Aptamers Binding to c-Met Inhibiting Tumor Cell Migration

The human receptor tyrosine kinase c-Met plays an important role in the control of critical cellular processes. Since c-Met is frequently over expressed or deregulated in human malignancies, blocking its activation is of special interest for therapy. In normal conditions, the c-Met receptor is activated by its bivalent ligand hepatocyte growth factor (HGF). Also bivalent antibodies can activate the receptor by cross linking, limiting therapeutic applications. We report the generation of the RNA aptamer CLN64 containing 2’-fluoro pyrimidine modifications by systematic evolution of ligands by exponential enrichment (SELEX). CLN64 and a previously described single-stranded DNA (ssDNA) aptamer CLN3 exhibited high specificities and affinities to recombinant and cellular expressed c-Met. Both aptamers effectively inhibited HGF-dependent c-Met activation, signaling and cell migration. We showed that these aptamers did not induce c-Met activation, revealing an advantage over bivalent therapeutic molecules. Both aptamers were shown to bind overlapping epitopes but only CLN3 competed with HGF binding to cMet. In addition to their therapeutic and diagnostic potential, CLN3 and CLN64 aptamers exhibit valuable tools to further understand the structural and functional basis for c-Met activation or inhibition by synthetic ligands and their interplay with HGF binding

Spontaneous Isopeptide Bond Formation as a Powerful Tool for Engineering Site-Specific Antibody-Drug Conjugates

Spontaneous isopeptide bond formation, a stabilizing posttranslational modification that can be found in gram-positive bacterial cell surface proteins, has previously been used to develop a peptide-peptide ligation technology that enables the polymerization of tagged-proteins catalyzed by SpyLigase. Here we adapted this technology to establish a novel modular antibody labeling approach which is based on isopeptide bond formation between two recognition peptides, SpyTag and KTag. Our labeling strategy allows the attachment of a reporting cargo of interest to an antibody scaffold by fusing it chemically to KTag, available via semi-automated solid-phase peptide synthesis (SPPS), while equipping the antibody with SpyTag. This strategy was successfully used to engineer site-specific antibody-drug conjugates (ADCs) that exhibit cytotoxicities in the subnanomolar range. Our approach may lead to a new class of antibody conjugates based on peptide-tags that have minimal effects on protein structure and function, thus expanding the toolbox of site-specific antibody conjugation

Aptamers Binding to c-Met Inhibiting Tumor Cell Migration.

The human receptor tyrosine kinase c-Met plays an important role in the control of critical cellular processes. Since c-Met is frequently over expressed or deregulated in human malignancies, blocking its activation is of special interest for therapy. In normal conditions, the c-Met receptor is activated by its bivalent ligand hepatocyte growth factor (HGF). Also bivalent antibodies can activate the receptor by cross linking, limiting therapeutic applications. We report the generation of the RNA aptamer CLN64 containing 2'-fluoro pyrimidine modifications by systematic evolution of ligands by exponential enrichment (SELEX). CLN64 and a previously described single-stranded DNA (ssDNA) aptamer CLN3 exhibited high specificities and affinities to recombinant and cellular expressed c-Met. Both aptamers effectively inhibited HGF-dependent c-Met activation, signaling and cell migration. We showed that these aptamers did not induce c-Met activation, revealing an advantage over bivalent therapeutic molecules. Both aptamers were shown to bind overlapping epitopes but only CLN3 competed with HGF binding to cMet. In addition to their therapeutic and diagnostic potential, CLN3 and CLN64 aptamers exhibit valuable tools to further understand the structural and functional basis for c-Met activation or inhibition by synthetic ligands and their interplay with HGF binding

Bi-specific Aptamers Mediating Tumor Cell Lysis

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

CLN3-T and CLN64-T influence on c-Met phosphorylation and signal transduction in A549 and EBC-1 cells.

<p>(A, B) A549 cells or (C) EBC-1 cells were serum-starved and treated with indicated amounts of c-Met-specific CLN3-T, CLN64-T; non-c-Met binding aptamers CLN-X and CLNF. (A, B) A549 cells were additionally stimulated with HGF for 5 min. (A–C) Cell lysates were analyzed by immunoblotting for the availability of phosphorylated c-Met (Phospho-Met), c-Met, phosphorylated Akt (Phospho-Akt), Akt, phosphorylated MAPK (Phospho-MAPK), MAPK and the actin-binding protein Cofilin as loading control. (A) Both aptamers CLN3-T, CLN64-T inhibited HGF-mediated phosphorylation of c-Met and downstream transducers at 3 μM concentrations. No effect was observed for CLN-X and CLN-F. (B) CLN3-T, CLN64-T did not activate c-Met without HGF stimulation. (C) No inhibition of receptor phosphorylation was observed on EBC-1 cells expressing activated c-Met. Dotted lines indicate unconnected spots on the same membrane. The respective anti c-Met and anti Phospho-Met images were derived from two different gels.</p

Analyses of the binding properties of CLN64.

<p>(A–C) Representative dotblot analyses of the ³²P-ATP-labeled aptamer CLN64. The unselected rRfY aptamer pool R0, IgG1-Fc and EGFR-Fc served as negative control. Enlarged symbols represent the calculated K_D value. (A) Specificity of CLN64 binding to c-Met in comparison to the unselected aptamer pool from 2’-fluoro pyrimidine composition (rRfY) analyzed with dotblot assays. (B) Cross-specificity of CLN64 binding to human c-Met and mouse c-Met by dotblot analyses. (C) Dotblot raw data; nitrocellulose readout for protein-bound CLN64 and PVDF readout for unbound CLN64. (D, E, F) Cellular binding analyses of truncated biotinylated aptamer CLN64 (CLN64-T). Previously described CLN3 was used as reference. CLN118 or CLN-F are unrelated aptamers with unmodified and 2’-fluoro modified compositions and served as negative controls. Biotinylated aptamers were detected with streptavidin phycoerythrin (SA-PE). Binding was tested on (D) EBC-1 cells with high c-Met surface levels, on (E) A549 cells with lower c-Met surface levels and on (F) MDA-MB-453 cells with no detectable c-Met surface levels. MDA-MB-453 cells express the related RON kinase on the cell surface. (G) For determination of the apparent cell surface binding constant, CLN64-T was titrated to EBC-1, A549 and Jurkat E6.1 cells (n = 2).</p

Epitope characterization of CLN64-T and CLN3-T on the c-Met ectodomain.

<p>(A, B) Competitive dotblot assays. (A) ³²P-ATP-labeled CLN64-T was almost completely displaced by 0.6–17.7 fold molar excess of non-labeled CLN3-T and not by the negative control aptamer with 2’-fluoro pyrimidine modifications CLN-F (n = 2). (B) ³²P-ATP-labeled CLN3-T was partially displaced by non-labeled CLN64-T and not by the negative control ssDNA aptamer CLN-X (n = 2). (C) In a competitive ELISA the microtiterplate-surface was coated with HGF and to this, c-Met-Fc pre-incubated with varying amounts of aptamer, was added. C-Met binding was subsequently analyzed. In a concentration range of 1.6×10–10 and 1×10-5 M, CLN3-T totally inhibited c-Met interaction with HGF at an IC₅₀ value of 72 nM. CLN64-T did not totally inhibit HGF binding to c-Met in the given concentration range. A non-cMet binding control ssDNA aptamer CLN-X achieved similar results (n = 2).</p

Effects of CLN3-T and CLN64-T on HGF-induced migration on NCI-H441 cells and matrigel invasion of MDA-MB-231 cells.

<p>NCI-H441 were grown until confluent and treated with CLN3-T, CLN64-T and CLN-X (negative control) at the indicated concentrations. A gap was introduced and cell migration was stimulated with 100 ng/ml HGF at maintained aptamer treatment. 1 μM of CLN3-T, CLN64-T inhibited HGF-mediated migration of NCI-H441 cells. Treatment of unrelated aptamer CLN-X did not show any effect on cell migration. Representative pictures of two independent experiments are shown.</p

Locked by Design: A Conformationally Constrained Transglutaminase Tag Enables Efficient Site‐Specific Conjugation

Based on the crystal structure of a natural protein substrate for microbial transglutaminase, an enzyme that catalyzes protein crosslinking, a recognition motif for site-specific conjugation was rationally designed. Conformationally locked by an intramolecular disulfide bond, this structural mimic of a native conjugation site ensured efficient conjugation of a reporter cargo to the therapeutic monoclonal antibody cetuximab without erosion of its binding properties