A recombinant triplebody with specificity for CD19 and HLA-DR mediates 
preferential binding to antigen double-positive cells by dual-targeting

Abstract

To test the hypothesis that dual-targeting confers the novel ability of selective binding to antigen double-positive over antigen single-positive cells, a single-chain triplebody (sctb), HLA-ds16-hu19, was produced and characterized. The molecule carries three single-chain Fv (scFv) antibody fragments in a single polypeptide chain, the two distal ones specific for the human histocompatibility protein HLA-DR and the B-lymphoid cell surface protein CD19, the central one for CD16, the human low affinity Fc-receptor FcγRIII. For comparison, the bispecific scFvs (bsscFv) hu19-ds16 and HLA-ds16 were also produced. All CD16 binding modules are disulfide-stabilized (ds). The sctb bound simultaneously to both CD19 and HLA-DR on the same cancer cell and, thus, showed functional dual-targeting. In a mixing-experiment with HLA-DR single-positive HUT-78 cells and (HLA-DR plus CD19) double-positive SEM cells, the triplebody showed preferential binding to the double-positive cells, even when the single-positive cells were present in a numerical excess of up to 20-fold. In antibody-dependent cellular cytotoxicity experiments with mononuclear cells as effector cells, the sctb promoted equal lysis of Raji cells, an antigen double-positive cell line, at 130-fold lower concentrations than the bsscFv hu19-ds16, indicating that both distal scFvs of the sctb contributed to tumor cell lysis. A panel of stably-transfected HEK293 cell lines was generated that included CD19- and HLA-DR single-positive and (HLA-DR plus CD19) double-positive lines with antigen-surface densities varying over a broad range. Using a pair of cell lines with matching densities, the sctb eliminated double-positive target cells preferentially single-positive cells. This ability of preferential or selective targeting of antigen double-positive over single-positive cells opens attractive new perspectives for the use of dual-targeting sctbs in cancer therapy.

Key words:

• triplebodies
• natural killer cells
• dual-targeting
• selective cytotoxicity

Acknowledgments

We thank Dr. R. Levy for the 4G7 hybridoma, Dr. J.G. van de Winkel for the CD16 transfected CHO cells, T. Mentz and B. Bock for excellent technical assistance and for support in the laboratory. Th. Lange is gratefully acknowledged for administrative assistance. The German José-Carreras Leukemia-Foundation is acknowledged for financial support, through grant No. DJCLS F07/03.

Financial Support

This research was supported by grants from the DFG (Deutsche Forschungsgemeinschaft; German Research Community) to G.H.F. and the late Wolfgang Hillen (SFB643/C3), a student fellowship from the Bayerische Eliteförderung (Bavarian Scholarship Foundation) to C.S., postdoctoral/Ph.D., fellowships from the German Jose-Carreras Leukemia-Foundation to M.S. and I.S., a research grant No. 2007.049.1 from the Wilhelm Sander Foundation, Neustadt, Germany to G.H.F. and B.S., and support from the Stiftung Deutsche Krebshilfe, the Beitlich Foundation, Tübingen and the Association “Kaminkehrer helfen krebskranken Kindern” (Chimney Sweeps support children with cancer) to G.H.F. Part of this work was funded by an intramural grant from the ELAN fond and the Training Grant GK592 from the German Research Community (DFG).

Figures and Tables

Figure 1 Design, purification and antigen binding of sctb HLA-ds16-hu19. (A) Block-structure of expression construct. I, secretion leader sequence from the murine Igkappa L chain; VL, VH, cDNA sequences coding for the V regions of Ig L-or H-chains; L, cDNA coding for a 20 amino acid flexible linker (Gly4Ser)4; S, m, H, cDNA coding for a strep-, c-myc- or a hexahistidine tag; S-S, stabilizing disulfide bond. (B) Integrity and purity of the sctb HLA-ds16-hu19 after affinity chromatography with Strep-tactin agarose beads as evaluated by reducing SDS-PAGE and staining with Coomassie blue. (C) Protein gel blot analysis using anti-his or (D) anti-strep antibodies for detection.

Figure 2 Specific antigen binding of sctb HLA-ds16-hu19. (A) FACS analysis of specific binding of sctb to CD19-transfected HEK 293 cells (i), untransfected HEK 293 cells (ii). (B) Specific binding to CD16-transfected CHO cells (i), an incubation with untransfected CHO cells showed no binding (ii). (C) Incubation of sctb with HLA-DR-transfected L66 cells resulted in binding (i), whereas incubation with untransfected L66 cells produced no binding (ii). Dark gray peak, signal from sctb; light gray peak, signal from control sctb.

Figure 3 HLA-ds16-hu19 selectively binds double-positive cells by dual-targeting. Stained SEM cells (CD19- and HLA-DR-positive) were mixed with unlabeled HUT-78 cells (HLA-DR positive) in an equal amount (A and B), and with a ratio of 1:10 (C) or 1:20 (D). Mixtures were incubated with PBA (A) or the sctb HLA-ds16-hu19 (B–D). Bound molecules were detected by flow cytometry (10,000 events collected) with an anti-His-antibody and a PE-conjugated Fab-fragment. SEM cells were detected in the upper left quadrant, the unstained HUT-78 cells in the lower left quadrant. Cells bound by the sctb shifted to the right quadrants. Numbers represent the percentage of the cells in the respective quadrants.

Figure 4 The dual-targeting sctb HLA-ds16-hu19 mediates potent lysis of CD19- and HLA-DR-positive Raji cells with isolated MNCs from healthy donors as effector cells. (A) At the concentration of 1 nM the sctb induced significant ADCC over a broad range of E:T ratios and the extent of specific lysis increased with increasing E:T ratios (black bars: sctb; gray bars: control sctb, white bars: no fusion protein). Data are presented as mean percentage lysis ± standard error of the mean (SEM) obtained with MNCs from four different healthy donors. *Statistically significant values, p ≤ 0.05. (B) Whole blood was fractionated into PMN and MNC to identify the relevant effector population for the sctb. Bars indicate sctb at a concentration of 1 nM. *Statistically significant (p ≤ 0.05) lysis. Data are acquired for four different donors. (C) Ag-specific induction of ADCC in the double-positive cell-line Raji by sctb HLA-ds16-hu19. The sctb induced potent lysis of Raji cells at a concentration of 1 nM. Simultaneous addition of a 500-fold molar excess of CD19- and HLA-DR-specific scFvs significantly blocked the ADCC reaction, but not the addition of a control scFv. Simultaneous incubation with a 125-fold molar excess of the CD16-specific monoclonal antibody 3G8, but not with a control IgG1, significantly reduced ADCC. Data points represent mean percent of specific lysis obtained with isolated MNCs from four different healthy donors at an E:T ratio of 40:1. Specific lysis measured for sctb HLA-ds16-hu19 was defined as 100%. Specific lysis is total lysis minus spontaneous lysis. *Statistically significant reduced (p ≤ 0.05) lysis.

Figure 5 Dose dependent induction of ADCC of the Raji tumor cell line by the sctb and the bsscFvs and test for selective lysis. The CD19- and HLA-DR-double-positive tumor cell line Raji (A) was used as target to compare efficacy of the fusion proteins at a constant E:T ratio of 40:1. The sctb (filled squares), the bsscFv hu19-ds16 (open triangles) and the bsscFv HLA-ds16 (filled inverted triangles) triggered ADCC in a dose-dependent manner. The non-relevant control bsscFv (filled diamonds) induced no significant killing. Data points represent mean percentage of lysis ± SEM obtained with isolated MNCs from 8 different healthy donors. *Statistically significant differences (p ≤ 0.05) between lysis induced by sctb and the bsscFvs. (B) For the first arm of the experiment (left bar), ag double-positive EZK 20 cells were labeled with ₅₁Cr and mixed with an equal number of unlabeled HLA-DR single-positive EZK-HLA 16 cells (Table 4). For the second arm (right bar), single-positive EZK HLA 16 cells were labeled with ₅₁Cr and mixed with an equal number of unlabeled double-positive EZK 20 cells. Sctb HLA-ds16-hu19 was added to a final concentration of 1 nM and MNCs were added to reach an E:T ratio of 40:1. The dual-targeting sctb (gray bar) induced statistically significant greater lysis of the double-positive cells in comparison to the lysis of the single-positive cells. Data points represent mean percentage of lysis ± SEM obtained with isolated MNCs from 4 different healthy donors. *Statistically significant (p ≤ 0.05) differences of lysis induced by the dual-targeting sctb.

Table 1 K_D-values and stability in human serum for sctb HLA-ds16-hu19 and bsscFvs hu19-ds16 and HLA-ds16

Table 2 EC₅₀ for ADCC by sctb HLA-ds16-hu19 and bsscFvs hu19-ds16, HLA-ds16a

Table 3 Ag-density of stably transfected HEK 293 clone EZK20 over 3 consecutive passages

Table 4 Antigen-density of the double- and single-positive single cell clonesa