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.

Immunotoxin: A new tool for cancer therapy

“…87 Stepanov and co-workers evaluated in vitro a panel of immunotoxins using the B-cell target moiety c-myc epitope. 88 Similar to hematological tumors, Ontak ™ showed promising results in clinical trials of psoriasis and rheumatoid arthritis treatment. 89 …”

Supramolecular agents for theranostics

“…For example, for elimination of pathogenic autoreactive B cells, a bifunctional immunotoxin was constructed, in which ribonuclease barnase was retained as an acting agent, and for a targeting compo nent, instead of antitumor antibodies, c myc epitope was used that selectively interacts with the receptor of pathogenic B cells and induces cell death due to the receptor mediated internalization of the immuno toxin [27]. As a prospective targeting component, an alternative to the anti HER2/neu mini antibody, an artificially manufactured polypeptide DARPin9 29 was used; this polypeptide belongs to a new class of targeting molecules of nonimmunoglobulin nature, possessing a high affinity to the same tumor marker [28].…”

Therapeutic targets and recent advances in protein immunotoxins

“…Very recently, a panel of ITs has been tested in vitro using the B cell target moiety c-myc epitope [43]. Another B cell auto antigen proteinase 3 (PR3) was fused with human toxin -the ribonuclease angiogenin to treat 304 Ecology and industrial microbiology Wegener's granulomatosis (WG) [44].…”