*3.6. mAbs Targeting Mucin-Like Domain*

The mucin-like domain (MLD) is a heavily glycosylated region located on the Cterminus of GP1 that shields the top of the GP trimer. The MLD has the most sequence variation among various species. Due to its highly flexible nature, the structure of MLD is not well characterized, and thus the mAbs targeting MLD are less understood. The residues required for binding of MLD mAbs were identified by peptide binding assays [47] or by

alanine scanning, which evaluates how mutations at individual residues affect binding to EBOV GP [74]. Several mAbs targeting EBOV MLD have been discovered, including the MB-003 cocktail mAbs 6D8 and 13F6, which exhibit low or no neutralization in vitro [47], yet protected in the mouse challenge model, and, collectively with 13C6, provide protection in the NHP model [49]. Efforts have been made to co-crystalize mAb Fab fragments in complex with peptides of identified epitopes. Such structures have been determined for 13F6 [94] and another mAb targeting the MLD, 14G7 [95] (Figure 3E).

In marburgviruses, the unique wing domain located at the N terminus of GP2 is also part of the MLD. Although this domain has not been fully characterized structurally and functionally, four mAbs targeting this region have shown 90–100% protection in the mouse challenge model [35]. Studies on two wing-specific antibodies, MR228 and MR235, identified from human survivors revealed more features of mAbs in this epitope group. MR228 is non-neutralizing but protective in the mouse model, and its protective activity is likely mediated by Fc effector functions, specifically the engagement of FcγRs [96]. MR235 does not protect in in vivo models of infections, yet cooperatively enhances binding of RBS-targeting neutralizing antibodies by facilitating the structural rearrangement of marburgvirus GP [96]. Overall, mAbs targeting the MLD are less likely to be neutralizing [31] but may offer protection through Fc-mediated functions [74,96].

#### *3.7. mAb Cocktail Immunotherapies*

For more than a decade, studies exploring mAbs against filoviruses have demonstrated the potential of using a single or a cocktail of mAbs as immunotherapy, leading to two approved mAb treatments, mAb114 and the cocktail REGN-EB3 in 2020. However, the uncertainty of the causative agents of the next viral outbreak requires a versatile toolbox. The usage of high quantities of mAbs to treat disease caused by filovirus infection presents challenges in production and in cost. Therefore, the development of mAb cocktails as immunotherapies aims to achieve broader reactivity and lower dosage.

First-generation cocktail immunotherapies such as MB-003, ZMAb, and ZMapp can protect against EBOV challenge in NHP [13,48,49]. MB-003 contains antibodies against the MLD and the glycan cap, whereas ZMAb is composed of antibodies against the glycan cap and the base domain. ZMapp is derived from both MB-003 and ZMAb, and combines one mAb from MB-003 with two mAbs from ZMAb, with one mAb (13C6) against the glycan cap and two against the base (2G4 and 4G7) [72,74]. ZMapp was the first antibody cocktail shown to reverse severe disease in the NHP model [13]. During the 2014–2016 outbreak in West Africa, the ZMapp and ZMAb cocktails were used to treat 25 EVD patients under compassionate use protocols in several countries [97]. However, the benefits of the cocktail therapeutics themselves could not be determined definitively since these patients also received other aggressive supportive measures [98]. In a randomized controlled clinical trial, administration of ZMapp was beneficial against human EVD but did not meet the efficacy threshold compared to patients who received the current standard of care alone [99].

REGN-EB3 is a second-generation cocktail of three mAbs, REGN3470, REGN3471, and REGN3479, each isolated from Velocimmune mice, which have human immunoglobulin variable regions [52]. REGN3470 targets the glycan cap from the side, with a binding angle parallel to the viral surface. REGN3471 targets the GP1 head region at the inner chalice, with an angle perpendicular to the viral surface, and REGN3479 targets the fusion loop [52]. REGN-EB3 was superior to ZMapp in reducing EVD mortality in a randomized clinical trial [20], and was approved by the FDA in 2020. An antibody against the head domain, mAb114 [14], was similarly effective as a monotherapy [20]. A two-antibody cocktail including rEBOV-520 that targets the fusion loop region/base area, and rEBOV-548, which targets the glycan cap, is also effective in protecting NHP from EBOV infection [60].

With the uncertainty of viral species responsible for the next outbreak, the next generation of immunotherapy ideally will offer a cross-protective cocktail. In recent years, several mAb cocktails have been characterized and investigated in NHPs to demonstrate

protection against viral infection. Two broadly neutralizing mAbs, FVM04 and CA45, have been evaluated as a cocktail in NHPs with EBOV and SUDV infections, and proved to be protective [58]. When supplemented with MR191, an anti-MARV mAb, the triple mAb cocktail exhibited full protection against death in MARV-infected NHPs [58]. In addition, antibody cocktail RIID F6-H2 is comprised of two SUDV specific mAbs, 16F6 and X10H2, targeting the base and glycan cap of GP, respectively [100,101]. This mAb cocktail protects macaques from the SUDV challenge with two doses on day 4 and day 6, at 25 mg/kg per mAb. The model is not fully lethal; 50% of the mock-treated exposed control animals survived the SUDV challenge [100].

A second cocktail named MBP134AF contains two non-competing IFL targeting mAbs, ADI-15878 (described in IFL region mAb section) and ADI-23774, which was selected after specificity maturation of ADI-15946 to bind SUDV GP using yeast-display technology [102,103]. The mAb pair was further optimized to improve their capacity to activate NK cell functions by adopting all afucosylated glycans (thus the AF in the cocktail name), in order to reach higher efficacy against EBOV [31,102]. The cocktail protects NHPs against EBOV, SUDV, and BDBV [59].

A third cocktail, which also comprises two mAbs, rEBOV-442 and rEBOV-515, was recently reported to protect NHP from disease caused by EBOV, BDBV, and SUDV [61]. These two mAbs exhibited synergy in neutralization by occupying non-overlapping epitopes, with rEBOV-442 targeting the glycan cap region [75], and rEBOV-515 targeting the conserved IFL region. Compared to the previously described ADI-15946 [89] and EBOV-520 [60], the footprint of rEBOV-515 is more conserved and thus provides better neutralizing breath against SUDV [61].

The fourth cocktail of two human survivor antibodies was recently described [62]. This cocktail includes antibodies isolated from survivors of EVD: 1C3 and 1C11, and also protects NHP against lethal challenge with EBOV or SUDV [62]. The 1C3 and 1C11 pair was chosen from a broad analysis of the VIC consortium, and has been tested in multiple animal models (mouse, guinea pig, and NHP). 1C3 uniquely targets the head region with one Fab anchoring into the GP chalice to bind all the three monomers of the GP trimer simultaneously (Figure 3F). This tripartite recognition mode leads to strong binding to the GP trimer, and no cross-reactivity to the dimeric shed sGP. The GP specificity of 1C3 is unique for an EBOV GP head-binding antibody and results from its particular quaternary epitope recognition. Interestingly, different parts of 1C3 target the identical GP residues on each monomer. For example, GP residues D117 in monomer A forms hydrogen bonds with CDRH3 of 1C3, in monomer B forms hydrogen bonds to FRL3, and contacts FRL1 in monomer C [62]. The broadly reactive 1C11 antibody targets the fusion loop/base region via an epitope similar to that of 6D6 [88] and ADI-15878 [85]. 1C11 binds with three copies of the Fab per GP trimer, with each individual Fab bridging two adjacent monomers together to link the fusion loop paddle of monomer A to the neighboring monomer B, including the N-linked glycan at position 563 at each of the three positions around the trimer.

These third-generation candidate therapeutic cocktails can all protect NHP against infection by multiple ebolaviruses, representing the direction of therapeutic development in the field.

For the two approved EVD treatments, the mAb114 monotherapy required a 50 mg/kg dose, whereas the REGN-EB3 triple cocktail required 50 mg/kg of each mAb component for a total 150 mg/kg dose [21,22]. Here we also compare the recently reported cocktails that are protective in NHPs. FVM04/CA45 was protective against EBOV when offered at 40 mg/kg (20 mg/kg each) on day 4, and protective against SUDV when offered at 40 mg/kg (20 mg/kg each) on day 4, plus a second dose at 13 mg/kg (8 mg/kg FVM04, 5 mg/kg CA45) on day 6. Against MARV, MR191 was administered in addition to the two-mAb FVM04/CA45 cocktail at 50 mg/kg, with the first dose (90 mg/kg total) on day 4 and the second dose on day 6 (70 mg/kg total). The second cocktail, MBP134AF, was tested in NHPs against EBOV, SUDV, and BDBV, and showed protection with a single 25 mg/kg dose. However, in both studies, the mock-treated exposed control animals

survived the SUDV challenge (one out of two in the FVM04/CA45 study, two out of four in the MBP134AF study), which limits the significance of the protection results. In the MBP134AF BDBV protection study, five out of six treated animals survived. The third cocktail, rEBOV-442/515, was tested in NHPs against EBOV, SUDV, and BDBV, and showed protection with a two-dose regimen at 30 mg/kg (10 mg/kg rEBOV-442, 20 mg/kg rEBOV-515). The fourth cocktail, 1C3/1C11, was protective against EBOV at 25 mg/kg, and protective against SUDV at 50 mg/kg, both with two doses on day 4 and day 7. The synergistic effect between mAbs described in several studies also hinted at the possibility that these dosages could be further optimized.

One limitation shared by these NHP studies is the relatively small number of animals per group, which results in lower statistical power for examination of the significance of the beneficial effect. Future studies will need to include additional NHPs to test the reported regimens and dosages and to explore the efficacy of single and lower dosages of the proposed cocktails.

#### **4. Conclusions**

After the neutralizing monoclonal antibody KZ52 was found not to protect NHPs infected with Ebola, it was initially thought to be an indication that mAbs may not be effective against rapidly progressing EVD [104]. The discovery that the three non-neutralizing antibodies of MB-003 could protect primates, and subsequent refinement and improvement of antibody cocktails to include neutralizing antibodies that targeted epitopes in the GP base allowed not only survival, but reversion of advanced disease symptoms, as demonstrated by ZMapp in NHPs [13], set a starting point for use of mAbs as therapeutics. The broad collaborative analysis of the VIC illuminated multiple antibody features that led to protection and proposed that antibody therapies ideally should offer potent neutralization, a lack of an un-neutralized viral fraction, and recruitment of Fc effector functions. Further, the analysis by the VIC indicated that the Fc effector function recruitment, particularly phagocytosis, could be strongest at the top of the molecule (e.g., head, glycan cap, and MLD), and that the head epitope, in particular, was a sweet spot that permits both effector function recruitment, as well as potent neutralization by blocking receptor binding [31]. The VIC work established standards, enabled cross-comparison, and allowed side-by-side evaluation of all the cocktail components thus far, including the second-generation mAb cocktails (MB-003, ZMAb, ZMapp, MBP134AF, REGN-EB3), and the proposed mAb cocktail (mAb 1C3 and 1C11) for the combination of complementary activities with antibodies of broad specificity.

Second-generation antibody treatments combine both neutralization and Fc functions, either by binding a single monotherapy at the head sweet spot (i.e., mAb114 [14]), or by combining antibodies having different epitopes and functions, as in REGN-EB3 [20]. The third generation of antibody treatments aims to confer protection against other diseasecausing ebolaviruses, and ideally, involve a lower dosage or a simpler therapeutic regimen. These cocktails, including MBP134AF, rEBOV-520/548, rEBOV-442/515, and 1C3/1C11, have reduced the three-antibody cocktail to two, and each contains at least one mAb targeting the fusion loop region, with the other mAb binding to IFL, glycan cap, or the apex/head region [59–62]. Mapping the structures and activities of antibodies effective against different ebolaviruses illuminates not only emergency post-exposure treatment options, but also illustrates the types of antibodies that vaccines should elicit.

The role of sGP in the immune system during viral infection still remains largely unclear. In the VIC systematic review, sGP cross-reactivity did not significantly affect in vivo protection [31]. The approved monotherapy mAb114 cross-reacts with sGP, and protects against EVD in NHP studies and in the clinical trial at a 50 mg/kg dosage. However, the currently lowest treatment dosage to protect NHP, 25 mg/kg, was achieved by mAb cocktails MBP134AF and 1C3/1C11, the components of which are specific to the GP trimer and do not bind to sGP [59,62]. This result could suggest that a lower effective dosage could be achieved using GP-specific antibodies alone. More studies are needed to better

understand whether sGP is an immunogen or a decoy, so that we can address whether crossreactivity against sGP is an advantageous feature or should be avoided for mAb candidates.

With the current vaccine only available for EBOV, there remains the need for the continued availability of antibodies as it is impractical to vaccinate all, and the frequency and unpredictable timing and location of outbreaks suggest continued treatment development is needed. Studies focusing on characterizing vaccine-elicited mAbs and comparison to those elicited from viral infection would also contribute to the next stage of broadly effective vaccine and immunotherapy development [56,57].

Lastly, platforms and expertise honed on the Ebola virus and the collaborative framework of the VIC [31] were both deployed in rapid development, advancement, and comparison of antibody therapeutics against SARS-CoV-2 [105], and will likely be called upon again against future emerging infections.

**Author Contributions:** Writing—original draft preparation, X.Y., E.O.S.; writing—review and editing, X.Y., E.O.S.; visualization, X.Y.; All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Institute of Allergy and Infectious Diseases (NIAID) U19 AI142790 and the National Institute of Health (NIH) R01 AI132204.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We gratefully acknowledge our funding from the National Institute of Allergy and Infectious Diseases (NIAID) U19 AI142790 and from the National Institute of Health (NIH) R01 AI132204. Figures 1 and 2B,D were created with BioRender.com.

**Conflicts of Interest:** The authors declare no conflict of interest.
