Novel fusion proteins for the antigen-specific staining and elimination of B cell receptor-positive cell populations demonstrated by a tetanus toxoid fragment C (TTC) model antigen

Diana Klose,Ute Saunders,Rainer Fischer,Annett Marita Jacobi,Thomas Nachreiner,Stefan Barth

doi:10.1186/s12896-016-0249-x

Abstract

BackgroundIn an earlier study we developed a unique strategy allowing us to specifically eliminate antigen-specific murine B cells via their distinct B cell receptors using a new class of fusion proteins. In the present work we elaborated our idea to demonstrate the feasibility of specifically addressing and eliminating human memory B cells.ResultsThe present study reveals efficient adaptation of the general approach to selectively target and eradicate human memory B cells. In order to demonstrate the feasibility we engineered a fusion protein following the principle of recombinant immunotoxins by combining a model antigen (tetanus toxoid fragment C, TTC) for B cell receptor targeting and a truncated version of Pseudomonas aeruginosa exotoxin A (ETA’) to induce apoptosis after cellular uptake. The TTC-ETA’ fusion protein not only selectively bound to a TTC-reactive murine B cell hybridoma cell line in vitro but also to freshly isolated human memory B cells from immunized donors ex vivo. Specific toxicity was confirmed on an antigen-specific population of human CD27+ memory B cells.ConclusionsThis protein engineering strategy can be used as a generalized platform approach for the construction of therapeutic fusion proteins with disease-relevant antigens as B cell receptor-binding domains, offering a promising approach for the specific depletion of autoreactive B-lymphocytes in B cell-driven autoimmune diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0249-x) contains supplementary material, which is available to authorized users.

Highlights

In an earlier study we developed a unique strategy allowing us to eliminate antigenspecific murine B cells via their distinct B cell receptors using a new class of fusion proteins
With the help of a model antigen well characterized in the human system we aim to demonstrate the antigen-specific targeting of human B-lymphocytes via their B cell receptor (BCR), which can offer a novel and promising alternative approach for the treatment of autoimmune diseases [9, 10]
The tetanus toxoid fragment C (TTC) is often used as a model antigen because many people worldwide are vaccinated with tetanus toxoid, and the well-established TTC fragment is characterized by a frequency of 0.01 % TTC-reactive memory B cells within the B cell pool without a recent booster vaccination [13]

Summary

Introduction

In an earlier study we developed a unique strategy allowing us to eliminate antigenspecific murine B cells via their distinct B cell receptors using a new class of fusion proteins. The first requirement for a functional toxic fusion protein is the specific binding to the BCR of self-reactive B cells, followed by receptor-mediated internalization, the release of the catalytic moiety from the endosomes for intracellular transport from the Golgi into the endoplasmic reticulum, and its cytosolic release. This allows ETA’ to exert its cytotoxic activity via ADP-ribosylation of eukaryotic elongation factor 2 (eEF2), leading to efficient inhibition of protein synthesis and to apoptosis [14, 15]. Based on the results of this study, we believe that this concept has a platform character and can be applied to generate powerful fusion proteins for immunotherapeutic approaches

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