Alanine scanning mutagenesis of the MEDI4839 (Suvratoxumab) epitope reduces alpha toxin lytic activity in vitro and S. aureus fitness in infection models

Alpha toxin (AT) is a cytolytic pore-forming toxin that plays a key role in Staphylococcus aureus pathogenesis, consequently extensive research was undertaken to understand the AT mechanism of action and its utility as a target for novel prophylaxis and treatment strategies against S. aureus infections. MEDI4893 (Suvratoxumab) is a human anti-AT IgG1 monoclonal antibody (mAb), which targets AT and is currently in Phase 2 clinical development. As shown previously, the MEDI4893-binding epitope on AT is comprised of the highly conserved amino acid regions 177-200 and 261-271, suggesting these amino acids are important for AT function. To test this hypothesis, and gain insight into the effect mutations in the epitope on AT neutralization by MEDI4893, nine MEDI4893 contact residues in AT were individually mutated to alanine. Consistent with our hypothesis, 8 out of 9 mutants exhibited >2-fold loss in lytic activity resulting from a defect in cell binding and pore formation. MEDI4893 binding affinity was reduced >2-fold (2 – 27-fold) for 7 out of 9 mutants and no binding detected for W187A mutant. MEDI4893 effectively neutralized all of the lytic mutants in vitro and in vivo. When the defective mutants were introduced into a S. aureus clinical isolate, the mutant-expressing strains exhibited less severe disease in mouse models and were effectively neutralized by MEDI4893. These results indicate the MEDI4893 epitope is highly conserved due, in part to its role in AT pore-formation and bacterial fitness, thus decreasing the likelihood for the emergence of mAb-resistant variants.

mutant. MEDI4893 effectively neutralized all of the lytic mutants in vitro and in vivo. When the 23 defective mutants were introduced into a S. aureus clinical isolate, the mutant-expressing strains 24 exhibited less severe disease in mouse models and were effectively neutralized by MEDI4893. 25 These results indicate the MEDI4893 epitope is highly conserved due, in part to its role in AT Increasing incidence of antibiotic resistant bacteria and desire to protect the healthy microbiome 33 raises the need for testing alternative strategies of antibacterial therapy such as immunotherapy. 34 We previously generated the human monoclonal antibody, MEDI4893 (Suvratoxumab), against 35 secreted alpha toxin (AT), a key virulence factor for Staphylococcus aureus pathogenesis, 36 currently in clinical testing for prevention of S. aureus pneumonia. The AT sequence is well 37 conserved among clinical isolates including the nine MEDI4893 contact residues on AT. To 38 better understand their role on AT function and mAb neutralizing activity, we tested the effect of 39 mutations in the nine contact residues in vitro and in vivo lytic activity and disease severity. 40 Several mutants exhibited reduced activity in vitro and in vivo, but none escaped MEDI4893 41 neutralization. These data demonstrate the conserved MEDI4893 epitope is essential for AT Introduction 52 The spread of antibiotic resistance along with a better understanding of the adverse 53 effects broad-spectrum antibacterial therapy has on the beneficial microbiome have led to the 54 exploration of alternative approaches to antibacterial therapy including pathogen-specific 55 monoclonal antibodies (mAbs) to prevent or treat serious bacterial infections (1, 2). MEDI4893 Previous studies 59 demonstrated that AT acts as a key virulence factor in numerous preclinical disease models 60 including dermonecrosis, lethal bacteremia and pneumonia (4-7). There is also evidence that AT 61 is important in human disease as high AT expression levels by colonizing isolates was linked to 62 progression to pneumonia in ventilated patients (8) and low serum anti-AT IgG levels correlate 63 with increased risk for recurrent skin infections in children (9). AT exerts its toxic effects by 64 forming pores in target cell membranes, leading to cell lysis at higher toxin levels (10). It also 65 has effects at sub-lytic levels, resulting in disruption of epithelial and endothelial tight-cell 66 junctions, a damaging hyper inflammatory response in the lung and evasion of killing by host 67 innate immune cells (11)(12)(13). Alpha toxin is secreted as a soluble monomer that binds 68 a disintegrin and metalloprotease 10, ADAM10, on cell membranes, oligomerizes into a 69 heptameric ring and undergoes a conformational change resulting in transmembrane pore 70 formation in host cells such as monocytes, lymphocytes, platelets, and endothelial and epithelial 71 cells (10, 14).

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Specifically, MEDI4893*, a non YTE version of MEDI4893, has been shown to reduce disease 75 severity in multiple animal models (13,17,20) and to exhibit synergy when administered in 76 adjunctive therapy with standard of care antibiotics (15,21,22

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Cytolytic activity of AT alanine mutants 94 The crystal structure of the MEDI4893 antigen-binding fragment (Fab) bound to AT 95 revealed a discontinuous epitope spanning amino acids (AA) 177-200 and 261-271 with direct 96 6 molecular contacts on the toxin at residues D183, S186, W187, N188, P189, V190, R200, T263 97 and K266 ( Fig. 1A and B) (23). These residues were highly conserved among ~1,250 diverse S. 98 aureus clinical isolates (24-26). Alanine scanning mutagenesis of these 9 contact residues was 99 conducted to determine their role in AT function and to gain insight into the effect these 100 mutations have on MEDI4893 neutralizing activity. Cytolytic activity of AT alanine mutants was 101 first examined on rabbit red blood cells ( Fig. 2A) and the A549 human lung epithelial cell line 102 (Fig. 2B). As shown in Figure 2B, W187A, N188A and R200A mutants exhibited little or no 103 cytolytic activity on A549 cells. All of the mutants, with the exception P189A on A549 cells and 104 D183A on red blood cells exhibited a >2-fold loss in cytolytic activity on both cell types (Fig. 2).

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When MEDI4893 was incubated with either the WT or mutant toxins (2:1 molar ratio mAb:AT) 106 prior to the assays, the mAb exhibited neutralizing activity (≥95%) against all mutants in the 107 hemolytic assay with the exception of K266A and W187A against which the mAb neutralized 108 60% and 0% of their activity, respectively (Fig. 3A). MEDI4893 neutralized ≥75% of the for AT function and that the mAb neutralizes epitope variants in vitro.

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AT is a key virulence factor in S. aureus skin and soft tissue infections (5, 6, 27, 28) and AAs in AT-MEDI4893 contact residues are essential for AT pore formation. with the mutants (W187A, N188A or R200A) most defective for A549 lysis (Fig. 6, Table S1), 148 indicating the residues comprising the MEDI4893 epitope are important for cell binding.

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Consistent with the MEDI4893 neutralization of AT mutant-mediated lysis ( Fig. 3), the mAb 150 effectively blocked detectable cell binding by all mutants (Fig. 6).

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The results above indicated MEDI4893 effectively neutralized the lytic alanine mutants 153 in vitro and in vivo. To further characterize the interaction of MEDI4893 with the epitope 154 mutants, mAb association (k on ) and dissociation (k off ) constants for each mutant were measured 155 by surface plasmon resonance and the affinity constant (K D ) for each variant was calculated.

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Although the mAb exhibited a modest drop in K D to D183A, S186A, V190A and T263A it 157 retained a sub-nanomolar K D , however antibody binding constants to N188A, P189A, R200A 158 and K266A were reduced >10-fold (11-to 26-fold) and no binding to W187A was detected in 159 the assay. These data showed a direct correlation between decreased neutralization of AT 160 mutants in the hemolytic assay and their loss for affinity to MEDI4893 ( aureus strains expressing the mutant toxins with the greatest loss in binding affinity, the alanine 168 mutants were introduced into the USA300 CA-MRSA SF8300 chromosome by allelic exchange.

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Each mutant was confirmed to express equivalent toxin levels in vitro (Fig. S3). Balb/c mice Despite a ~16-fold drop in binding affinity for N188A, MEDI4893* prophylaxis significantly 180 reduced death following infection with the N188A mutant strain (Fig. 8). Collectively, these 181 data demonstrate that the AT AAs comprising the MEDI4893-binding epitope are essential not 182 only for AT function, but also for S. aureus fitness in vivo and that MEDI4893* can neutralize 183 the toxic effects of AT even when its epitope is mutated and its binding affinity reduced.  In the current study, we mutated 9 AA contact residues in the MEDI4893 epitope to  Erythrocyte ghosts were prepared as described previously (20) from rabbit blood (Peel Freez).   Washed RBCs were incubated with serial dilutions of WT-AT or AT mutants (10 to 0.005g/ml). intradermal infection with S. aureus SF8300 or the SF8300 hla knock-in mutants (5e7cfu).

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Lesion sizes were measured 24h post infection.   Table 1: Association and dissociation rate constant and apparent binding constants of MEDI4893 to AT alanine mutants. Association (k on ) and dissociation (k off ) rate constants were measured using a BIAcore instrument, and apparent binding constant (K D ) was calculated as k on /k off