Aptamer BC 007's Affinity to Specific and Less-Specific Anti-SARS-CoV-2 Neutralizing Antibodies

Abstract

COVID-19 is a pandemic respiratory disease that is caused by the highly infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Anti-SARS-CoV-2 antibodies are essential weapons that a patient with COVID-19 has to combat the disease. When now repurposing a drug, namely an aptamer that interacts with SARS-CoV-2 proteins for COVID-19 treatment (BC 007), which is, however, a neutralizer of pathogenic autoantibodies in its original indication, the possibility of also binding and neutralizing anti-SARS-CoV-2 antibodies must be considered. Here, the highly specific virus-neutralizing antibodies have to be distinguished from the ones that also show cross-reactivity to tissues. The last-mentioned could be the origin of the widely reported SARS-CoV-2-induced autoimmunity, which should also become a target of therapy. We, therefore, used enzyme-linked immunosorbent assay (ELISA) technology to assess the binding of well-characterized publicly accessible anti-SARS-CoV-2 antibodies (CV07-209 and CV07-270) with BC 007. Nuclear magnetic resonance spectroscopy, isothermal calorimetric titration, and circular dichroism spectroscopy were additionally used to test the binding of BC 007 to DNA-binding sequence segments of these antibodies. BC 007 did not bind to the highly specific neutralizing anti-SARS-CoV-2 antibody but did bind to the less specific one. This, however, was a lot less compared to an autoantibody of its original indication (14.2%, range 11.0-21.5%). It was also interesting to see that the less-specific anti-SARS-CoV-2 antibody also showed a high background signal in the ELISA (binding on NeutrAvidin-coated or activated but noncoated plastic plate). These initial experiments suggest that the risk of binding and neutralizing highly specific anti-SARS CoV-2 antibodies by BC 007 should be low.

Keywords: BC 007; COVID-19; SARS-CoV-2 antibody; aptamer; autoantibody; re-purposing.

Figure 1

NMR spectroscopic evaluation of the molecular interaction of BC 007 with published CDR-H3 sequences of the anti-SARS-CoV-2 mABs of patients. (A) Upper spectrum (purple): The upper NMR spectrum of BC 007 (1 mM) in combination with ARARGSSGWYRIGTRWGNWFDP (published CDR-H3 of CV07-270 [21]) (1:1) shows BC 007-folding-specific imino signals in the range of 11.5–12.5 ppm. Second from top (green): The spectrum of 1 mM BC 007 in the presence of 1 mM AGSDNYGFPYNGMDV (published CDR-H3 of CV07-250 [21]) does not show any folding of BC 007, excluding any binding or molecular interactions. Third from top (blue): Here, 1 mM ARDGVIPPRFDY (published CDR-H3 of CV07-209 [21]) showed a very small amount of quadruplex-specific imino signals between 11.5 and 12.5 ppm. Bottom spectrum (black): For comparison of the visualization of the quadruplex signal as binding and folding success: SARS-CoV-2 spike protein occurring sequence YRLFRK-caused fold of BC 007 as discussed before in detail by Weisshoff et al. [1]. (B) Upper spectrum: Peptide sequence of the published CDR-H3 region of CV07-209 (ARDGVIPPRFDY, 1 mM) alone. Lower spectrum: Here, 1 mM ARDGVIPPRFDY in the presence of 1 mM BC 007 does not show significant shifts in the aromatic protons of Phe and Tyr. Charge-, pH-, and ionic strength-dependent amide protons of all amino acids are showing only small changes in the chemical shift but not in the coupling constants, again excluding any strong interaction of the CDR-H3 region of CV07-209 with BC 007.

Figure 2

CD spectroscopic evaluation of the molecular interaction of BC 007 with sequence sections of the CDR-H3 regions of anti-SARS-CoV-2 antibodies. The BC 007 (21.5 µM) folding success in presence of no peptides (control, black curves) and increasing concentrations of peptides (A) ARARGSSGWYRIGTRWGNWFDP (source: CV07-270, PDB ID 6XKP), (B) ARDGVIPPRFDY (source: CV07-209 of [21]) and (C) AGSDNYGFPYNGMDV (source: CV-250, PDB ID: 6XKQ) up to a molar excess as indicated by the color of the curve was measured. Green, red, and blue curves show the BC 007 spectra with additional presence of 6.25, 12.6, and 25.0 µM peptide, respectively.

Figure 3

Binding of monoclonal antibodies onto immobilized BC 007. Upper row presents raw data of (A1) neutralizing anti-SARS-CoV-2 antibody CV07-209 (highly specific) and (B1) neutralizing SARS-CoV-2 antibody CV07-270 (showing also cross-reactivity [21]) in comparison to (C1) a BC 007-affine monoclonal antibody that activates beta1-adrenoceptors (clone 011-138, origin: Kreye et al., unpublished). Lower row data are in percent, with the highest value of each experiment and clone set to 100%: (A2) CV07-209, (B2) CV07-270, and (C2) 011-138. Shown in each case is the specific binding onto immobilized BC 007 (blue line), the nonspecific binding onto immobilized NeutrAvidin (the carrier for BC 007–biotin, control, black line), and the nonspecific binding of the mABs onto the carbonate buffer activated but noncoated plastic plate (control, red line). Note the different axis scales with the raw data (A1,B1,C1), which were chosen to show details clearly.