Bi-specific aptamers mediating tumor cell lysis

Abstract

Antibody-dependent cellular cytotoxicity plays a pivotal role in antibody-based tumor therapies and is based on the recruitment of natural killer cells to antibody-bound tumor cells via binding of the Fcγ receptor III (CD16). Here we describe the generation of chimeric DNA aptamers that simultaneously bind to CD16α and c-Met, a receptor that is overexpressed in many tumors. By application of the systematic evolution of ligands by exponential enrichment (SELEX) method, CD16α specific DNA aptamers were isolated that bound with high specificity and affinity (91 pm-195 nm) to their respective recombinant and cellularly expressed target proteins. Two optimized CD16α specific aptamers were coupled to each of two c-Met specific aptamers using different linkers. Bi-specific aptamers retained suitable binding properties and displayed simultaneous binding to both antigens. Moreover, they mediated cellular cytotoxicity dependent on aptamer and effector cell concentration. Displacement of a bi-specific aptamer from CD16α by competing antibody 3G8 reduced cytotoxicity and confirmed the proposed mode of action. These results represent the first gain of a tumor-effective function of two distinct oligonucleotides by linkage into a bi-specific aptamer mediating cellular cytotoxicity.

FIGURE 1.

Concept of bi-specific aptamers mediating tumor cell lysis. Bi-specific aptamers mimic ADCC by recruitment of natural killer cells via FcγRIIIα (CD16α) to c-Met-overexpressing tumor cells.

FIGURE 2.

Biochemical and cellular binding characterization of CD16α and c-Met specific aptamers. A, representative dot blot raw data of three DNA aptamers CLN0003, CLN0004, and CLN0020. NC, nitrocellulose membrane readout; PVDF, polyvinylidene difluoride membrane readout. B, fitted binding curves for CLN0003 and CLN0004 to c-Met or Fc. C, fitted CLN0020 binding to both CD16α allotypes and minor binding to CD16β. Enlarged symbols represent calculated K_D values. D, FACS analysis of CLN0003 binding to c-Met-positive GTL-16, MKN-45, and EBC-1 cells as compared with c-Met-negative control Jurkat E6.1 cells. E, CLN0004 binding to Met-positive GTL-16, MKN-45, and EBC-1 cells as compared with Jurkat E6.1. F, CLN0020 binding to NK cells and to both alloforms of CD16α presented on recombinant Jurkat cells, in comparison with the parental, CD16-negative Jurkat E6.1 cell line.

FIGURE 3.

Structure prediction and dot blot-based minimization. A, CLN0020 structure prediction indicating nucleotides 20–53 to be essential for structure formation. Numbering indicates base numbers of the full-length aptamer. B, affinity alteration upon removal of C20 in CLN0020 as determined in a dot blot. C, dot blot minimization studies of c-Met specific CLN0003 and CLN0004 as well as CD16α specific CLN0020 and CLN0123. Aptamers could be minimized individually up to a 34-mer CLN0020 core sequence. Asterisks mark the truncation variants used for the design of bi-specific aptamers.

FIGURE 4.

Simultaneous binding of bi-specific aptamers bsA17 and bsA31 to CD16α and c-Met. A, CLN0003-derived bsA17 exhibited binding to CD16α-6His (additional band in lane 2) or c-Met-Fc fusion proteins (additional band in lane 3). This c-Met-Fc bound aptamer band shifted again upon the addition of CD16α-6His (lane 4). B, negative control parental single aptamer CLN0003 did not show a migration shift. Additional bands in all lanes could be due to unspecific aggregation. C and D, bsA31 and original single c-Met specific aptamer CLN0004 exhibited the same pattern as in A and B. E, negative control aptamer (CLNC) did not bind to any protein, as expected. Application of a gradient gel and size differences between CD16α-6His and c-Met-Fc fusion protein led to differently extended migration (lanes 2 and 3) and an expectedly minor but clearly present migration shift upon the addition of both target proteins (from lane 3 to 4). Arrows indicate the lowest migration frontier of specific aptamer bands.

FIGURE 5.

Serum stability of major DNA aptamers. A and B, PAGE of bsA17 after incubation in FCS or murine serum, respectively. Bands at the migration level of the 0-h sample represent intact aptamer, whereas increasing signals at lower positions depict breakdown products. C, degradation of bsA17 in PBS was evaluated similarly but could not be observed within 48 h. D, intensity values were extracted from gels, the percentage of intact aptamer was calculated, and a curve was fitted to the resulting time course. Half-lives in FCS were determined as 9.8 (CLN0004), 14.5 (CLN0020), 6.4 (bsA3), and 20.3 h (bsA17), as well as 11 h for bsA17 in murine serum. Gel raw data of stability curves of additional aptamers are not shown.

FIGURE 6.

Functional ADCC assays of bi-specific aptamers. A, bi-specific aptamer bsA17-mediated specific GTL-16 cell lysis as compared with background levels of non-binding negative control aptamer (CLNC) and reference with medium only. B, similar bsA17-mediated concentration-dependent specific EBC-1 cell lysis. C, PBMC:target cell ratio reduction diminished cytotoxicity of both bsA17 and cetuximab at 50 nm. D, influence of linker lengths on bsA-mediated cell lysis. Estimated linker lengths were 49 Å for bsA17, 105 Å for bsA22 and 217 Å for bsA11. E and F, bsA22-induced specific cytotoxicity dependent on aptamer concentration and effector cell amount. G, the addition of 20-fold molar excess of antibody 3G8 resulted in a decrease of bsA17-mediated lysis due to inhibition of bsA17-binding to CD16α. H, bsA31 with lower c-Met affinity induced weaker but distinct lysis at higher concentrations. I, bsA15, composed of CLN0123 as a lower affinity CD16α binding entity, mediated weak but significant cytotoxicity as well. GTL-16 cells were applied in all measurements, except for B. Maximal lysis varied between individual experiments due to donor and CD16α allotype dependence. ADCC assays were performed 5 times with n = 4 (A), 4 times with n = 4 (B), 3 times with n = 3 (C), 3 times with n = 9 (D), 4 times with n = 4 (E), 1 time with n = 3 (F), 3 times with n = 9 (G), 2 times with n = 4 (H), and 1 time with n = 4 (I), and representative measurements are shown as mean ± S.D.