Article Title: Cornering an Ever-Evolving Coronavirus: TATX-03, a fully human synergistic multi-antibody cocktail targeting the SARS-CoV-2 Spike Protein with in vivo efficacy
Figure Lengend Snippet: IPA’s library-to-leads triage process. (a) High-level schematic of the workflow. (b) Heat map for a pairwise analysis of 19 library-derived anti-SARS-CoV-2 S1-specific Abs merged with a panel of ten structural benchmarks (9 literature Abs and ACE2). The red, yellow, and green colored cells indicate Ab pairs that are blocked (b), partially blocked (pb), or not blocked (nb), respectively. Colored cells with a designation of “b, pb, or nb” were measured empirically, whereas those without a designation are “inferred”. The black boxed cells along the diagonal indicate the “self-blocked” pairs. In our bin-definition, bin-members block one another and show similar blocking behaviors when tested against other Abs in the panel. RBD-specific clones were assigned to five bins (1-5). RBD binders that did not block ACE2 were assigned bin “1”, which was split into sub-bins (bin 1a, 1b, and 1c) based on their nuanced blockade towards the structural benchmarks ( Supplemental Fig S1 ). A cluster of S1 non-RBD binders blocked bin 1 (but not the sub-bins) and did not block any of the literature Abs, so were assigned to bin “C”, representing the C-terminal nub of the S1 fragment, between the RBD and the furin cleavage site, distinct from the N-terminal domain (NTD); hence, bin C’s specificity is assigned as S1-nonRBD-nonNTD. One RBD-binder Ab (23-H7) blocked bin 1, REGN10987 and uniquely perturbed/partially blocked ACE2 ( Supplemental Fig S2 ) , so was assigned to bin 2. Within the ACE2 fully blocking clones, we identified two discrete sets of Abs (bin 4 and bin 5); bin 4 co-located with REGN10933 and CB6, while bin 5 co-located with the “cryptic” epitope of CR3022, VHH-72 and SB68. Bin 3 blocked both bin 2 (23-H7) and bin 4. (c) Image of the spike trimer and zoomed in view of the RBD in grey (PDB ID: 7BNM residues 330-520) showing the epitope contacts for benchmark Abs CR3022 (red, PDB ID: 6YMO) REGN10987 (orange, PDB ID: 6XDG), REGN10933 (blue, PDB ID: 6XDG), CB6 (yellow, PDB ID: 7C01), and REGN10933/CB6 shared residues in green. Depiction of the benchmark “bald spot” present on the RBD, an area of the Spike where none of the available literature controls bound. The C terminus of S1-nonRBD is shaded darker grey (residues 320-329; 521-593). Predicted epitope regions for Abs assigned to bin C, bin 1 and sub-bins 1a, 1b, and 1c (dotted ovals) are also indicated. RBD and benchmark antibody structures were imported from PDB to Maestro. Proteins were aligned via Protein Structure Alignment. (d) Color wheel showing the composition of TATX-03 blends comprised from six lead Abs distributed across four distinct bins.
Article Snippet: Various spike protein subunits including S1-mFc, S1-hFc, S2-hFc, NTD-hFc, S1-S2-His, S1-His, and mutants S1-His(D614G), S. African B.1.351 lineage S1-His(K417N, E484K, N501Y, D614G), S1-His(HV69-70del, N501Y, D614G), UK S1-His(HV69-70del, Y144del, N501Y, A570D, D614G, P681H) were purchased from Sino Biological (cat# 40591-V05H1, 40591-V02H, 40590-V02H, 40591-V41H, V0589-V08B1, 40591-VO8H, 40591-V08H3, 40591-V08H10, 40591-V08H7, 40591-V08H12, respectively) as well as RBD single point mutants (A435S, F342L, G476S, K458R, N354D, N439K, S477N, V367F, V483A, W436R, E484K, K417N, Y453F, N501Y; cat# 40592-V08H4, 40592-V08H6, 40592-V08H8, 40592-V08H7, 40592-V08H2, 40592-V08H14, 40592-V08H46, 40592-V08H1, 40592-V08H5, 40592-V08H9, 40592-V08H84, 40592-V08H59, 40592-V08H80, 40592-V08H82, respectively).
Techniques: Indirect Immunoperoxidase Assay, Derivative Assay, Blocking Assay, Clone Assay