key: cord-1038429-dudn3nhq
authors: Ren, Ping; Peng, Lei; Fang, Zhenhao; Suzuki, Kazushi; Renauer, Paul; Lin, Qianqian; Bai, Meizhu; Yang, Luojia; Li, Tongqing; Clark, Paul; Klein, Daryl; Chen, Sidi
title: Potent and specific human monoclonal antibodies against SARS-CoV-2 Omicron variant by rapid mRNA immunization of humanized mice
date: 2022-03-18
journal: bioRxiv
DOI: 10.1101/2022.03.17.484817
sha: 0880da7adcf8cb47650ef7baf4f60fec26daed47
doc_id: 1038429
cord_uid: dudn3nhq

The Omicron variant (B.1.1.529) of SARS-CoV-2 rapidly becomes dominant globally. Its extensive mutations confer severe efficacy reduction to most of existing antibodies or vaccines. Here, we developed RAMIHM, a highly efficient strategy to generate fully human monoclonal antibodies (mAbs), directly applied it with Omicron-mRNA immunization, and isolated three potent and specific clones against Omicron. Rapid mRNA immunization elicited strong anti-Omicron antibody response in humanized mice, along with broader anti-coronavirus activity. Customized single cell BCR sequencing mapped the clonal repertoires. Top-ranked clones collectively from peripheral blood, plasma B and memory B cell populations showed high rate of Omicron-specificity (93.3%) from RAMIHM-scBCRseq. Clone-screening identified three highly potent neutralizing antibodies that have low nanomolar affinity for Omicron RBD, and low ng/mL level IC50 in neutralization, more potent than majority of currently approved or authorized clinical RBD-targeting mAbs. These lead mAbs are fully human and ready for downstream IND-enabling and/or translational studies.

numbers of hospitalizations and deaths daily. Therefore, it is essential to develop next-generation 93 neutralizing mAbs that retain potency and limit SARS-CoV-2 virus transmission when current vaccines and 94 therapeutic antibodies are compromised (Cele et al., 2021a) . 95

In this study, we developed RApid mRNA Immunization of Humanized Mice (RAMIHM), an accelerated 97 animal immunization approach for neutralizing mAb discovery. The principle of this approach is to utilize 98 the high doses of antigen-specific LNP-mRNA to frequently immunize immunoglobulin (Ig) humanized 99 mice within 2 weeks, for isolation of high potency neutralizing mAbs against the targeted antigen. We 100 applied this approach directly with Omicron spike-encoding mRNA, used customized single cell BCR 101 sequencing (scBCR-seq) to obtain the human variable region sequences from enriched B cell clonotypes, 102 and generated potent and specific fully human antibodies against the Omicron variant. CoV-2 Omicron variant has substantial changes in its genome, especially the spike protein (Fig. 1A) therapeutics. We thus developed a novel antibody discovery approach named RAMIHM, with repetitive 123 intramuscular injections using high doses of LNP-mRNA, followed by B cell isolation, antigen enrichment 124 and single B cell sequencing (Fig. 1C) . We applied this directly with Omicron-spike-encoding LNP-mRNA 125 to induce Omicron-specific immune responses for isolation of Omicron-targeting mAbs. 126 127 Using Omicron-specific LNP-mRNA that contains lipid nanoparticle formulated mRNA encoding the 128

HexaPro engineered full length of Omicron spike glycoprotein (Methods), we first characterized the 129 biophysical integrity of these LNP-mRNAs (Fig. S1A, S1B) , and validated the expression of functional 130

Omicron spike protein surface expression via human ACE2 (hACE2) staining of LNP-mRNA transfected 131 HEK293 cells (Fig. S1C) . Next, we performed administration of four 10μg doses and one 20μg dose of 132

Omicron specific-mRNA LNP in 3 IgG-humanized mice, collected retro-orbital blood samples from each 133 humanized mouse before and after booster immunization. Blood samples were labeled as pre-, 1 st -, or 2 nd 134 immunization draw depend on collection sequence (Fig. 1C) . Antibody titers were measured using serial 135 plasma dilutions on ELISA plates coated with recombinant Omicron RBD protein. Binding activity was 136 visualized using anti-mouse IgG antibodies at 450nm optical density (OD). Three sequential plasma 137 samples showed increasing vaccine-elicited antibody responses during each blood collection (Fig. 1D) . All 138 post-immunized plasma samples (2 nd blood) showed strong reactivity to the recombinant SARS-CoV-2 139

Omicron RBD protein antigen (Fig. 1D) . In addition, all these samples also showed strong cross-reactivity 140 to recombinant SARS-CoV-2 Delta RBD protein, and intermediately cross-reactivity to recombinant 141 SARS-CoV RBD protein, but no cross-binding to recombinant MERS-CoV RBD protein (Fig. 1D) . 142

Together, these results demonstrated that Omicron-specific rapid mRNA immunization (Omicron-143 RAMIHM) elicited strong anti-Omicron plasma in IgG humanized mice in two weeks, which also contains 144 broader reactive antibodies against other variant and coronavirus species such as SARS-CoV-2 Delta and 145 SARS-CoV. 146 memory B cells enriched library, we used mouse memory B cell isolation kit to obtain total memory B cells 154 from fresh spleen and lymph nodes, and baited SARS-CoV-2 Omicron RBD specific memory B cells by 155 enrichment using recombinant Omicron-RBD proteins from isolated memory B cell subsets (Memory B 156 library). To generate plasma B cells enriched library, we applied anti-mouse CD138 + plasma cell isolation 157 to isolate CD138 + plasma B cells from freshly isolated raw bone marrow cells (Plasma B library). To 158 generate peripheral blood mononuclear cells library, we isolated peripheral blood mononuclear cells 159 (PBMCs) by centrifugation using PBMC isolation method from whole blood (PBMC / Peripheral B library). 160

We subjected each single cell BCR sequencing library with input of approximately 10,000 fresh cells from 161 above. After sequencing, we analyzed a total of 3,502 single B cells, and obtained 2,558 paired heavy-and 162 light-chain variable regions of antibody sequences (Fig. 1E) . To examine the IgG clonal repertoires from 163 the scBCRseq data, we first examined B cell clonotypes, by calculating the frequencies of cells observed 164 for the clonotype and distributions of identical CDR3 region for both heavy and light chains in pairs. By The SARS-CoV-2 Omicron RBD-specific antibodies had a relative enrichment for IGVH3-7, IGVH3-15, 169 IGVH3-20, IGVH3-23, IGVH3-30, IGVH3-33, IGHV3-43, and IGVH4-59, analyzed from 3 individual 170 BCR libraries (Fig. 1E) . A range of lengths between 8-24 aa was observed for these BCR CDRH3s (Fig.  171   1E) . Interestingly, a large portion of IgG2B-expressing B cells were identified from three B cell type 172 isolations (Fig. 1F) , a signature of potential involvement of Th2 cells in B cells maturation and class switch 173 in these mice undergoing the Omicron-RAMIHM procedure. By analyzing the Ig heavy chain (IGH) and 174 light chain (IGK) paring, we also mapped out the overall, enriched and the top 10 heavy-and light-chain 175 V/J segment recombination in these B cell populations ( Fig. 2A-B, Fig. S3 , Dataset S1). In summary, 176 scBCRseq data mapped the clonal repertoires and revealed enriched IgG clonotypes in the peripheral blood, To test whether the most enriched BCRs in these B cell populations are Omicron-reactive, we selected a 182 panel of BCRs for recombinant mAb expression, including 3 from peripheral blood, 3 from plasma B and 183 9 from memory B cell populations (Fig. 2C) . In order to functionally analyze the antibody response to 184 using transfected culture supernatants that contain secreted antibodies. As a result, almost all of the top-188 enriched antibody clones collectively from peripheral blood, plasma B cell and memory B cell populations 189 are reactive to Omicron RBD (14/15 reactive, 1/15 slightly reactive), showing a high rate of antigen-190 specificity (14/15, 93.3%) from Omicron-RAMIHM-scBCRseq (Fig. 2C) . Ten out of fifteen (10/15) 191 selected clones showed potent binding capacity against recombinant SARS-CoV-2 Omicron RBD proteins, 192 4/15 showed moderate binding, an 1/15 showed relatively weak binding (Fig. 2C) . These results indicated 193 that RAMIHM is a highly effective approach for generating and isolating antigen-specific mAbs. 194

To further screen for highly potent functional mAbs, we recombinantly expressed these 15 mAb candidate 196 clones in mammalian system and tested their neutralization ability against the Omicron variant. By 197 screening the mAbs from culture supernatants by neutralizing assay using a spike-based SARS-CoV-2 198

Omicron pseudovirus system, we found 3 clones with obvious neutralization activity against Omicron 199 pseudovirus ( Fig. S4A-B) . We chose these top 3 clones (named as PC.03, MB.02, and MB.08) for further 

We purified the three leading clones, PC.03, MB.02, and MB.08, by affinity chromatography using Protein 204 A beads and examined antibody purity by SDS-PAGE (Fig. 3B) . Thereafter, purified leading mAbs were 205 tested for SARS-CoV-2 Omicron RBD reactivity by ELISA and monitored real-time association and 206 dissociation to recombinant SARS-CoV-2 Omicron RBD proteins using the Octet system. The ELISA 207 titration result of lead mAb clones vs. recombinant SARS-CoV-2 Omicron RBD proteins showed that these 208 three mAb clones have EC50s at the level of ~0.01 µg/mL, suggesting that these mAbs can indeed tightly 209 bind to Omicron RBD (EC50<16ng/mL for all 3 clones) (Fig. 3C) . Octet results with his-tag Omicron RBD 210 antigen immobilization showed ultra-strong binding (KD at 0.8nM for MB.02, and KD <1pM for PC.03 and 211 MB.08) (Fig. 3D) . Noted that this might be contributed by avidity effect due to multi-valent binding, we 212 also performed the reverse Octet assay with antibody immobilization, which measured the single-mAb 213 binding affinity (Fig. 3D) , and showed that the affinity between these clones to Omicron RBD are at the 214 level of low nanomolar range (Fig. 3D) . These KD values (Fig. 3E) showed that the binding strengths of 215

To further determine whether these leading mAbs compete for similar epitopes, we performed epitope 220 binning experiments by Octet using an in-tandem assay (Fig. S5A) . The results have exhibited that PC.03, 221 MB.02, and MB.08 likely share overlapping epitopes ( Fig. S5B-C) . We next measured antibody 222 competition with ACE2, which was quantified as reduction in ACE2 and RBD binding. Consistent with 223 binding affinity findings, these three leading clones showed competitive binding with ACE2 against 224

Omicron RBD (Fig. S6A-D) . 225 226

We then performed neutralization assays for the 3 lead mAbs in purified form, along with other mAbs. We and 0.04 µg/mL (MB.08) ( Fig. 4B; Fig. S7A ). The neutralization potency of the 3 lead Omicron-specific 236 mAb clones are much stronger than those of our prior mAbs and those under prior regulatory approval or 237 EUAs (Fig. 4, Table S1 ). These 3 mAbs however showed no neutralization against the Delta variant ( Fig.  238 S7B), further suggesting that they are Omicron-specific. 239

In order to test if these clones can be used in combination, we again performed neutralization assays by 241 combining two clones. Interestingly, despite epitope overlap, these mAb clones can still enhance each 242 other's neutralization capacity, with the best combination being an antibody cocktail of MB.02 + MB.08 243 (IC50 = 0.03 µg/mL) against pseudotyped SARS-CoV-2 Omicron variant (Fig. 4C) . In summary, these 244 lead neutralizing mAbs showed that they have high affinity vs Omicron RBD, and strong potency in 245 pseudovirus neutralization, which are at least 2 orders of magnitude more potent than existing clinically 246 approved or authorized SARS-CoV-2 mAbs, where their cocktail combinations can also further enhance 247 the neutralization potency (Table S1) . We thank various members from our labs for discussions and support. We thank staffs from various Yale 292 core facilities (Keck, YCGA, HPC, YARC, CBDS and others) for technical support. We thank Drs. The full-length Omicron spike sequence used in mRNA immunization was based on two North America 314 patients identified on Nov23 rd , 2021. The LNP-mRNA was generated as previously described (Fang et al., 315 2022) . Humanized mice with human IgG and IgK transgene knock-ins (ATX-GK, Alloy Therapeutics) were 316 used for rapid mRNA immunization, according to an accelerated (two-week) vaccination schedule. Pre-317 immune sera were collected from the mice prior to the initiation of immunization. The mice were primed 318 with intramuscular injection of 10μg Omicron LNP-mRNA and boosted on days 2, 4, 7 with the same dose 319 as prime. On day 11, three days prior to sacrifice, mice received a final boost with 20μg Omicron LNP-320 mRNA. All mice were retro-orbital bled on days 7, 14 and anti-plasma titers were evaluated using an 321 immunoassay as described below. was diluted with PBS to 20μg/mL, and was captured by anti-Penta-His (HIS1K) sensors (Sartorius, 18-430 5120). The primary antibody was diluted to 150nM with a running buffer in wells, and then sensors were 431 firstly subjected to an association phase for 500s, the response value was recorded. Followed by sensors 432 were subjected to the secondary antibody mixture, and the response value was recorded again. Competition 433 tolerance was calculated by the percentage increase of response after the secondary antibody was added. 434

The column indicates the primary antibody, and the row indicates secondary antibodies. Competition 435 tolerance less than 25% indicates a high possibility of closely-situated epitope. 

All data generated or analyzed during this study are included in this article and its supplementary 479 information files. Specifically, source data and statistics for non-high-throughput experiments are provided 480 in a supplementary table excel file. The ATX humanized mice are available via Alloy Therapeutics. 481

Additional information related to this study are available from the corresponding author(s) upon reasonable 482 request. 483 given for each mouse on day0, day2 and day4 and day7, and followed by 20μg of Omicron LNP-mRNA 498 were injected on day11. Retro-orbital blood was collected on day0, day7 and day14. Plasma was isolated 499 from blood for downstream experiments. Statistics: One-way ANOVA was used to assess statistical significance. Each mAb clone was compared to 531 control. Multiple testing correction was made to correct the p values. Two-sided tests were performed. The 532

p-values are indicated in the plots. Statistical significance labels: * p < 0.05; ** p < 0.01; *** p < 0.001; 533 **** p < 0.0001. Non-significant comparisons are not shown, unless otherwise noted as n.s., not significant. S371L S373P S375F  K417N  N440K G446S S477N T478K E484A Q493R G496S  Q498R  N501Y  Y505H  L981F  N969K  Q954H  N856K  D796Y  N764K  P681H  N679K  H655Y  D614G  T547K  G142D  T95I  A67V 69- Clonal freq%  #02  IGHV5-51  #03  IGHV1-2  #04  IGHV3-30  #05  IGHV3-20  #06  IGHV3-7  #07  IGHV3-9  #08  IGHV4-59  #09  IGHV3-9  #10  IGHV3-20   CARGGYEDVFDIW  CASHSGSYYDYYYYMDVW  CAKGGSYSYYYYMDVW  CARGSGSEDYW  CARDPLYSSFDAFDIW  CAKERWGPFDYW  CARGLTGGDAFDIW  CAKDYYGSGSFDAFDIW  CARGSGSLDYW   CQQYGSSPPTF  CQQSYSTPLTF  CQQYDNLPLTF  CQQYGSSPYTF  CHQSSSLPHTF  CQQYNIWPITF  CQQYYSTPLTF  CQQANSFPLTF IGKV3-20  IGKV1-39  IGKV1-33  IGKV3-20  IGKV6-21  IGKV3-15  IGKV4-1  IGKV1-12  IGKV3-20   IGHG2B  IGHG2B  IGHG2B  IGHG2B  IGHG2B  IGHG2B  IGHG2B  IGHG2B 

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