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. 2016 Feb 17:16:18.
doi: 10.1186/s12896-016-0249-x.

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  1   2 , Ute Saunders  3 , Stefan Barth  4   5 , Rainer Fischer  6   7 , Annett Marita Jacobi  8   9 , Thomas Nachreiner  10
Affiliations

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 et al. BMC Biotechnol. .

Abstract

Background: In 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.

Results: The 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.

Conclusions: This 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.

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Figures

Fig. 1
Fig. 1
Illustration of the prokaryotic and eukaryotic expression vectors. The synthetic tetanus toxoid fragment C (TTC) DNA sequence was cloned into the prokaryotic expression vector pBM and the eukaryotic expression vector pMS using the restriction sites SfiI and NotI. a Prokaryotic expression vector pBM-TTC-ETA’. pelB = signal sequence for protein secretion into the periplasm; 10xHis-tag = polyhistidin-sequence for detection and purification of recombinant proteins; A = antigen fragment; ETA’ = deletion mutant of Pseudomonas aeruginosa exotoxin A; f1 ori = origin of replication for production of single-stranded DNA by M13-helper phage; kanR = kanamycin resistance gene for he selection of transformed cells; ori(3331) = origin of replication; lacI = Lac repressor; T7 prom + Lac op. = IPTG-inducible promotor + Lac-Operator. b Eukaryotic expression vector pMS-L-SNAP-TTC. pCMV = constitutive active promotor of the cytomegalovirus; Ig-κ-Leader = murine signal sequence for protein secretion into the cell culture supernatant; Myc/His-tag = c-myc-epitope for detection/polyhistidin-tag for detection and purification; eGFP = enhanced green fluorescent protein; BGH = Bovine growth hormone (BGH) polyadenylation signal, ZeoR = Zeocin® resistance gene for the selection of transfected cells, pSV40 = early SV40-promotor, SV40 replication origin (ORI); polyA = polyadenylation signal, ColE1 origin = bacterial origin of replication; AmpR = ampicillin resistance gene for the selection of transformed Escherichia coli
Fig. 2
Fig. 2
Fluorescent in-gel detection of SNAP-TTC labeled with different dyes. a SDS-PAGE of SNAP-TTC fusion protein labeled with SNAP-Surface® Alexa Fluor® 488 (2) or BG-647 (3), respectively. Fluorescence signals were visualized using the Maestro CRi in vivo imaging system with the appropriate filter set. b Coomassie-stained SDS gel from (a). The stained protein bands correspond to the measured fluorescence signals from (a). (1) prestained protein marker, broad range (NEB), (2) SNAP-TTC-SNAP-Surface® Alexa Fluor® 488, (3) SNAP-TTC-BG647, (4) uncoupled SNAP-TTC protein
Fig. 3
Fig. 3
Binding analysis of recombinant TTC-based proteins to TTC-reactive hybridoma cells. Equimolar amounts (100 nM) of TTC (c) and TTC-ETA’ (d) were used for binding analysis to the TTC-reactive hybridoma cell line 5E4 (a) compared to the control hybridoma cell line 8.18-C5 (b). Detection of bound proteins was carried out using an Alexa Fluor® 488-coupled anti-His5 antibody. Staining with Alexa Fluor 488-coupled anti-His5 antibody (b) and unstained cells (a) served as controls. Binding analysis of 100 nM SNAP-TTC coupled to the SNAP-Surface® 647 fluorescence dye (b) to 5E4 hybridoma cells (c) and to the control hybridoma cell line 8.18-C5 (d). Unstained cells served as control (a)
Fig. 4
Fig. 4
Dose-dependent binding analysis of the recombinant fusion protein TTC-ETA’ on hybridoma cells. Various concentrations (1–400 nM) of TTC-ETA’ were used to determine a dose-dependent binding activity on TTC-reactive hybridoma cell line 5E4 (a) and to exclude specific binding to the control hybridoma cell line 8.18-C5 (b). The detection of bound protein was carried out by flow cytometry using a Penta-His Alexa Fluor 488 Conjugate antibody. Measurements were performed in triplicates (n = 3); error bars indicate SD. The recombinant TTC-ETA’ exhibits a dosedependent binding on the target hybridoma cell line 5E4, whereas no binding could be determined on the control hybridoma cell line 8.18-C5
Fig. 5
Fig. 5
Cytotoxic activity of recombinant TTC-ETA’ on hybridoma cells. The hybridoma cell lines 5E4 (▼; n = 4) and 8.18-C5 (■; n = 3) were incubated with serial dilutions of TTC-ETA’ (a) and TTC (b) in complete RPMI 1640 cell culture medium. After incubation at 37 °C and 5 % CO2 for 72 h, the cells were refreshed with 50 μl RPMI 1640 medium containing XTT/phenazine methosulfate and incubated for another 4 h. Absorbance was measured at 450 nm and 630 nm on an ELISA reader. The half maximal inhibitory protein concentration (EC50) relative to untreated control cells was calculated using GraphPad Prism software. The recombinant TTC-ETA’ exhibits a dose-dependent cytotoxicity on the target hybridoma cell line 5E4 with an EC50of 1.3 nM ± 0.4 nM but has no effect on the control cell line 8.18-C5. These data represent the mean ± SD of three independent experiments performed in triplicates. *, P < 0.05; ** , P < 0.01; P < 0.001 versus control cells
Fig. 6
Fig. 6
Binding analysis of recombinant SNAP-TTC-BG647 on polyclonally activated CD19+ B cells. CD19+ B cells were isolated from leukocyte filters by density gradient centrifugation and magnetic cell separation with anti-CD19 beads. Isolated B cells were activated by adding 2.5 μg/ml CpG and 50 ng/ml IL-21 to the cell culture medium. On day 4 of cultivation, the cells were analyzed by flow cytometry. Surface a and intracellular b staining with the recombinant SNAP-TTC-BG 647 (1:25) was performed
Fig. 7
Fig. 7
Binding analysis of recombinant TTC and TTC-FITC on isolated PBMCs. Surface staining of CD27+ memory B cells (a) and intracellular staining of CD27++CD38++ plasma cells (c) using recombinant TTC (10 nM) and anti-His5 Alexa Fluor 488 antibody (1:100). Surface staining of CD27+ memory B cells (b) and intracellular staining of CD27++CD38++ plasma cells (d) using a FITC-coupled TTC peptide (1:25)
Fig. 8
Fig. 8
Cytotoxic activity of TTC-ETA’ on TTC-specific polyclonally activated memory B cells. Frequencies of IgG (a, c), of tetanus toxoid (TT)- (b) and of TTC-specific IgG (d) antibody-secreting cells (ASC) incubated with recombinant TTC (10 nM) or TTC-ETA’ (10 nM) protein on day 2 of polyclonal activation. The data are the results of 5 independent experiments using TT-coated plates (b) and 7 experiments using TTC-coated plates (d) and the corresponding total IgG ELISPOT assay (a, c). Statistical analysis was performed using the Wilcoxon matched-pairs signed rank test
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References

    1. Engel P, Gomez-Puerta JA, Ramos-Casals M, Lozano F, Bosch X. Therapeutic targeting of B cells for rheumatic autoimmune diseases. Pharmacol Rev. 2011;63(1):127–56. doi: 10.1124/pr.109.002006. - DOI - PubMed
    1. Davidson A, Diamond B. Autoimmune diseases. N Engl J Med. 2001;345(5):340–50. doi: 10.1056/NEJM200108023450506. - DOI - PubMed
    1. Dorner T, Jacobi AM, Lipsky PE. B cells in autoimmunity. Arthritis Res Ther. 2009;11(5):247. doi: 10.1186/ar2780. - DOI - PMC - PubMed
    1. Mamula MJ. B lymphocyte biology in SLE. In: Systemic Lupus Erythematosus. 5th ed., Edited by R.G. Lahita TK, J. Buyon, G. Tsokos. Boston: Elsevier/Academic Press, 2010.
    1. Hampe CS. B cells in autoimmune diseases. Scientifica. 2012;2012:18. doi: 10.6064/2012/215308. - DOI - PMC - PubMed

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