title: Design of targeted B cell killing agents.
abstract: B cells play an important role in the pathogenesis of both systemic and organ-specific autoimmune diseases. Autoreactive B cells not only produce autoantibodies, but also are capable to efficiently present specific autoantigens to T cells. Furthermore, B cells can secrete proinflammatory cytokines and amplify the vicious process of self-destruction. B cell-directed therapy is a potentially important approach for treatment of various autoimmune diseases. The depletion of B cells by anti-CD20/19 monoclonal antibody Retuximab® used in autoimmune diseases therapy leads to systemic side effects and should be significantly improved. In this study we designed a repertoire of genetically engineered B cell killers that specifically affected one kind of cells carrying a respective B cell receptor. We constructed immunotoxins (ITs), fused with c-myc epitope as a model targeting sequence, based on barnase, Pseudomonas toxin, Shiga-like toxin E.coli and Fc domain of human antibody IgGγ1. C-MYC hybridoma cell line producing anti-c-myc IgG was chosen as a model for targeted cell depletion. C-myc sequence fused with toxins provided addressed delivery of the toxic agent to the target cells. We demonstrated functional activity of designed ITs in vitro and showed recognition of the fusion molecules by antibodies produced by targeted hybridoma. To study specificity of the proposed B cells killing molecules, we tested a set of created ITs ex vivo, using C-MYC and irrelevant hybridoma cell lines. Pseudomonas-containing IT showed one of the highest cytotoxic effects on the model cells, however, possessed promiscuous specificity. Shiga-like toxin construct demonstrated mild both cytotoxicity and specificity. Barnase and Fc-containing ITs revealed excellent balance between their legibility and toxic properties. Moreover, barnase and Fc molecules fused with c-myc epitope were able to selectively deplete c-myc-specific B cells and decrease production of anti-c-myc antibodies in culture of native splenocytes, suggesting their highest therapeutic potential as targeted B cell killing agents.
authors.affiliation: M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.
authors.forename: Alexey V
authors.initials: AV
authors.lastname: Stepanov
authors.forename: Alexey A
authors.initials: AA
authors.lastname: Belogurov
authors.forename: Natalia A
authors.initials: NA
authors.lastname: Ponomarenko
authors.forename: Oleg A
authors.initials: OA
authors.lastname: Stremovskiy
authors.forename: Leonid V
authors.initials: LV
authors.lastname: Kozlov
authors.forename: Anna M
authors.initials: AM
authors.lastname: Bichucher
authors.forename: Sergey E
authors.initials: SE
authors.lastname: Dmitriev
authors.forename: Ivan V
authors.initials: IV
authors.lastname: Smirnov
authors.forename: Olga G
authors.initials: OG
authors.lastname: Shamborant
authors.forename: Dmitry S
authors.initials: DS
authors.lastname: Balabashin
authors.forename: Lidia P
authors.initials: LP
authors.lastname: Sashchenko
authors.forename: Alexander G
authors.initials: AG
authors.lastname: Tonevitsky
authors.forename: Alain
authors.initials: A
authors.lastname: Friboulet
authors.forename: Alexander G
authors.initials: AG
authors.lastname: Gabibov
authors.forename: Sergey M
authors.initials: SM
authors.lastname: Deyev
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000494:pharmacology* / Q000627:therapeutic use; D001324:Autoantigens / Q000276:immunology; D001327:Autoimmune Diseases / Q000188:drug therapy / Q000276:immunology*; D001402:B-Lymphocytes / Q000187:drug effects* / Q000276:immunology*; D001426:Bacterial Proteins; D016466:CHO Cells; D016923:Cell Death / Q000276:immunology*; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D000939:Epitopes / Q000276:immunology; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D012260:Ribonucleases / Q000235:genetics / Q000276:immunology; D013154:Spleen / Q000166:cytology
chemical_list: D000911:Antibodies, Monoclonal; D001324:Autoantigens; D001426:Bacterial Proteins; D000939:Epitopes; D016271:Proto-Oncogene Proteins c-myc; D011994:Recombinant Proteins; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease