sep;34(9):1009-20.
    doi: 10.1016/j.dci.2010.05.003pubmed: 20466019google scholar: lookup
  15. Walther S, Rusitzka TV, Diesterbeck US, Czerny CP. Equine immunoglobulins and organization of immunoglobulin genes.. Dev Comp Immunol 2015 Dec;53(2):303-19.
    doi: 10.1016/j.dci.2015.07.017pubmed: 26219564google scholar: lookup
  16. Roth KDR, Wenzel EV, Ruschig M, Steinke S, Langreder N, Heine PA, Schneider KT, Ballmann R, Fühner V, Kuhn P, Schirrmann T, Frenzel A, Dübel S, Schubert M, Moreira GMSG, Bertoglio F, Russo G, Hust M. Developing Recombinant Antibodies by Phage Display Against Infectious Diseases and Toxins for Diagnostics and Therapy.. Front Cell Infect Microbiol 2021;11:697876.
    doi: 10.3389/fcimb.2021.697876pmc: PMC8294040pubmed: 34307196google scholar: lookup
  17. Torgeman A, Mador N, Dorozko M, Lifshitz A, Eschar N, White MD, Wolf DG, Epstein E. Efficacy of inactivation of viral contaminants in hyperimmune horse plasma against botulinum toxin by low pH alone and combined with pepsin digestion.. Biologicals 2017 Jul;48:24-27.
    doi: 10.1016/j.biologicals.2017.06.003pubmed: 28633975google scholar: lookup
  18. Ben David A, Barnea A, Diamant E, Dor E, Schwartz A, Torgeman A, Zichel R. Small Molecule Receptor Binding Inhibitors with In Vivo Efficacy against Botulinum Neurotoxin Serotypes A and E.. Int J Mol Sci 2021 Aug 9;22(16).
    doi: 10.3390/ijms22168577pmc: PMC8395308pubmed: 34445283google scholar: lookup
  19. Ben David A, Diamant E, Barnea A, Rosen O, Torgeman A, Zichel R. The receptor binding domain of botulinum neurotoxin serotype A (BoNT/A) inhibits BoNT/A and BoNT/E intoxications in vivo.. Clin Vaccine Immunol 2013 Aug;20(8):1266-73.
    doi: 10.1128/CVI.00268-13pmc: PMC3754506pubmed: 23761665google scholar: lookup
  20. Ben David A, Torgeman A, Barnea A, Zichel R. Expression, purification and characterization of the receptor-binding domain of botulinum neurotoxin serotype B as a vaccine candidate.. Protein Expr Purif 2015 Jun;110:122-9.
    doi: 10.1016/j.pep.2015.02.008pubmed: 25727047google scholar: lookup
  21. Diamant E, Lachmi BE, Keren A, Barnea A, Marcus H, Cohen S, David AB, Zichel R. Evaluating the synergistic neutralizing effect of anti-botulinum oligoclonal antibody preparations.. PLoS One 2014;9(1):e87089.
    doi: 10.1371/journal.pone.0087089pmc: PMC3903612pubmed: 24475231google scholar: lookup
  22. Azriel-Rosenfeld R, Valensi M, Benhar I. A human synthetic combinatorial library of arrayable single-chain antibodies based on shuffling in vivo formed CDRs into general framework regions.. J Mol Biol 2004 Jan 2;335(1):177-92.
    doi: 10.1016/j.jmb.2003.10.053pubmed: 14659749google scholar: lookup
  23. Berdichevsky Y, Ben-Zeev E, Lamed R, Benhar I. Phage display of a cellulose binding domain from Clostridium thermocellum and its application as a tool for antibody engineering.. J Immunol Methods 1999 Aug 31;228(1-2):151-62.
    doi: 10.1016/S0022-1759(99)00096-4pubmed: 10556552google scholar: lookup
  24. Vander Heiden JA, Yaari G, Uduman M, Stern JN, O'Connor KC, Hafler DA, Vigneault F, Kleinstein SH. pRESTO: a toolkit for processing high-throughput sequencing raw reads of lymphocyte receptor repertoires.. Bioinformatics 2014 Jul 1;30(13):1930-2.
    doi: 10.1093/bioinformatics/btu138pmc: PMC4071206pubmed: 24618469google scholar: lookup
  25. Alamyar E, Giudicelli V, Duroux P, Lefranc M. IMGT/HighV-QUEST: A high-throughput system and web portal for the analysis of rearranged nucleotide sequences of antigen receptors-high-throughput version of IMGT/V-QUEST. V-QUEST 11èmes Journées Ouvertes En Biologie Informatique Mathématiques (JOBIM) 2010, 7–9.
  26. Gupta NT, Vander Heiden JA, Uduman M, Gadala-Maria D, Yaari G, Kleinstein SH. Change-O: a toolkit for analyzing large-scale B cell immunoglobulin repertoire sequencing data.. Bioinformatics 2015 Oct 15;31(20):3356-8.
    doi: 10.1093/bioinformatics/btv359pmc: PMC4793929pubmed: 26069265google scholar: lookup
  27. Noy-Porat T, Makdasi E, Alcalay R, Mechaly A, Levy Y, Bercovich-Kinori A, Zauberman A, Tamir H, Yahalom-Ronen Y, Israeli M, Epstein E, Achdout H, Melamed S, Chitlaru T, Weiss S, Peretz E, Rosen O, Paran N, Yitzhaki S, Shapira SC, Israely T, Mazor O, Rosenfeld R. A panel of human neutralizing mAbs targeting SARS-CoV-2 spike at multiple epitopes.. Nat Commun 2020 Aug 27;11(1):4303.
    doi: 10.1038/s41467-020-18159-4pmc: PMC7452893pubmed: 32855401google scholar: lookup
  28. Zichel R, Mimran A, Keren A, Barnea A, Steinberger-Levy I, Marcus D, Turgeman A, Reuveny S. Efficacy of a potential trivalent vaccine based on Hc fragments of botulinum toxins A, B, and E produced in a cell-free expression system.. Clin Vaccine Immunol 2010 May;17(5):784-92.
    doi: 10.1128/CVI.00496-09pmc: PMC2863383pubmed: 20357058google scholar: lookup
  29. European Directorate for the Quality of Medicines and Healthcare. Botulinum toxin type a / type b for injection. Strasbourg, France: European Pharmacopoeia; (2019).
  30. Irwin JO, Cheeseman EA. On an approximate method of determining the median effective dose and its error, in the case of a quantal response.. J Hyg (Lond) 1939 Sep;39(5):574-80.
    doi: 10.1017/S0022172400012213pmc: PMC2199486pubmed: 20475518google scholar: lookup
  31. Noy-Porat T, Rosenfeld R, Ariel N, Epstein E, Alcalay R, Zvi A, Kronman C, Ordentlich A, Mazor O. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates.. Toxins (Basel) 2016 Mar 3;8(3).
    doi: 10.3390/toxins8030064pmc: PMC4810209pubmed: 26950154google scholar: lookup
  32. Arun SS, Breuer W, Hermanns W. Immunohistochemical examination of light-chain expression (lambda/kappa ratio) in canine, feline, equine, bovine and porcine plasma cells.. Zentralbl Veterinarmed A 1996 Nov;43(9):573-6.
    doi: 10.1111/j.1439-0442.1996.tb00489.xpubmed: 8968166google scholar: lookup
  33. Hood L, Campbell JH, Elgin SC. The organization, expression, and evolution of antibody genes and other multigene families.. Annu Rev Genet 1975;9:305-53.
    doi: 10.1146/annurev.ge.09.120175.001513pubmed: 813561google scholar: lookup
  34. Rosenfeld R, Zvi A, Winter E, Hope R, Israeli O, Mazor O, Yaari G. A primer set for comprehensive amplification of V-genes from rhesus macaque origin based on repertoire sequencing.. J Immunol Methods 2019 Feb;465:67-71.
    doi: 10.1016/j.jim.2018.11.011pubmed: 30471299google scholar: lookup
  35. Li D, Mattoo P, Keller JE. New equine antitoxins to botulinum neurotoxins serotypes A and B.. Biologicals 2012 Jul;40(4):240-6.
    doi: 10.1016/j.biologicals.2012.03.004pmc: PMC3393819pubmed: 22560800google scholar: lookup
  36. Kabat EA, Wu TT, Perry HM, Gottesman KS, Foeller C. Sequences of proteins of immunological interest. Bethesda, MD, USA: Public Health Service National Institutes of Health US Department of Health and Human Services; (1991).
  37. Noy-Porat T, Alcalay R, Mechaly A, Peretz E, Makdasi E, Rosenfeld R, Mazor O. Characterization of antibody-antigen interactions using biolayer interferometry.. STAR Protoc 2021 Dec 17;2(4):100836.
    doi: 10.1016/j.xpro.2021.100836pmc: PMC8449132pubmed: 34568849google scholar: lookup
  38. Froude JW, Stiles B, Pelat T, Thullier P. Antibodies for biodefense.. MAbs 2011 Nov-Dec;3(6):517-27.
    doi: 10.4161/mabs.3.6.17621pmc: PMC3242838pubmed: 22123065google scholar: lookup
  39. Noy-Porat T, Alcalay R, Epstein E, Sabo T, Kronman C, Mazor O. Extended therapeutic window for post-exposure treatment of ricin intoxication conferred by the use of high-affinity antibodies.. Toxicon 2017 Mar 1;127:100-105.
    doi: 10.1016/j.toxicon.2017.01.009pubmed: 28089771google scholar: lookup
  40. Rosenfeld R, Marcus H, Ben-Arie E, Lachmi BE, Mechaly A, Reuveny S, Gat O, Mazor O, Ordentlich A. Isolation and chimerization of a highly neutralizing antibody conferring passive protection against lethal Bacillus anthracis infection.. PLoS One 2009 Jul 24;4(7):e6351.
    doi: 10.1371/journal.pone.0006351pmc: PMC2710523pubmed: 19629185google scholar: lookup
  41. de los Rios M, Criscitiello MF, Smider VV. Structural and genetic diversity in antibody repertoires from diverse species.. Curr Opin Struct Biol 2015 Aug;33:27-41.
    doi: 10.1016/j.sbi.2015.06.002pmc: PMC7039331pubmed: 26188469google scholar: lookup
  42. Sun Y, Liu Z, Ren L, Wei Z, Wang P, Li N, Zhao Y. Immunoglobulin genes and diversity: what we have learned from domestic animals.. J Anim Sci Biotechnol 2012 Jun 20;3(1):18.
    doi: 10.1186/2049-1891-3-18pmc: PMC3487963pubmed: 22958617google scholar: lookup
  43. Manso TC, Groenner-Penna M, Minozzo JC, Antunes BC, Ippolito GC, Molina F, Felicori LF. Next-generation sequencing reveals new insights about gene usage and CDR-H3 composition in the horse antibody repertoire.. Mol Immunol 2019 Jan;105:251-259.
    doi: 10.1016/j.molimm.2018.11.017pubmed: 30562645google scholar: lookup
  44. Galson JD, Pollard AJ, Trück J, Kelly DF. Studying the antibody repertoire after vaccination: practical applications.. Trends Immunol 2014 Jul;35(7):319-31.
    doi: 10.1016/j.it.2014.04.005pubmed: 24856924google scholar: lookup
  45. Poulsen TR, Jensen A, Haurum JS, Andersen PS. Limits for antibody affinity maturation and repertoire diversification in hypervaccinated humans.. J Immunol 2011 Oct 15;187(8):4229-35.
    doi: 10.4049/jimmunol.1000928pubmed: 21930965google scholar: lookup
  46. McIntire KR, Rouse AM. Mouse immunoglobulin light chains, alteration of kappa, lamba ratio. Abstr 1970 29(2):A704.
  47. Diamant E, Torgeman A, Ozeri E, Zichel R. Monoclonal Antibody Combinations that Present Synergistic Neutralizing Activity: A Platform for Next-Generation Anti-Toxin Drugs.. Toxins (Basel) 2015 May 29;7(6):1854-81.
    doi: 10.3390/toxins7061854pmc: PMC4488679pubmed: 26035486google scholar: lookup
  48. Miethe S, Mazuet C, Liu Y, Tierney R, Rasetti-Escargueil C, Avril A, Frenzel A, Thullier P, Pelat T, Urbain R, Fontayne A, Sesardic D, Hust M, Popoff MR. Development of Germline-Humanized Antibodies Neutralizing Botulinum Neurotoxin A and B.. PLoS One 2016;11(8):e0161446.
    doi: 10.1371/journal.pone.0161446pmc: PMC4999263pubmed: 27560688google scholar: lookup

Citations

This article has been cited 1 times.
  1. Wibmer CK, Mashilo P. Exploiting V-Gene Bias for Rapid, High-Throughput Monoclonal Antibody Isolation from Horses.. Viruses 2022 Sep 30;14(10).
    doi: 10.3390/v14102172pubmed: 36298728google scholar: lookup
Subscribe to our newsletter
Join 99,357 horse owners like you who receive our email updates on horse health and nutrition.