Probing Affinity, Avidity, Anticooperativity, and Competition in Antibody and Receptor Binding to the SARS-CoV-2 Spike by Single Particle Mass Analyses

Abstract

Determining how antibodies interact with the spike (S) protein of the SARS-CoV-2 virus is critical for combating COVID-19. Structural studies typically employ simplified, truncated constructs that may not fully recapitulate the behavior of the original complexes. Here, we combine two single particle mass analysis techniques (mass photometry and charge-detection mass spectrometry) to enable the measurement of full IgG binding to the trimeric SARS-CoV-2 S ectodomain. Our experiments reveal that antibodies targeting the S-trimer typically prefer stoichiometries lower than the symmetry-predicted 3:1 binding. We determine that this behavior arises from the interplay of steric clashes and avidity effects that are not reflected in common antibody constructs (i.e., Fabs). Surprisingly, these substoichiometric complexes are fully effective at blocking ACE2 binding despite containing free receptor binding sites. Our results highlight the importance of studying antibody/antigen interactions using complete, multimeric constructs and showcase the utility of single particle mass analyses in unraveling these complex interactions.

Figure 1

Representative mass histograms of the SARS-CoV-2 S-trimer. (A) MP histogram. (B) 1D CD-MS histogram, with the 2D CD-MS histogram shown in the inset. The measured masses and abundances related to these data are provided in Table S1. In both cases, the S-trimer is the predominant species, with a minor contribution of S-monomer. A low population of particles corresponding to S-dimer (∼300 kDa) could also be detected. The total concentration of S-trimer is 100 nM.

Figure 2

Measurement of IgG binding stoichiometries to the S-trimer by MP. MP histograms of the S-trimer following incubation with (A) COVA2-15 or (B) COVA1-18. The vertical dashed lines indicate the theoretical peak positions of each IgG-bound species. MP histograms of each of the Abs alone show a single major distribution at ∼150 kDa, in line with the expected IgG mass (Figure S2). The data clearly reveal that the “complete” 3:1 binding is not achieved for either Ab. COVA2-15 preferably binds two IgGs, whereas just one COVA1-18 binds to the S-trimer. Increasing concentrations of Ab do not change the preferred binding stoichiometries (Figure S3). Binding of both Abs to the S-trimer was also measured by CD-MS, and very similar binding behavior was observed, further illustrating the complementarity between MP and CD-MS (Figure S4). The low-abundance signals observed between 1200 and 1600 kDa originate from Ab-binding-induced S-trimer dimers. (C) Fractional occupancies of each IgG-bound S-trimer species for a panel of 12 monoclonal Abs. A large diversity of binding stoichiometries are observed, ranging from 0 to 2. None of the tested Abs exhibited a preference for 3:1 binding. Additional representative MP histograms are depicted in Figure S1. A tabulation of binding stoichiometries related to these data are provided in Table S2. The concentration of S-trimer in each measurement is 50 nM.

Figure 3

Stoichiometry of Fab binding to the S-trimer. (A–F) MP histograms of COVA2-15 and COVA1-18 Fab binding to the SARS-CoV-2 S-trimer at different mixing ratios. The vertical dashed lines indicate the theoretical peak positions of each Fab-bound stoichiometry. The data reveal that the S-trimer readily binds 3 COVA2-15 Fabs, whereas even in excess not a single COVA1-18 Fab binds to the S-trimer. (G, H) 2D and 1D CD-MS histograms of COVA2-15 Fab binding with excess ratio of Fab (green) as well as SARS-CoV-2 S-trimer only (blue). The observed shift in mass of ∼135 kDa confirms that the S-trimer predominantly binds 3 COVA2-15 Fabs. The concentration of S-trimer in each measurement is 50 nM.

Figure 4

Proposed binding modes of COVA1-18 and COVA2-15 to the S-trimer. (A) For COVA1-18, its Fab has too low an affinity to effectively bind the S-trimer (violet). (B) In its native IgG format, bivalent interactions of the two Fabs enable effective binding with a dominant stoichiometry of 1:1. (C) For COVA2-15, its Fab possesses sufficient affinity alone to bind the S-trimer and occupies all three binding sites due to the lack of steric interactions. While the COVA2-15 IgG should theoretically be able to also bind with a 3:1 ratio, a combination of steric clashes (D) and/or bivalent binding (E) prevents this stoichiometry from being preferred.

Figure 5

Substoichiometric Ab binding to the S-trimer is sufficient to neutralize receptor binding. (A–C) MP and CD-MS histograms of ACE2 alone, revealing the dimeric nature of the utilized ACE2 construct and (D–F) ACE2 binding to the S-trimer. These results show that ACE2 is largely dimeric, and only the ACE2-dimer binds to S-trimer, whereby the S-trimer can accommodate either one or two ACE2. (G–L) MP and CD-MS histograms of ACE2 binding to the S-trimer following preincubation with either (G–I) COVA2-15 or (J–L) COVA1-18. The observed mass shifts of ∼150 kDa (and not 200 kDa) indicate that both Abs fully prevent ACE2 binding to the S-trimer. Mixing ratios of 4:1 and 4:4:1 (ACE2-dimer:S-trimer and Ab:ACE2-dimer:S-trimer, respectively) were used for the CD-MS experiments, while 1:1 and 3:1:1 were used for the MP experiments. Note the similarities between the data presented in panel G and Figure 2A, and panel J and Figure 2B.

Figure 6

MP histograms of COVA2-15 and COVA1-18 binding to the SARS-CoV-2 variant N501Y.V2 S-trimer. (A) Variant S-trimer alone. S-trimer incubated with (B) COVA2-15 or (C) COVA1-18. In stark contrast to the original lineage (Figure 2), essentially no Ab binding is observed.