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Engineering SARS-CoV-2 cocktail antibodies into a bispecific format improves neutralizing potency and breadth

One major limitation of neutralizing antibody-based COVID-19 therapy is the requirement of costly cocktails to reduce antibody resistance. We engineered two bispecific antibodies (bsAbs) using distinct designs and compared them with parental antibodies and their cocktail. Single molecules of both bsAbs block the two epitopes targeted by parental antibodies on the receptor-binding domain (RBD). However, bsAb with the IgG-(scFv)2 design (14-H-06) but not the CrossMAb design (14-crs-06) increases antigen-binding and virus-neutralizing activities and spectrum against multiple SARS-CoV-2 variants including the Omicron, than the cocktail. X-ray crystallography and computational simulations reveal distinct neutralizing mechanisms for individual cocktail antibodies and suggest higher inter-spike crosslinking potentials by 14-H-06 than 14-crs-06. In mouse models of infections by SARS-CoV-2 and the Beta, Gamma, and Delta variants, 14-H-06 exhibits higher or equivalent therapeutic efficacy than the cocktail. Rationally engineered bsAbs represent a cost-effective alternative to antibody cocktails and a promising strategy to improve potency and breadth.


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The COVID-19 pandemic has ravaged the world with unprecedented health, social and 40 economic losses 1 . Vaccination is among the most effective countermeasures but not sufficient 41 to end the pandemic due to challenges such as limited global access, vaccine hesitancy, and 6 from CDR-L2 interact with RBD residues T345 and R346, respectively (Fig. 3a). The 148 interactions revealed by X-ray crystallography are fully consistent with epitope mapping results 149 reported in our previous study 9 . Next, we used the Fab06/RBD crystal structure (this work) to 150 perform a superposition with two cryo-EM structures of the spike trimer: one where the three 151 RBD adopt the down conformation and the other with one RBD up and two RBD down. As no 152 steric hindrance was observed, this superposition suggests that Fab06 could bind to RBDs 153 regardless of their down/up conformation in the spike trimer (Fig. 3b, left), which supports the 154 ability of inter-spike crosslinking for bispecific antibodies incorporating the CoV2-06 155 paratope 24 . Although we were able to obtain the structure of the free Fab14 (Extended Data 156 Table 1), so far, our attempts to use X-ray diffraction to determine a Fab14/RBD crystal 157 structure have not been successful and work with Cryo-EM to determine this structure is in 158 progress. Meanwhile, we used the X-ray structures of Fab14 and RBD for docking using 159 Haddock 2.4 guided by previous epitope mapping results 9 . Docking suggests that Fab14 can 160 only bind RBD in the up confirmation as, in the down conformation, Fab14 would clash with 161 an adjacent RBD domain (Fig. 3b, right). Interestingly, Fab06 has little while Fab14 has large 162 steric clash with ACE2 (Fig.3c). The binding epitopes and orientations indicate that the major 163 neutralizing mechanisms for individual cocktail antibodies are different: CoV2-06 through 164 crosslinking of spikes and CoV2-14 through ACE2 blocking. We used the molecular dynamics 165 (MD) method to model the structures of bsAbs and superposed them with RBDs in the spike. 166 The result shows that both bsAbs could simultaneously engage multiple RBDs in different spike 167 trimer (Fig. 3d). However, the maximum number of spikes can be crosslinked by the two bsAbs 168 were different when binding to RBDs adopting various combinations of up and down 169 conformations. As summarized in Fig. 3e, the tetravalent 14-H-06 can crosslink more spikes 170 than the bivalent 14-crs-06 and parental antibodies in all possible scenarios. 7 were significantly different. The NT50s for 14-crs-06 against the K444R and E484A viruses 179 were 2.29 µg/ml and 0.83 µg/ml, respectively, which were slightly less potent compared with 180 the NT50S of the cocktail against the K444R (1.02 µg/ml) and E484A (0.59 µg/ml) viruses. In 181 contrast, 14-H-06 neutralized the K444R and E484A viruses with NT50s of 0.23 µg/ml and 182 0.096 µg/ml, which were 4.4-fold and 6.1-fold more potent compared with the cocktail (Fig.   183 4a-b). Consistent with the neutralization results, the bsAbs bound to the K444R and E484A 184 mutant RBD proteins, while CoV2-06 and CoV2-14 bound to E484A and K444R mutant RBDs,185 respectively (Extended Data Fig. 3c). 186 We focused on 14-H-06 and evaluated its neutralizing activities against seven SARS-187 CoV-2 variants using the plaque reduction neutralization test (PRNT) Fig. 3d). Notably, 14-H-06 potently neutralized 194 all the tested variants with the NT50s between 0.009 µg/ml and 0.176 µg/ml, which were in a 195 close range compared with the NT50 (0.037 µg/ml) against the US-WA1 strain (Fig. 4c) which is more than 10-fold more potent than the cocktail (Fig. 4d). 201 We further used a collection of 20 mutant RBD proteins to compare the epitope 202 coverages by the two bsAbs, the cocktail, and individual parental antibodies through ELISA 203 titrations (Extended Data Fig. 4). These RBDs contain mutations in naturally emerging 204 variants or mutations in escaping viruses selected from two FDA approved antibodies:  10987 and REGN-10933 23 . Selected RBD mutations reduced the binding activities of 206 individual parental antibodies, such as K444R and K444Q mutations for CoV2-06 and E484A, 207 E484K, and F486V mutations for CoV2-14. Expectedly, the cocktail and the two bsAbs had 208 good coverages of these RBD variants (Fig. 4e). Across all the mutants, 14-H-06, but not 14-209 crs-06, exhibited significantly higher binding activities than the cocktail (Fig. 4f), indicating 210 that the IgG-(scFv)2 design, but not the CrossMAb design, provides additional advantage for 211 binding to RBD mutants over the cocktail. Together, these data demonstrate that engineering 212 an antibody cocktail into a bsAb using the IgG-(scFv)2 design increases the neutralizing potency 213 against SARS-CoV-2 variants and broadens the epitope coverages of RBD mutants.

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In vivo protection by 14-H-06 216 We focused on 14-H-06 to evaluate the in vivo efficacy against SARS-CoV-2 and its variants.

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First, we performed dose range evaluations of 14-H-06 in the Balb/c mice infection model by 218 the CMA4 strain, a mouse-adapted SARS-CoV-2 containing the spike N501Y mutation which 219 represented the Alpha variant 23 (Fig. 5a). Three dose levels (2.5, 0.83 and 0.27 mg/kg) for 220 prophylactic treatment and two dose levels for therapeutic treatment (2.5 and 0.83 mg/kg) were 221 tested. For prophylactic treatment, 14-H-06 reduced the viral loads in the lungs to undetectable 222 levels in 100% (10/10) and 40% (4/10) of mice in the 2.5 and 0.83 mg/kg groups, respectively.

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Even with the 0.27 mg/kg dose, the geometric mean viral load (4.79-log) was 8.2-fold lower 224 than that from the isotype control group (5.70-log). For 14-H-06 therapy, the geometric mean 225 viral loads (excluding the mice with undetectable viruses) were reduced by 72,766-and 669-226 fold in the 2.5 and 0.83 mg/kg groups, respectively (Fig. 5b). These data demonstrate that 14-227 H-06 is highly effective for prophylactic and therapeutic treatment against SARS-CoV-2. 228 Next, we compared the therapeutic effects of 14-H-06, the cocktail, and individual 229 parental antibodies against the CMA4 strain. The geometric mean viral load for the cocktail 230 group was 1.79-log, significantly lower than was in the CoV2-14 group (4.21-log) and slightly 231 lower than was in the CoV2-06 group (2.51-log). In contrast, 14-H-06 showed substantially 232 better efficacy than the cocktail and individual parental antibodies, reducing viral loads to 233 undetectable levels for all mice (10/10) (Fig. 5c). We also compared the therapeutic effects of and Gamma variants, respectively ( Fig. 5d-e). We further compared the therapeutic efficacy of 240 14-H-06 and the cocktail against the Delta variant in the human ACE2 transgenic mouse (K18-241 hACE2) model. We performed two sets of experiments to evaluate the therapeutic effects on 242 viral replication (experiment set 1) and mice body weight change (experiment set 2) (Fig.5f).

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In experiment set 1, we treated the mice with one dose of antibodies at 6 h after infection with 244 the Delta variant, and the pfu assay measured viral loads in the lungs. Compared with the 245 isotype group, 14-H-06 reduced the viral load by 278-fold, significantly more potent than the 246 cocktail treatment, which reduced the viral load by 27.8-fold (Fig.5g). In experiment set 2, we 14-H-06 and the cocktail at day 7 (Fig. 5i). 254 Neutralizing antibody levels predict the protection from SARS-CoV-2 infection 28 . We  rationally designed bsAbs with suitable formats and distinct epitope specificities represent a 281 promising alternative to antibody cocktails for developing COVID-19 therapeutic antibodies. 282 We directly compared the therapeutic efficacy of 14-H-06 and the cocktail against the 283 spike N501Y mutation-containing CMA4 strain, the Beta, Gamma, and Delta variants in vivo.

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Across all these tested viruses, 14-H-06 has better efficacy than the cocktail regimen. These 285 results support bsAbs as a promising alternative to cocktails for COVID-19 treatment. Although  The IgG-(scFv)2 design is a promising platform and has been used for developing 302 more than ten-clinical stage bsAb candidates 33 . Antibody 14-H-06 expresses in high yield (>0.5 303 g/L) in transient expression and assembles homogenously, suggesting suitable early 304 developmentability profiles. It is noted that 14-H-06 has a shorter half-life than the parental 305 antibodies in mice, which may have limited its therapeutic advantage over the cocktail in vivo.

Antibody blocking of RBD and ACE2 interaction 641
The Fc-tagged RBD proteins (4 μg/ml) were captured on the protein A biosensor for 300s. Then,

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The system was simulated in a water box where the minimal distance between the solute and 690 the box boundary was 20 Å along all three axes. The charges of the solvated system were 691 neutralized with counter-ions, and the ionic strength of the solvent was set to 150 mM NaCl 692 using VMD 47 . The final system contains over 1.2 million atoms, including proteins, water 693 molecules, and ions. It was subjected to conjugate gradient minimization for 10,000 steps, 694 subsequently heated to 310 K in steps of 5 ps. The system was equilibrated for 5 ns with the 695 backbone atoms constrained by a harmonic potential of the form U(x)=k(x-xref) 2 , where k is 1 696 kcal mol -1 Å -2 and xref is the initial atom coordinates. For mouse serum collection, the blood samples were stored at room temperature for 1 hour, and