key: cord-0277127-jy71ydm8
authors: Gilchuk, Pavlo; Murin, Charles D.; Cross, Robert W.; Ilinykh, Philipp A.; Huang, Kai; Kuzmina, Natalia; Borisevich, Viktoriya; Agans, Krystle N.; Geisbert, Joan B.; Carnahan, Robert H.; Nargi, Rachel S.; Sutton, Rachel E; Suryadevara, Naveenchandra; Zost, Seth J.; Bombardi, Robin G.; Bukreyev, Alexander; Geisbert, Thomas W.; Ward, Andrew B.; Crowe, James E.
title: Protective pan-ebolavirus combination therapy by two multifunctional human antibodies
date: 2021-05-02
journal: bioRxiv
DOI: 10.1101/2021.05.02.442324
sha: 7ead413cd237551f36054c33c6b17705ce698279
doc_id: 277127
cord_uid: jy71ydm8

Ebolaviruses cause a severe and often fatal illness with the potential for global spread. Monoclonal antibody-based treatments that have become available recently have a narrow therapeutic spectrum and are ineffective against ebolaviruses other than Ebola virus (EBOV), including medically important Bundibugyo (BDBV) and Sudan (SUDV) viruses. Here we report the development of a therapeutic cocktail comprising two broadly neutralizing human antibodies rEBOV-515 and rEBOV-442 that recognize non-overlapping sites on the ebolavirus glycoprotein (GP). Antibodies in the cocktail exhibited synergistic neutralizing activity and resisted viral escape, and they were optimized for their Fc-mediated effector function activities. The cocktail protected non-human primates from ebolavirus disease caused by EBOV, BDBV, or SUDV with high therapeutic effectiveness. High-resolution structures of the cocktail antibodies in complex with GP revealed the molecular determinants for neutralization breadth and potency. This study provides advanced preclinical data to support clinical development of this cocktail for pan-ebolavirus therapy.

The Filoviridae family consists of six antigenically distinct species, including Zaire = 5 PFU/mL), and viral genomes were not detected in 12 of those NHPs 3.7 log10 GEQ/mL at 275 that time (Figures 4, 5) . The three remaining NHPs that were from the BDBV inoculation cohort 276 and that showed slightly delayed clearance of viral genomes in plasma no longer had detectable 277 virus by day 15 after viral challenge. Analysis of various tissues harvested at study endpoint 278 from treated animals (day 28 after inoculation with EBOV and SUDV or day 35 after inoculation with BDBV) and untreated animals (day 6, 14, or 10 after inoculation with EBOV, BDBV, or 280 SUDV, respectively) confirmed virologic protection mediated by the cocktail treatment ( Figure   281 S3). 282 Together these results showed a high therapeutic efficacy of the cocktail of rEBOV-515 283 LALA-PG + rEBOV-442 IgG1 to treat and revert disease caused by primary ebolaviruses that 284 are responsible for outbreaks in humans -EBOV, BDBV, and SUDV.

Structural basis for the efficacy and broad ebolavirus neutralization by the cocktail. 287 We previously reported the molecular determinants of the GP binding for several glycan (Table S7 ; Figure 6A and S4). 296 The interface between GP and rEBOV-515 in this structure was resolved to ~3 to 3.5 Å 297 resolution ( Figure S4) , allowing for confident modeling of most of the side-chain residues. 298 rEBOV-515 binds to the base of the internal fusion loop (IFL), directly below the glycan cap in 299 GP1 ( Figure 6A ). There is significant overlap of the rEBOV-515 epitope with that of rEBOV-300 520, but rEBOV-515 has more extensive contacts in GP2 and less in GP1 ( Figure 6B ). Most and several residues at the base of the IFL, using long CDRH3 loops that mimic and replace 323 the β17-β18 loop of the GP1 in apo-GP structure ( Figure 6E) . However, characterization of the 324 rEBOV-515 binding site revealed several unique features. rEBOV-515 uses a single CDR loop to 325 simultaneously mimic and displace the β17-β18 loop and to bind to R136 in GP1. This feature 326 allows rEBOV-515 to access a region that is occupied by a glycerol cryoprotectant molecule in 327 the unliganded crystal structure of GP (Protein Data Base [PDB] 5JQ3; 5JQB) that we defined as 328 "glycerol pocket" (Figure 6E ). In addition, this mechanism facilitates greater interaction of 329 EBOV-515 with the cleavage loop compared to that caused by rEBOV-520, which may explain 330 the high GP cleavage-inhibiting activity of EBOV-515 ( Figure S7B ). Further, a distinct pose of 331 rEBOV-515 binding into the 3 10 pocket avoids clashes with the a2 helix of the glycan cap 332 (Figure 6F) , unlike the binding of rEBOV-520 to the GP, which requires the a2 helix shift 333 (Gilchuk et al., 2020b). This finding suggested that the binding site of rEBOV-515 on the intact 334 GP molecule is more accessible than the rEBOV-520 binding site, which could explain the 335 higher neutralizing potency of rEBOV-515 when compared to the potency of rEBOV-520 336 against the virus carrying the intact GP. Of note, we have shown previously that GP cleavage, 337 which removes the glycan cap along with the β17-β18 loop, resulted in enhanced binding to 338 cell-surface-displayed cleaved GP and increased neutralizing potency for both rEBOV-515 and between the three major ebolavirus (Figure 7; S6) , providing insight that may help explain 349 differences in neutralization breadth. ADI-15946 makes fewer contacts with GP1 but more 350 contacts to the N-terminus of GP2, a region that is less conserved that may explain weak SUDV 351 neutralization. Conversely, rEBOV-520 makes more extensive contacts with GP1, as this region 352 is generally less conserved. rEBOV-515 relies on minimal contacts with GP1, except in regions 353 that are completely conserved, including R136 and E106. Taken together with a more conserved 354 footprint, these observations provide a molecular basis for the superior breadth of neutralization During acute EVD, circulating infectious virus sometimes seeds immune-privileged 416 tissues, including the brain, eyes, and testes, and persist after clearance from the blood and In summary, these studies highlight the power of implementing a rational mAb cocktail 432 development program using structure-function-guided principles (e.g., knowledge of binding 433 sites, neutralization breadth, resistance to escape, multifunctionality, synergy etc.). We identified 434 a pan-ebolavirus biologic comprising a two-antibody cocktail that exhibits high efficacy for 435 treatment of all three medically important ebolaviruses with only two doses of mAb. These See also Tables S1-6. Non-overlapping contact surfaces for rEBOV-515 or rEBOV-520 are shown, respectively, in 590 maroon or orange, and overlapping contact surface of both antibodies is shown in yellow.

(C) Contact residue details of the rEBOV-515 heavy chain Fab interactions with the base of GP.

CDRH3 contacts include a backbone-mediated hydrogen bond at S102H3 to N512GP2, a key 593 contact that links the β17-β18 loop to the base of the IFL via W291 in unliganded GP1. Contacts 594 near the 3 10 pocket include a potential hydrogen bond between S105H3 and E106GP1 that forms 595 when a large portion of the CDRH3 loop displaces the β17-β18 loop. Within the CDRH2 loop, 596 Y52H2 and Y55H2 make potential hydrogen bonds via their hydroxyl groups to H549GP2 or 597 H516GP2 on GP2, respectively. One of the most extensive rEBOV-515 contacts is from W103H3, 598 which forms a strong cation-pi bond with R136GP1, allowing R136 to make additional hydrogen 599 bonds with Y34H1 and a salt bridge with E106H3. 

The corresponding antibody footprints are highlighted. On the right are aligned sequences of the 625 interacting regions on GP from the three major ebolaviruses, EBOV, BDBV, and SUDV. Total 626 contacts for each residue at 4 Å distance or less were determined (see Table S7 ) and residues are 872 We used RTCA assay to assess neutralizing activity from a pairwise combinatorial matrix of two 873 antibodies in the mixture. Serially-diluted rEBOV-442 (2-fold dilutions) was titrated into twice daily from 0 to 14 dpi for illness, survival, and weight loss, followed by once daily monitoring 926 from 15 dpi to the end of the study at 28 dpi. The extent of disease was scored using the following 927 parameters: score 1 -healthy; score 2 -ruffled fur and hunched posture; score 3 -a score of 2 plus 928 one additional clinical sign such as orbital tightening and/or >15% weight loss; score 4 -a score of 3 929 plus one additional clinical sign such as reluctance to move when stimulated, or any neurologic signs 930 (seizures, tremors, head tilt, paralysis, etc.), or >20% weight loss. Animals reaching a score of 4 931 were euthanized as per the IACUC-approved protocol. All mice were euthanized on day 28 after 932 EBOV challenge. Table S7 998 for additional details. Note that ADI-16061 Fab was added to assist in angular sampling and 999 orientations of the complexes in ice as we described previously (Gilchuk et al., 2020b).

Cryo-EM data were collected according to Table S1 . Micrographs were aligned and dose- Table. the SARS-CoV-2 spike receptor-binding domain that escape antibody recognition. Cell Host 

Privateer: software for the conformational validation of carbohydrate structures