METHODS AND COMPOSITIONS RELATING TO COVID ANTIBODY EPITOPES

Abstract

Provided herein are methods and compositions relating to libraries of optimized antibodies having nucleic acids encoding for an antibody comprising modified sequences. Libraries described herein comprise nucleic acids encoding SARS-CoV-2 or ACE2 antibodies. Further described herein are protein libraries generated when the nucleic acid libraries are translated. Further described herein are cell libraries expressing variegated nucleic acid libraries described herein.

Description

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 17, 2022, is named 44854-830_201_SL.txt and is 1,448,782 bytes in size.

BACKGROUND

Coronaviruses like severe acute respiratory coronavirus 2 (SARS-CoV-2) can cause severe respiratory problems. Therapies are needed for treating and preventing viral infection caused by coronaviruses like SARS-CoV-2. Antibodies possess the capability to bind with high specificity and affinity to biological targets. However, the design of therapeutic antibodies is challenging due to balancing of immunological effects with efficacy. Thus, there is a need to develop compositions and methods for the optimization of antibody properties in order to develop effective therapies for treating coronavirus infections.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF SUMMARY

Provided herein are antibodies that bind to a region consisting of amino acids 380 to 430 of SARS-Cov-2 S receptor binding domain (RBD). Further provided herein are antibodies, wherein the antibody binds one, two, three, or four residues of V382, S383, P384, or T430. Further provided herein are antibodies, wherein the antibody binds to at least V382. Further provided herein are antibodies, wherein the antibody binds to at least S383. Further provided herein are antibodies, wherein the antibody binds to at least P384. Further provided herein are antibodies, wherein the antibody binds to at least T430. Further provided herein are antibodies, wherein the antibody binds to all residues of the following residues: V382, S383, P384, or T430. Further provided herein are antibodies, wherein the antibody binds one or two residues K378 or P384. Further provided herein are antibodies, wherein the antibody binds to at least K378. Further provided herein are antibodies, wherein the antibody binds to at least P384. Further provided herein are antibodies, wherein the antibody binds K378 and P384.

Provided herein are antibodies that bind to a region consisting of amino acids 100 to 300 of SARS-Cov-2 S receptor binding domain (RBD). Further provided herein are antibodies, wherein the antibody binds one, two, three, four, five, six, seven, or eight residues of R102, N125, F157, S172, F175, L176, R190, and Y265. Further provided herein are antibodies, wherein the antibody binds to at least R102. Further provided herein are antibodies, wherein the antibody binds to at least N125. Further provided herein are antibodies, wherein the antibody binds to at least F157. Further provided herein are antibodies, wherein the antibody binds to at least 5172. Further provided herein are antibodies, wherein the antibody binds to at least F175. Further provided herein are antibodies, wherein the antibody binds to at least L176. Further provided herein are antibodies, wherein the antibody binds to at least R190. Further provided herein are antibodies, wherein the antibody binds to at least Y265. Further provided herein are antibodies, wherein the antibody binds to all residues of the following residues: R102, N125, F157, S172, F175, L176, R190, and Y265.

Provided herein are antibodies that bind to a region consisting of amino acids 400 to 500 of SARS-Cov-2 S receptor binding domain (RBD). Further provided herein are antibodies, wherein the antibody binds to one, two, three, four, five, or six residues of K417, F456, G476, F486, N487, or Y489. Further provided herein are antibodies, wherein the antibody binds to at least K417. Further provided herein are antibodies, wherein the antibody binds to at least F456. Further provided herein are antibodies, wherein the antibody binds to at least G476. Further provided herein are antibodies, wherein the antibody binds to at least F486. Further provided herein are antibodies, wherein the antibody binds to at least N487. Further provided herein are antibodies, wherein the antibody binds to at least Y489. Further provided herein are antibodies, wherein the antibody binds to all residues of the following residues: K417, F456, G476, F486, N487, or Y489. Further provided herein are antibodies, wherein the antibody binds to one, two, or three residues of N450, 1472, or F490. Further provided herein are antibodies, wherein the antibody binds to at least N450. Further provided herein are antibodies, wherein the antibody binds to at least 1472. Further provided herein are antibodies, wherein the antibody binds to at least F490. Further provided herein are antibodies, wherein the antibody binds to all residues of the following residues: N450, 1472, or F490. Further provided herein are antibodies, wherein the antibody binds to one, two, or three residues of L452, 1468, or F490. Further provided herein are antibodies, wherein the antibody binds to at least L452. Further provided herein are antibodies, wherein the antibody binds to at least 1468. Further provided herein are antibodies, wherein the antibody binds to all residues of the following residues: L452, 1468, or F490.

Provided herein are antibodies that bind to a region consisting of amino acids 300 to 600 of SARS-Cov-2 S receptor binding domain (RBD). Further provided herein are antibodies, wherein the antibody binds to one, two, three, four, five, six, seven, eight, nine, or then residues of I326, R328, T531, N532, L533, F543, L552, S555, F559, or F562. Further provided herein are antibodies, wherein the antibody binds to at least 1326. Further provided herein are antibodies, wherein the antibody binds to at least R328. Further provided herein are antibodies, wherein the antibody binds to at least T531. Further provided herein are antibodies, wherein the antibody binds to at least N532. Further provided herein are antibodies, wherein the antibody binds to at least L533. Further provided herein are antibodies, wherein the antibody binds to at least F543. Further provided herein are antibodies, wherein the antibody binds to at least L552. Further provided herein are antibodies, wherein the antibody binds to at least S555. Further provided herein are antibodies, wherein the antibody binds to at least F559. Further provided herein are antibodies, wherein the antibody binds to at F562. Further provided herein are antibodies, wherein the antibody binds to all residues of the following residues: I326, R328, T531, N532, L533, F543, L552, S555, F559, or F562.

Provided herein are bispecific antibodies for use in treatment of SARS-CoV-2. Further provided herein are bispecific antibodies with at least 90% similarity to SEQ ID NO: 2670. Further provided herein are bispecific antibodies with at least 95% similarity to SEQ ID NO: 2670. Further provided herein are bispecific antibodies which have a sequence of SEQ ID NO: 2670. Further provided herein are bispecific antibodies which are derived from the sequence at least 90% similar to SEQ ID NO: 2669. Further provided herein are bispecific antibodies which are derived from the sequence at least 95% similar to SEQ ID NO: 2669. Further provided herein are bispecific antibodies which are derived from the sequence of SEQ ID NO: 2669.

Provided herein is a method of treating SARS-CoV-2, the method comprising administering an antibody to a subject wherein the antibody is at least 90% similar to SEQ ID NO: 2670.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 depicts a workflow for antibody optimization.

FIG. 2 presents a diagram of steps demonstrating an exemplary process workflow for gene synthesis as disclosed herein.

FIG. 3 illustrates an example of a computer system.

FIG. 4 is a block diagram illustrating an architecture of a computer system.

FIG. 5 is a diagram demonstrating a network configured to incorporate a plurality of computer systems, a plurality of cell phones and personal data assistants, and Network Attached Storage (NAS).

FIG. 6 is a block diagram of a multiprocessor computer system using a shared virtual address memory space.

FIGS. 7A-7C illustrate epitope mapping of SARS-CoV-2 S1-binding antibodies. FIG. 7A illustrates solvent-accessible surface representation of spike protein trimer in closed (PDB: 6VXX) and open (PDB: 6VSB) conformations. VHH nanobodies (Antibody 5, Antibody 6) binding sites overlap with that of ACE2 in both conformations, while Antibody 1 and Antibody 2 IgGs access a more occluded region. FIG. 7B illustrates cartoon representations of SARS-CoV-2 S protein receptor binding domain (RBD) with critical residues highlighted as spheres for each monoclonal antibody. FIG. 7C illustrates negative-staining electron microscopy analysis which shows the distinct binding regions of antibodies identified from the distinct antibody libraries utilized in this study (colored surface). The SARS-CoV-2 spike protein N-terminal domain (NTD), C-terminal domain (CTD), RBD, and bound ACE2 are shown as cartoon representations.

FIG. 8 illustrates tables showing which mutations are located at the receptor binding domain (RBD). These mutations include G22813T, G23012A, A23063T, A23403g, K417N, E484K, N501Y, D641G for the 501Y.V2 variant (S. African), and A23063T and N501Y for the B.1.1.7, 501Y.V1 variant (UK).

FIGS. 9A-9B illustrate SPR kinetics measured for SARS-COV-2 variant antibodies 6-3 and 6-63 against different SARS-COV-2 variant strains.

FIG. 10 illustrate IC50 data of neutralizing antibodies against pseudoviruses with single mutations relative to the G614-parent virus was tested.

FIGS. 11A-11F illustrate neutralization data of 1-12, 6-3 and 6-63 measured against single mutations and variant pseudovirus strains such as strain alpha (FIG. 11A), strain beta (FIG. 11B), strain delta (FIG. 11C), strain epsilon (B.1,427) (FIG. 11D), and strain epsilon (B.1.429) (FIG. 11E). FIG. 11F shows IC50s of variant antibodies against several variant strains.

FIGS. 12A-12D illustrate neutralization data of SARS-CoV-2 variants AZ1 (FIG. 12A) and B.1.351 (FIG. 12B). Replicates of the neutralization experiments for AZ1 (FIG. 12C), and B.1.351 (FIG. 12D) are also shown.

FIG. 13 shows Fc-gamma Receptor 1 (FcγR1) as an analyte was titrated (0.37-30 nM, 3-fold dilutions, 5-membered series) over antibody 493-004 (275RU) as ligand (Panel A) and anti-RBD isotype control (278RU) as ligand (Panel B), tethered via RBD-coated chip surface. The sensorgram view shows an example of an overlay plot of the measured data superposed with the global kinetic fit from a single experiment used to deduce the KD value.

FIG. 14 shows FcγR2a R167 as an analyte was titrated (4.1-1000 nM, 3-fold dilutions, 6-membered series) over antibody 493-004 (510RU) as ligand, tethered via RBD-coated chip surface. Example of KD values determined via alternate fitting methods; (Panel A) kinetic model and (Panel B) steady-state binding isotherm.

FIG. 15 shows FcγR2a H167 as an analyte was titrated (12.3-1000 nM, 3-fold dilutions, 5-membered series) over antibody 493-004 (488RU) as ligand, tethered via RBD-coated chip surface. Example of KD values determined via alternate fitting methods; (Panel A) kinetic model and (Panel B) steady-state binding isotherm.

FIG. 16 shows FcγR2B/C as analyte was titrated (37-3000 nM, 3 fold dilutions, 5-membered series) over antibody 493-004 (510RU) as ligand, tethered via RBD-coated chip surface. Example of KD values determined via alternate fitting methods; (Panel A) kinetic model and (Panel B) steady-state binding isotherm.

FIG. 17 shows FcγR3A 176F as analyte was titrated (12.3-1000 nM, 3-fold dilutions, 5-membered series) over antibody 493-004 (79RU) as ligand, tethered via an RBD-coated chip surface. Example of KD values determined via alternate fitting methods; (Panel A) kinetic model and (Panel B) steady-state binding isotherm.

FIG. 18 shows FcγR3A 176V as analyte was titrated (12.3-1000 nM, 3-fold dilutions) over antibody 493-004 (79RU) as ligand, tethered via RBD-coated chip surface. Example of KD values determined via alternate fitting methods; (Panel A) kinetic model and (Panel B) steady-state binding isotherm.

FIG. 19 shows the neonatal Fc receptor (FcRn) as analyte was titrated (1.4-300 nM, 3-fold dilutions, 6-membered series) over antibody 493-004 (216RU) as ligand, tethered via RBD-coated chip surface. Example of KD values determined via alternate fitting methods; (Panel A) kinetic model and (Panel B) steady-state binding isotherm.

FIG. 20 shows a direct comparison of complement component C1q binding to ELISA plates adsorbed with antibody 493-004 (IgG1) and control antibodies of various isotypes (IgG1, IgG2, and IgG4).

FIG. 21 shows signal-to-background (‘signal-to-noise’ or S/N) ratio data for complex formation between ACE2-muFc and biotinylated SARS-CoV2 spike RBD. The optimal concentrations of ACE2 (highlighted portion of the graph) and spike RBD (highlighted portion of the legend) for a robust S/N of binding are indicated.

FIG. 22 shows dose dependent inhibition of ACE2-muFc/SARS-CoV-2 Spike protein interactions by antibody 493-004 and Controls (indicated in the legends). The SARS-CoV-2 Spike proteins used in these assays were (Panel A) RBD (Ancestral), (Panel B) Trimer (D614), (Panel C) Trimer (Delta), and (Panel D) Trimer (Omicron).

FIG. 23 depicts a schematic of the bispecific monoclonal antibody 493-004 which is constructed with two individual, single domain VHH antibodies: antibody 202-03 (top VHH domains) and antibody 339-031 (bottom VHH domains) linked together with the constant heavy chain 2 (CH2) and the constant heavy chain 3 (CH3) Fc regions of the antibody.

FIG. 24 depicts a schematic representation of the upstream manufacturing process of antibody 493-004 antibodies.

FIG. 25 depicts a schematic representation of the downstream manufacturing process of antibody 493-004 antibodies.

FIG. 26 depicts a flow diagram of the drug product process.

FIG. 27A depicts a schematic design of a Phase 1 clinical trial in humans. FIG. 27B depicts an schematic design of a Phase 2A clinical trial in humans.

FIG. 28 depicts VHH antibody 202-03 in SPR Assays with SARS-CoV-2 variants of concern. Panel A shows the wildtype variant, Panel B shows the beta variant, Panel C shows the gamma variant, and Panel D shows the kappa variant. 339-031 demonstrated a higher apparent binding affinity for variants containing the L452R mutation (e.g., Delta and Kappa), compared to the initial 202-03 VHH antibody.

FIGS. 29A-29C depict SPR using 202-03 and 339-031. From the SPR data, the VHH leads were further assessed for binding ability against the SARS-CoV-2 variants of concern.

FIG. 30 depicts a summary of the kinetic data for individual VHH antibody variants 202-03 and 339-031. Equilibrium association (e.g., Ka) and dissociation (e.g., Kd) constants, as well as the affinity of the antibody to the respective receptors (e.g., KD), were calculated. Low calculated KD values are suggestive of a high apparent binding affinity and high calculated KD values are suggestive of low apparent binding affinity. Based on these data, the two VHH antibodies were selected for construction of the bispecific product.

FIG. 31 depicts epitope bin heat maps for WA1 S trimer and Delta S Trimer.

FIG. 32 describes SARS-CoV-2 spike mutations used in pseudovirus experiments.

FIG. 33 shows plots of VHH antibody neutralization potential across SARS-CoV-2 variants of concern using representative pseudovirus.

FIG. 34 shows neutralization activity of the antibodies 202-03 and 339-031 with the bispecific antibody 493-004 against the two Epsilon variants (L452R mutations).

FIG. 35 shows pseudovirus for Delta (B.1.617.2) Neutralization Potential Using the VHH antibody 339-031 and the bispecific antibody 493-004.

FIG. 36 shows live virus assays with the VHH antibody 339-031, the bispecific antibody 493-004, and a control h2165, using cells infected with SARS-CoV-2 (wild-type [AZ1] (Panel A), Beta [B.1.351] (Panel B), and Delta (Panel C)) variants.

FIG. 37 shows neutralization potential of antibody constructs using FRNT₅₀ measures.

FIG. 38 shows that the bispecific antibody administered at either 1 mg/kg or 5 mg/kg by intraperitoneal injection and 1 mg/kg intranasally, resulted in improved body weights in the animals starting 4-days after start of the challenge.

FIG. 39 shows that the bispecific antibody appears to demonstrate a therapeutic response and animal weights increased after administration of the antibody on each of the days administered (e.g., day 1, 2, 3, or 4 post-infection).

FIGS. 40A-40B show preliminary weight data from all treated animal groups, by day (−1, +1, +2, +3, and +4) post-infection.

FIGS. 41A-41F show a complete double stranded DNA and amino acid sequence of bispecific monoclonal antibody 493-004 showing the location of VHH antibodies, G4S linkers (SEQ ID NO: 2671), IgG1 Fc, and N-link glycosylation. Figure discloses SEQ ID NOs: 2677 and 2678, respectively, in order of appearance. FIG. 41A depicts the top left portion of the sequence. FIG. 41B depicts the top middle portion of the sequence. FIG. 41C depicts the top right portion of the sequence. FIG. 41D depicts the bottom left portion of the sequence. FIG. 41E depicts the bottom middle portion of the sequence. FIG. 41F depicts the bottom right portion of the sequence.

FIG. 42 shows the neutralization assessment of antibody 493-004 in a live virus plaque reduction assay using dose response curves, EC₅₀, and EC₉₀ determinations for antibody 493-004 against ancestral, delta, and omicron variants of the SARS-CoV-2 virus.

FIGS. 43A-43B depict the mean two-week plasma expires in a rat following a single intravenous infusion of 30 mg/kg (FIG. 43A) and following IV infusion dosing at 30 mg/kg across the entire 42-week study FIG. 43B).

FIG. 44 depicts results of alanine mutational analysis of the individual VHH antibodies 339-031 and 202-03 and identified critical contact points.

FIG. 45 shows graphical display, EC₅₀, and EC₉₀ results of virus neutralization of omicron lineages BA.1, BA.2, and BA.3 with bispecific antibody 493-004.

FIGS. 46A-46C show the results of the top ten clones (FIG. 46A), the top two clones (FIG. 46B), and the top clone (FIG. 46C) used in development of the antibody 493-004 stable cell line.

FIG. 47 shows the drug substance specifications for the pharmaceutical formulation of monoclonal antibody 493-004.

FIG. 48A shows the experimental design for a pharmacokinetic study in rats.

FIG. 48B shows a sample collection schedule.

FIG. 49 shows an overview atlas of a measurement grid for CryoEM analysis.

FIG. 50 shows an example motion corrected micrograph from data collection during CryoEM experiments.

FIGS. 51A-51D depict the 3D classification models from the results of the CryoEM experiments. Shown here are the 3.2 Å resolution “initial consensus map” (FIG. 51A); the 3.4 Å resolution M4.3 map, used to build the majority of the VHH1 epitope/paratope (FIG. 51B); the 3.7 Å resolution M4.4 map, used to build the N-terminal chain epitope of VHH in position 1 (FIG. 51C); and the 3.3 Å resolution M4.5 map, used to build the VHH2 epitope/paratope (FIG. 51D).

FIGS. 52A-52D show a mashup of the reconstructed maps such that each part of the spike trimer-bispecific antibody structure is represented in the best resolution obtained. Magenta/Red/Green represent monomers of the spike trimer, further denoted as chain A, chain B, and chain C, respectively. Grey density represents the bispecific antibody constant fragment. VHH1 is depicted in gold, VHH2 is blue, and VHH3 is orange. FIG. 52A shows an overview of the bispecific antibody layout at a side view. VHH1 is bound to the RBD down domain and VHH2 is bound to the neighboring RBD up domain. The constant fragment is located above the spike trimer, close to the spike center. FIG. 52B shows the front view (rotated 90 degrees vertically from FIG. 52A). VHH2 is bound to one of the RBD up domains while VHH3 is bound flexibly to the second RBD up domain. Both VHH2 and VHH3 share a strong constant fragment density. FIG. 52C shows the back view (rotated 180 degrees vertically from FIG. 52B). VHH3 is bound flexibly to the second RBD up domain while VHH1 is bound to the RBD down domain. FIG. 52D shows the top view of the spike trimer-bispecific antibody structure. VHH1 is located at the bottom and VHH2 is located in the upper right corner. Both VHH1 and VHH2 are partially covered by the constant fragment, while VHH3 is located on the left.

FIG. 53A shows an epitope/paratope overview of epitope 1. FIG. 53B shows a list of explicit bonds. FIG. 53C shows a comparison to mutagenesis studies where residues 450 and 490 were found to directly interact with the VHH. Residue 472 does not directly interact with the VHH but possibly serves as a stabilizer of interacting RBD loops, thereby contributing to the VHH/RBD interaction indirectly.

FIG. 54 shows a breakdown of the sequence of epitope 1. Figure discloses SEQ ID NO: 2679.

FIG. 55A shows a cartoon representation of the atomic model of the SARS-CoV-2 spike protein with an N-terminal VHH at epitope 1 (orange) on RBD down domain (red).

FIG. 55B shows a surface representation of the atomic model of SARS-CoV-2 spike protein with an N-terminal VHH at epitope 1 (orange) on RBD down domain (red).

FIG. 56A shows glycosylation of the ASN 234 residue of the spike protein. The interaction of TYR54 of the VHH with the OH carbohydrate group was also verified. This suggests the importance of the ASN234 residue of the spike protein, which is glycosylated, in VHH binding. FIG. 56B shows verification of the interaction between the ASN164 residue of the spike protein sidechain and the backbone of the THR28 residue of the VHH using computational interfacing methods. FIG. 56C shows the possible stacking interaction between the PHE490 residue of the spike protein and the PHE37 an PHE47 residues from VHH, which were suggested during manual model inspection. These findings are in agreement with results from mutagenesis studies of N-terminal VHH Ab. FIG. 56D shows that residues ARG45 and TRP105 from the VHH interact with the sidechain (ARG45) and backbone (TRP105) of the ASN450 residue of the spike protein. These findings are in agreement with results from mutagenesis studies of N-terminal VHH Ab and highlights the importance of the ARG45:ASN450 interaction in the integrity of the spike:Ab complex. FIG. 56E shows that in epitope 1 the ILE472 reside of the spike protein does not interact with the VHH, but the GLU471 residue of the spike protein does interact with the VHH my means of a hydrogen bond with the ASN58 residue sidechain (FIG. 56F). The ILE472 residue of the spike protein strongly interacts with the PHE456 residue of the spike protein. This interaction could help maintain local spike protein folding.

FIG. 57A shows an epitope/paratope overview of epitope 2. FIG. 57B shows a list of explicit bonds. FIG. 57C shows a comparison to mutagenesis studies residue 472 was found to interact with the VHH based on manual inspection of the map and strong surrounding densities.

FIG. 58 shows a breakdown of the sequence of epitope 2. Figure discloses SEQ ID NO: 2679.

FIG. 59A shows a cartoon representation of the atomic model of the SARS-CoV-2 spike protein with an N-terminal VHH at epitope 2 (orange) on RBD up domain (red). FIG. 59B shows a surface representation of the atomic model of SARS-CoV-2 spike protein with an N-terminal VHH at epitope 2 (orange) on RBD up domain (red).

FIG. 60A shows that residue ASN450 of the spike protein interacts with both ARG45 and TRP105, both residues of VHH (high confidence interval). As only ARG45 is interacting with the ASN 450 sidechain, this suggests the ARG45-ASN450 bond is the more important bond. This data verifies the importance of ANS450 in interactions with SARS-CoV-2.

FIG. 60B shows that residue THR470 from the spike protein interacts with the backbone of residue TYR59. For residue ILE472, even though there was no discovered interaction, the cryEM map suggests that there is a strong interaction with VHH, probably also with ASN58. Therefore, THR470, PRO479, and IL472 can contribute to the interaction with VHH binding. FIG. 60C shows that residue PHE490 fo the spike protein has no automatically verified interaction. PHE490 appears to interact with PHE37 and PHE47 of the VHH using a stacking interaction and possibly with LEU492 through a hydrophobic interaction. The CryoEM studies verify PHE490 importance in VHH Ab binding. FIG. 60D shows that two hydrogen bonds were confirmed between ARG346 of the spike proteins and ASP103 of the VHH. Probably stacking interaction was also discovered between ARG346 and PHE102. ARG346 contributes to the interaction between the spike protein and the VHH antibody.

FIG. 61 shows a comparison of the VHH1 and VHH2 epitopes/paratopes.

FIG. 62A shows that VHH3 (orange) assigned to the low-resolution density. It is positioned on top of the RBD up domain in a distinctly different position from the VHH1 or the VHH2. The blue portion shows the position as if VHH2 was at that location, supporting the different binding epitope. Pointing toward the constant fragment is the N-terminal of the VHH (i.e., GLU1). FIG. 62B shows further detail of the VHH3 from a side view. Mutagenesis studies suggest that for the C-term VHH, residues 476-489 are key for its interaction. These residues are marked red. Note, how these residues form a loop protruding up and sideways from the main body of the RBD (similar to the epitopes of VHH1 and 2) and how the VHH3 is very close to these residues while being a good distance away from the rest of the RBD up domain. We speculate that this is the reason for its more flexible binding compared to the VHH1 and VHH2—it interacts primarily with a probably flexible, protruding loop of the RBD, lacking more stabilizing contacts. Also this is further evidence supporting it being the C-term VHH. However, this assignment has been done only as a rigid body fitting of the RBD up domain modeled adjacent to position 2.

DETAILED DESCRIPTION

The present disclosure employs, unless otherwise indicated, conventional molecular biology techniques, which are within the skill of the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art.

Definitions

Throughout this disclosure, various embodiments are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of any embodiments. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range to the tenth of the unit of the lower limit unless the context clearly dictates otherwise. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual values within that range, for example, 1.1, 2, 2.3, 5, and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, unless the context clearly dictates otherwise.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of any embodiment. As used herein, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and/or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.

Unless specifically stated or obvious from context, as used herein, the term “about” in reference to a number or range of numbers is understood to mean the stated number and numbers+/−10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.

Unless specifically stated, as used herein, the term “nucleic acid” encompasses double- or triple-stranded nucleic acids, as well as single-stranded molecules. In double- or triple-stranded nucleic acids, the nucleic acid strands need not be coextensive (i.e., a double-stranded nucleic acid need not be double-stranded along the entire length of both strands). Nucleic acid sequences, when provided, are listed in the 5′ to 3′ direction, unless stated otherwise. Methods described herein provide for the generation of isolated nucleic acids. Methods described herein additionally provide for the generation of isolated and purified nucleic acids. A “nucleic acid” as referred to herein can comprise at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, or more bases in length. Moreover, provided herein are methods for the synthesis of any number of polypeptide-segments encoding nucleotide sequences, including sequences encoding non-ribosomal peptides (NRPs), sequences encoding non-ribosomal peptide-synthetase (NRPS) modules and synthetic variants, polypeptide segments of other modular proteins, such as antibodies, polypeptide segments from other protein families, including non-coding DNA or RNA, such as regulatory sequences e.g. promoters, transcription factors, enhancers, siRNA, shRNA, RNAi, miRNA, small nucleolar RNA derived from microRNA, or any functional or structural DNA or RNA unit of interest. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, intergenic DNA, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), small nucleolar RNA, ribozymes, complementary DNA (cDNA), which is a DNA representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification; DNA molecules produced synthetically or by amplification, genomic DNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. cDNA encoding for a gene or gene fragment referred herein may comprise at least one region encoding for exon sequences without an intervening intron sequence in the genomic equivalent sequence. cDNA described herein may be generated by de novo synthesis.

Antibody Optimization Library for Coronavirus

Provided herein are methods, compositions, and systems for the optimization of antibodies for coronavirus. In some embodiments, the antibodies are optimized for SARS-CoV, MERS-CoV, CoV-229E, HCoV-NL63, HCoV-OC43, or HCoV-HKU1. In some embodiments, the antibodies are optimized for SARS-CoV-2. In some embodiments, the antibodies are optimized for a receptor that binds to the coronavirus. In some embodiments, the receptor of the coronavirus is ACE2 or dipeptidyl peptidase 4 (DPP4). In some embodiments, the antibodies are optimized based on interactions between the coronavirus and the receptor that binds the coronavirus. In some embodiments, the antibodies are optimized for angiotensin-converting enzyme 2 (ACE2). In some embodiments, the antibodies are optimized based on interactions between SARS-CoV-2 and ACE2.

Antibodies are in some instances optimized by the design of in-silico libraries comprising variant sequences of an input antibody sequence (FIG. 1). Input sequences 100 are in some instances modified in-silico 102 with one or more mutations or variants to generate libraries of optimized sequences 103. In some instances, such libraries are synthesized, cloned into expression vectors, and translation products (antibodies) evaluated for activity. In some instances, fragments of sequences are synthesized and subsequently assembled. In some instances, expression vectors are used to display and enrich desired antibodies, such as phage display. Selection pressures used during enrichment in some instances includes, but is not limited to, binding affinity, toxicity, immunological tolerance, stability, receptor-ligand competition, or developability. Such expression vectors allow antibodies with specific properties to be selected (“panning”), and subsequent propagation or amplification of such sequences enriches the library with these sequences. Panning rounds can be repeated any number of times, such as 1, 2, 3, 4, 5, 6, 7, or more than 7 rounds. Sequencing at one or more rounds is in some instances used to identify which sequences 105 have been enriched in the library.

Described herein are methods and systems of in-silico library design. For example, an antibody or antibody fragment sequence is used as input. In some instances, the antibody sequence used as input is an antibody or antibody fragment sequence that binds SARS-CoV-2. In some instances, the input is an antibody or antibody fragment sequence that binds a protein of SARS-CoV-2. In some instances, the protein is a spike glycoprotein, a membrane protein, an envelope protein, a nucleocapsid protein, or combinations thereof. In some instances, the protein is a spike glycoprotein of SARS-CoV-2. In some instances, the protein is a receptor binding domain of SARS-CoV-2. In some instances, the input sequence is an antibody or antibody fragment sequence that binds angiotensin-converting enzyme 2 (ACE2). In some instances, the input sequence is an antibody or antibody fragment sequence that binds an extracellular domain of the angiotensin-converting enzyme 2 (ACE2).

A database 102 comprising known mutations or variants of one or more viruses is queried 101, and a library 103 of sequences comprising combinations of these mutations or variants are generated. In some instances, the database comprises known mutations or variants of SARS-CoV-like coronaviruses, SARS-CoV-2, SARS-CoV, or combinations thereof. In some instances, the database comprises known mutations or variants of the spike protein of SARS-CoV-like coronaviruses, SARS-CoV-2, SARS-CoV, or combinations thereof. In some instances, the database comprises known mutations or variants of the receptor binding domain of SARS-CoV-like coronaviruses, SARS-CoV-2, SARS-CoV, or combinations thereof. In some instances, the database comprises mutations or variants of a protein of SARS-CoV-like coronaviruses, SARS-CoV-2, SARS-CoV, or combinations thereof that binds to ACE2.

In some instances, the input sequence is a heavy chain sequence of an antibody or antibody fragment that binds SARS-CoV-like coronaviruses, SARS-CoV-2, SARS-CoV, or combinations thereof. In some instances, the input sequence is a light chain sequence of an antibody or antibody fragment that binds SARS-CoV-like coronaviruses, SARS-CoV-2, SARS-CoV, or combinations thereof. In some instances, the heavy chain sequence comprises varied CDR regions. In some instances, the light chain sequence comprises varied CDR regions. In some instances, known mutations or variants from CDRs are used to build the sequence library. Filters 104, or exclusion criteria, are in some instances used to select specific types of variants for members of the sequence library. For example, sequences having a mutation or variant are added if a minimum number of organisms in the database have the mutation or variant. In some instances, additional CDRs are specified for inclusion in the database. In some instances, specific mutations or variants or combinations of mutations or variants are excluded from the library (e.g., known immunogenic sites, structure sites, etc.). In some instances, specific sites in the input sequence are systematically replaced with histidine, aspartic acid, glutamic acid, or combinations thereof. In some instances, the maximum or minimum number of mutations or variants allowed for each region of an antibody are specified. Mutations or variants in some instances are described relative to the input sequence or the input sequence's corresponding germline sequence. For example, sequences generated by the optimization comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more than 16 mutations or variants from the input sequence. In some instances, sequences generated by the optimization comprise no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or no more than 18 mutations or variants from the input sequence. In some instances, sequences generated by the optimization comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or about 18 mutations or variants relative to the input sequence. In some instances, sequences generated by the optimization comprise about 1, 2, 3, 4, 5, 6, or 7 mutations or variants from the input sequence in a first CDR region. In some instances, sequences generated by the optimization comprise about 1, 2, 3, 4, 5, 6, or 7 mutations or variants from the input sequence in a second CDR region. In some instances, sequences generated by the optimization comprise about 1, 2, 3, 4, 5, 6, or 7 mutations or variants from the input sequence in a third CDR region. In some instances, sequences generated by the optimization comprise about 1, 2, 3, 4, 5, 6, or 7 mutations or variants from the input sequence in a first CDR region of a heavy chain. In some instances, sequences generated by the optimization comprise about 1, 2, 3, 4, 5, 6, or 7 mutations or variants from the input sequence in a second CDR region of a heavy chain. In some instances, sequences generated by the optimization comprise about 1, 2, 3, 4, 5, 6, or 7 mutations or variants from the input sequence in a third CDR region of a heavy chain. In some instances, sequences generated by the optimization comprise about 1, 2, 3, 4, 5, 6, or 7 mutations or variants from the input sequence in a first CDR region of a light chain. In some instances, sequences generated by the optimization comprise about 1, 2, 3, 4, 5, 6, or 7 mutations or variants from the input sequence in a second CDR region of a light chain. In some instances, sequences generated by the optimization comprise about 1, 2, 3, 4, 5, 6, or 7 mutations or variants from the input sequence in a third CDR region of a light chain. In some instances, a first CDR region is CDR1. In some instances, a second CDR region is CDR2. In some instances, a third CDR region is CDR3. In-silico antibodies libraries are in some instances synthesized, assembled, and enriched for desired sequences.

The germline sequences corresponding to an input sequence may also be modified to generate sequences in a library. For example, sequences generated by the optimization methods described herein comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more than 16 mutations or variants from the germline sequence. In some instances, sequences generated by the optimization comprise no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or no more than 18 mutations or variants from the germline sequence. In some instances, sequences generated by the optimization comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or about 18 mutations or variants relative to the germline sequence.

Provided herein are methods, systems, and compositions for antibody optimization, wherein the input sequence comprises mutations or variants in an antibody region. Exemplary regions of the antibody include, but are not limited to, a complementarity-determining region (CDR), a variable domain, or a constant domain. In some instances, the CDR is CDR1, CDR2, or CDR3. In some instances, the CDR is a heavy domain including, but not limited to, CDRH1, CDRH2, and CDRH3. In some instances, the CDR is a light domain including, but not limited to, CDRL1, CDRL2, and CDRL3. In some instances, the variable domain is variable domain, light chain (VL) or variable domain, heavy chain (VH). In some instances, the VL domain comprises kappa or lambda chains. In some instances, the constant domain is constant domain, light chain (CL) or constant domain, heavy chain (CH). In some instances, sequences generated by the optimization comprise about 1, 2, 3, 4, 5, 6, or 7 mutations or variants from the germline sequence in a first CDR region. In some instances, sequences generated by the optimization comprise about 1, 2, 3, 4, 5, 6, or 7 mutations or variants from the germline sequence in a second CDR region. In some instances, sequences generated by the optimization comprise about 1, 2, 3, 4, 5, 6, or 7 mutations or variants from the germline sequence in a third CDR region. In some instances, sequences generated by the optimization comprise about 1, 2, 3, 4, 5, 6, or 7 mutations or variants from the germline sequence in a first CDR region of a heavy chain. In some instances, sequences generated by the optimization comprise about 1, 2, 3, 4, 5, 6, or 7 mutations or variants from the germline sequence in a second CDR region of a heavy chain. In some instances, sequences generated by the optimization comprise about 1, 2, 3, 4, 5, 6, or 7 mutations or variants from the germline sequence in a third CDR region of a heavy chain. In some instances, sequences generated by the optimization comprise about 1, 2, 3, 4, 5, 6, or 7 mutations or variants from the germline sequence in a first CDR region of a light chain. In some instances, sequences generated by the optimization comprise about 1, 2, 3, 4, 5, 6, or 7 mutations or variants from the germline sequence in a second CDR region of a light chain. In some instances, sequences generated by the optimization comprise about 1, 2, 3, 4, 5, 6, or 7 mutations or variants from the germline sequence in a third CDR region of a light chain. In some instances, a first CDR region is CDR1. In some instances, a second CDR region is CDR2. In some instances, a third CDR region is CDR3.

VHH Libraries

Provided herein are methods, compositions, and systems for generation of antibodies or antibody fragments. In some instances, the antibodies or antibody fragments are single domain antibodies. Methods, compositions, and systems described herein for the optimization of antibodies comprise a ratio-variant approach that mirror the natural diversity of antibody sequences. In some instances, libraries of optimized antibodies comprise variant antibody sequences. In some instances, the variant antibody sequences are designed comprising variant CDR regions. In some instances, the variant antibody sequences comprising variant CDR regions are generated by shuffling the natural CDR sequences in a llama, humanized, or chimeric framework. In some instances, such libraries are synthesized, cloned into expression vectors, and translation products (antibodies) evaluated for activity. In some instances, fragments of sequences are synthesized and subsequently assembled. In some instances, expression vectors are used to display and enrich desired antibodies, such as phage display. In some instances, the phage vector is a Fab phagemid vector. Selection pressures used during enrichment in some instances includes, but is not limited to, binding affinity, toxicity, immunological tolerance, stability, receptor-ligand competition, or developability. Such expression vectors allow antibodies with specific properties to be selected (“panning”), and subsequent propagation or amplification of such sequences enriches the library with these sequences. Panning rounds can be repeated any number of times, such as 1, 2, 3, 4, 5, 6, 7, or more than 7 rounds. In some instances, each round of panning involves a number of washes. In some instances, each round of panning involves at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more than 16 washes.

Described herein are methods and systems of in-silico library design. Libraries as described herein, in some instances, are designed based on a database comprising a variety of antibody sequences. In some instances, the database comprises a plurality of variant antibody sequences against various targets. In some instances, the database comprises at least 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more than 5000 antibody sequences. An exemplary database is an iCAN database. In some instances, the database comprises naïve and memory B-cell receptor sequences. In some instances, the naïve and memory B-cell receptor sequences are human, mouse, or primate sequences. In some instances, the naïve and memory B-cell receptor sequences are human sequences. In some instances, the database is analyzed for position specific variation. In some instances, antibodies described herein comprise position specific variations in CDR regions. In some instances, the CDR regions comprise multiple sites for variation.

Described herein are libraries comprising variation in a CDR region. In some instances, the CDR is CDR1, CDR2, or CDR3 of a variable heavy chain. In some instances, the CDR is CDR1, CDR2, or CDR3 of a variable light chain. In some instances, the libraries comprise multiple variants encoding for CDR1, CDR2, or CDR3. In some instances, the libraries as described herein encode for at least 50, 100, 200, 300, 400, 500, 1000, 1200, 1500, 1700, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more than 5000 CDR1 sequences. In some instances, the libraries as described herein encode for at least 50, 100, 200, 300, 400, 500, 1000, 1200, 1500, 1700, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more than 5000 CDR2 sequences. In some instances, the libraries as described herein encode for at least 50, 100, 200, 300, 400, 500, 1000, 1200, 1500, 1700, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more than 5000 CDR3 sequences. In-silico antibodies libraries are in some instances synthesized, assembled, and enriched for desired sequences.

Following synthesis of CDR1 variants, CDR2 variants, and CDR3 variants, in some instances, the CDR1 variants, the CDR2 variants, and the CDR3 variants are shuffled to generate a diverse library. In some instances, the diversity of the libraries generated by methods described herein have a theoretical diversity of at least or about 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, or more than 10¹⁸ sequences. In some instances, the library has a final library diversity of at least or about 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, or more than 10¹⁸ sequences.

The germline sequences corresponding to a variant sequence may also be modified to generate sequences in a library. For example, sequences generated by methods described herein comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more than 16 mutations or variants from the germline sequence. In some instances, sequences generated comprise no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or no more than 18 mutations or variants from the germline sequence. In some instances, sequences generated comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or about 18 mutations or variants relative to the germline sequence.

Coronavirus Antibody Libraries

Provided herein are libraries generated from antibody optimization methods described herein. Antibodies described herein result in improved functional activity, structural stability, expression, specificity, or a combination thereof.

Provided herein are methods and compositions relating to SARS-CoV-2 binding libraries comprising nucleic acids encoding for a SARS-CoV-2 antibody. Further provided herein are methods and compositions relating to ACE2 binding libraries comprising nucleic acids encoding for an ACE2 antibody. Such methods and compositions in some instances are generated by the antibody optimization methods and systems described herein. Libraries as described herein may be further variegated to provide for variant libraries comprising nucleic acids each encoding for a predetermined variant of at least one predetermined reference nucleic acid sequence. Further described herein are protein libraries that may be generated when the nucleic acid libraries are translated. In some instances, nucleic acid libraries as described herein are transferred into cells to generate a cell library. Also provided herein are downstream applications for the libraries synthesized using methods described herein. Downstream applications include identification of variant nucleic acids or protein sequences with enhanced biologically relevant functions, e.g., improved stability, affinity, binding, functional activity, and for the treatment or prevention of an infection caused by a coronavirus such as SARS-CoV-2.

In some instances, an antibody or antibody fragment described herein comprises a sequence of any one of SEQ ID NOs: 1-2668. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 80% identical to a sequence of any one of SEQ ID NOs: 1-2668. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 85% identical to a sequence of any one of SEQ ID NOs: 1-2668. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-2668. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 95% identical to a sequence of any one of SEQ ID NOs: 1-2668.

In some instances, an antibody or antibody fragment described herein comprises a CDRH1 sequence of any one of SEQ ID NOs: 151-165, 241-255, 331-357, and 547-575. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 80% identical to a CDRH1 sequence of any one of SEQ ID NOs: 151-165, 241-255, 331-357, and 547-575. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 85% identical to a CDRH1 sequence of any one of SEQ ID NOs: 151-165, 241-255, 331-357, and 547-575. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 90% identical to a CDRH1 sequence of any one of SEQ ID NOs: 151-165, 241-255, 331-357, and 547-575. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 95% identical to a CDRH1 sequence of any one of SEQ ID NOs: 151-165, 241-255, 331-357, and 547-575. In some instances, an antibody or antibody fragment described herein comprises a CDRH2 sequence of any one of SEQ ID NOs: 166-180, 256-270, 358-384, and 576-604. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 80% identical to a CDRH2 sequence of any one of SEQ ID NOs: 166-180, 256-270, 358-384, and 576-604. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 85% identical to a CDRH2 sequence of any one of SEQ ID NOs: 166-180, 256-270, 358-384, and 576-604. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 90% identical to a CDRH2 sequence of any one of SEQ ID NOs: 166-180, 256-270, 358-384, and 576-604. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 95% identical to a CDRH2 sequence of any one of SEQ ID NOs: 166-180, 256-270, 358-384, and 576-604. In some instances, an antibody or antibody fragment described herein comprises a CDRH3 sequence of any one of SEQ ID NOs: 181-195, 271-285, 385-411, and 605-633. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 80% identical to a CDRH3 sequence of any one of SEQ ID NOs: 181-195, 271-285, 385-411, and 605-633. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 85% identical to a CDRH3 sequence of any one of SEQ ID NOs: 181-195, 271-285, 385-411, and 605-633. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 90% identical to a CDRH3 sequence of any one of SEQ ID NOs: 181-195, 271-285, 385-411, and 605-633. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 95% identical to a CDRH3 sequence of any one of SEQ ID NOs: 181-195, 271-285, 385-411, and 605-633.

In some instances, an antibody or antibody fragment described herein comprises a CDRH1 sequence of any one of SEQ ID NOs: 1-50, 779-919, 1344-1523, and 2381-2452. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 80% identical to a CDRH1 sequence of any one of SEQ ID NOs: 1-50, 779-919, 1344-1523, and 2381-2452. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 85% identical to a CDRH1 sequence of any one of SEQ ID NOs: 1-50, 779-919, 1344-1523, and 2381-2452. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 90% identical to a CDRH1 sequence of any one of SEQ ID NOs: 1-50, 779-919, 1344-1523, and 2381-2452. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 95% identical to a CDRH1 sequence of any one of SEQ ID NOs: 1-50, 779-919, 1344-1523, and 2381-2452. In some instances, an antibody or antibody fragment described herein comprises a CDRH2 sequence of any one of SEQ ID NOs: 51-100, 920-1061, 1524-1703, and 2453-2524. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 80% identical to a CDRH2 sequence of any one of SEQ ID NOs: 51-100, 920-1061, 1524-1703, and 2453-2524. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 85% identical to a CDRH2 sequence of any one of SEQ ID NOs: 51-100, 920-1061, 1524-1703, and 2453-2524. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 90% identical to a CDRH2 sequence of any one of SEQ ID NOs: 51-100, 920-1061, 1524-1703, and 2453-2524. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 95% identical to a CDRH2 sequence of any one of SEQ ID NOs: 51-100, 920-1061, 1524-1703, and 2453-2524. In some instances, an antibody or antibody fragment described herein comprises a CDRH3 sequence of any one of SEQ ID NOs: 101-150, 1062-1202, 1704-1883, and 2525-2596. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 80% identical to a CDRH3 sequence of any one of SEQ ID NOs: 101-150, 1062-1202, 1704-1883, and 2525-2596. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 85% identical to a CDRH3 sequence of any one of SEQ ID NOs: 101-150, 1062-1202, 1704-1883, and 2525-2596. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 90% identical to a CDRH3 sequence of any one of SEQ ID NOs: 101-150, 1062-1202, 1704-1883, and 2525-2596. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 95% identical to a CDRH3 sequence of any one of SEQ ID NOs: 101-150, 1062-1202, 1704-1883, and 2525-2596.

In some instances, an antibody or antibody fragment described herein comprises a CDRL1 sequence of any one of SEQ ID NOs: 196-210, 286-300, 412-438, and 634-662. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 80% identical to a CDRL1 sequence of any one of SEQ ID NOs: 196-210, 286-300, 412-438, and 634-662. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 85% identical to a CDRL1 sequence of any one of SEQ ID NOs: 196-210, 286-300, 412-438, and 634-662. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 90% identical to a CDRL1 sequence of any one of SEQ ID NOs: 1196-210, 286-300, 412-438, and 634-662. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 95% identical to a CDRL1 sequence of any one of SEQ ID NOs: 196-210, 286-300, 412-438, and 634-662. In some instances, an antibody or antibody fragment described herein comprises a CDRL2 sequence of any one of SEQ ID NOs: 211-225, 301-315, 439-465, and 663-691. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 80% identical to a CDRL2 sequence of any one of SEQ ID NOs: 211-225, 301-315, 439-465, and 663-691. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 85% identical to a CDRL2 sequence of any one of SEQ ID NOs: 211-225, 301-315, 439-465, and 663-691. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 90% identical to a CDRL2 sequence of any one of SEQ ID NOs: 211-225, 301-315, 439-465, and 663-691. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 95% identical to a CDRL2 sequence of any one of SEQ ID NOs: 211-225, 301-315, 439-465, and 663-691. In some instances, an antibody or antibody fragment described herein comprises a CDRL3 sequence of any one of SEQ ID NOs: 226-240, 316-330, 466-492, and 692-720. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 80% identical to a CDRL3 sequence of any one of SEQ ID NOs: 226-240, 316-330, 466-492, and 692-720. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 85% identical to a CDRL3 sequence of any one of SEQ ID NOs: 226-240, 316-330, 466-492, and 692-720. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 90% identical to a CDRL3 sequence of any one of SEQ ID NOs: 226-240, 316-330, 466-492, and 692-720. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 95% identical to a CDRL3 sequence of any one of SEQ ID NOs: 226-240, 316-330, 466-492, and 692-720.

In some embodiments, the antibody or antibody fragment comprising a variable domain, heavy chain region (VH) and a variable domain, light chain region (VL), wherein VH comprises complementarily determining regions CDRH1, CDRH2, and CDRH3, wherein VL comprises complementarily determining regions CDRL1, CDRL2, and CDRL3, and wherein (a) an amino acid sequence of CDRH1 is as set forth in any one of SEQ ID NOs: 151-165, 241-255, 331-357, and 547-575; (b) an amino acid sequence of CDRH2 is as set forth in any one of SEQ ID NOs: 166-180, 256-270, 358-384, and 576-604; (c) an amino acid sequence of CDRH3 is as set forth in any one of SEQ ID NOs: 181-195, 271-285, 385-411, and 605-633; (d) an amino acid sequence of CDRL1 is as set forth in any one of SEQ ID NOs: 196-210, 286-300, 412-438, and 634-662; (e) an amino acid sequence of CDRL2 is as set forth in any one of SEQ ID NOs: 211-225, 301-315, 439-465, and 663-691; and (0 an amino acid sequence of CDRL3 is as set forth in any one of SEQ ID NOs: 226-240, 316-330, 466-492, and 692-720. In some embodiments, the antibody or antibody fragment comprising a variable domain, heavy chain region (VH) and a variable domain, light chain region (VL), wherein VH comprises complementarity determining regions CDRH1, CDRH2, and CDRH3, wherein VL comprises complementarity determining regions CDRL1, CDRL2, and CDRL3, and wherein (a) an amino acid sequence of CDRH1 is at least or about 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs: 151-165, 241-255, 331-357, and 547-575; (b) an amino acid sequence of CDRH2 is at least or about 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs: 166-180, 256-270, 358-384, and 576-604; (c) an amino acid sequence of CDRH3 is at least or about 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs: 181-195, 271-285, 385-411, and 605-633; (d) an amino acid sequence of CDRL1 is at least or about 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs: 196-210, 286-300, 412-438, and 634-662; (e) an amino acid sequence of CDRL2 is at least or about 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs: 211-225, 301-315, 439-465, and 663-691; and (f) an amino acid sequence of CDRL3 is at least or about 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs: 226-240, 316-330, 466-492, and 692-720.

Described herein, in some embodiments, are antibodies or antibody fragments comprising a variable domain, heavy chain region (VH) and a variable domain, light chain region (VL), wherein the VH comprises an amino acid sequence at least about 90% identical to a sequence as set forth in any one of SEQ ID NOs: 493-519 and 721-749, and wherein the VL comprises an amino acid sequence at least about 90% identical to a sequence as set forth in any one of SEQ ID NOs: 520-546 and 750-778. In some instances, the antibodies or antibody fragments comprise VH comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 493-519 and 721-749, and VL comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 520-546 and 750-778.

Described herein, in some embodiments, are antibodies or antibody fragments comprising a variable domain, heavy chain region (VH), wherein the VH comprises an amino acid sequence at least about 90% identical to a sequence as set forth in any one of SEQ ID NOs: 1884-2063, 2302-2380, and 2597-2668. In some instances, the antibodies or antibody fragments comprise a heavy chain variable domain comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 1884-2063, 2302-2380, and 2597-2668.

The term “sequence identity” means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.

The term “homology” or “similarity” between two proteins is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one protein sequence to the second protein sequence. Similarity may be determined by procedures which are well-known in the art, for example, a BLAST program (Basic Local Alignment Search Tool at the National Center for Biological Information).

The term “epitope” includes any determinant capable of being bound by an antigen binding protein, such as an antibody. An epitope is a region of an antigen that is bound by an antigen binding protein that targets that antigen, and when the antigen is a protein, includes specific amino acids that directly contact the antigen binding protein. Most often, epitopes reside on proteins, but in some instances can reside on other kinds of molecules, such as saccharides or lipids. Epitope determinants can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three dimensional structural characteristics, and/or specific charge characteristics. Generally, antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of proteins and/or macromolecules.

Provided herein are libraries comprising nucleic acids encoding for SARS-CoV-2 antibodies. Antibodies described herein allow for improved stability for a range of SARS-CoV-2 or ACE2 binding domain encoding sequences. In some instances, the binding domain encoding sequences are determined by interactions between SARS-CoV-2 and ACE2.

Sequences of binding domains based on surface interactions between SARS-CoV-2 and ACE2 are analyzed using various methods. For example, multispecies computational analysis is performed. In some instances, a structure analysis is performed. In some instances, a sequence analysis is performed. Sequence analysis can be performed using a database known in the art. Non-limiting examples of databases include, but are not limited to, NCBI BLAST (blast.ncbi.nlm.nih.gov/Blast.cgi), UCSC Genome Browser (genome.ucsc.edu/), UniProt (www.uniprot.org/), and IUPHAR/BPS Guide to PHARMACOLOGY (guidetopharmacology.org/).

Described herein are SARS-CoV-2 or ACE2 binding domains designed based on sequence analysis among various organisms. For example, sequence analysis is performed to identify homologous sequences in different organisms. Exemplary organisms include, but are not limited to, mouse, rat, equine, sheep, cow, primate (e.g., chimpanzee, baboon, gorilla, orangutan, monkey), dog, cat, pig, donkey, rabbit, fish, fly, and human. In some instances, homologous sequences are identified in the same organism, across individuals.

Following identification of SARS-CoV-2 or ACE2 binding domains, libraries comprising nucleic acids encoding for the SARS-CoV-2 or ACE2 binding domains may be generated. In some instances, libraries of SARS-CoV-2 or ACE2 binding domains comprise sequences of SARS-CoV-2 or ACE2 binding domains designed based on conformational ligand interactions, peptide ligand interactions, small molecule ligand interactions, extracellular domains of SARS-CoV-2 or ACE2, or antibodies that target SARS-CoV-2 or ACE2. Libraries of SARS-CoV-2 or ACE2 binding domains may be translated to generate protein libraries. In some instances, libraries of SARS-CoV-2 or ACE2 binding domains are translated to generate peptide libraries, immunoglobulin libraries, derivatives thereof, or combinations thereof. In some instances, libraries of SARS-CoV-2 or ACE2 binding domains are translated to generate protein libraries that are further modified to generate peptidomimetic libraries. In some instances, libraries of SARS-CoV-2 or ACE2 binding domains are translated to generate protein libraries that are used to generate small molecules.

Methods described herein provide for synthesis of libraries of SARS-CoV-2 or ACE2 binding domains comprising nucleic acids each encoding for a predetermined variant of at least one predetermined reference nucleic acid sequence. In some cases, the predetermined reference sequence is a nucleic acid sequence encoding for a protein, and the variant library comprises sequences encoding for variation of at least a single codon such that a plurality of different variants of a single residue in the subsequent protein encoded by the synthesized nucleic acid are generated by standard translation processes. In some instances, the libraries of SARS-CoV-2 or ACE2 binding domains comprise varied nucleic acids collectively encoding variations at multiple positions. In some instances, the variant library comprises sequences encoding for variation of at least a single codon in a SARS-CoV-2 or ACE2 binding domain. In some instances, the variant library comprises sequences encoding for variation of multiple codons in a SARS-CoV-2 or ACE2 binding domain. An exemplary number of codons for variation include, but are not limited to, at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275, 300, or more than 300 codons.

Methods described herein provide for synthesis of libraries comprising nucleic acids encoding for the SARS-CoV-2 or ACE2 binding domains, wherein the libraries comprise sequences encoding for variation of length of the SARS-CoV-2 or ACE2 binding domains. In some instances, the library comprises sequences encoding for variation of length of at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275, 300, or more than 300 codons less as compared to a predetermined reference sequence. In some instances, the library comprises sequences encoding for variation of length of at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, or more than 300 codons more as compared to a predetermined reference sequence.

Following identification of SARS-CoV-2 or ACE2 binding domains, antibodies may be designed and synthesized to comprise the SARS-CoV-2 or ACE2 binding domains. Antibodies comprising SARS-CoV-2 or ACE2 binding domains may be designed based on binding, specificity, stability, expression, folding, or downstream activity. In some instances, the antibodies comprising SARS-CoV-2 or ACE2 binding domains enable contact with the SARS-CoV-2 or ACE2. In some instances, the antibodies comprising SARS-CoV-2 or ACE2 binding domains enables high affinity binding with the SARS-CoV-2 or ACE2. Exemplary amino acid sequences of SARS-CoV-2 or ACE2 binding domains comprise any one of SEQ ID NOs: 1-2668.

In some instances, the SARS-CoV-2 antibody comprises a binding affinity (e.g., K_D) to SARS-CoV-2 of less than 1 nM, less than 1.2 nM, less than 2 nM, less than 5 nM, less than 10 nM, less than 11 nm, less than 13.5 nM, less than 15 nM, less than 20 nM, less than 25 nM, or less than 30 nM. In some instances, the SARS-CoV-2 antibody comprises a K_D of less than 1 nM. In some instances, the SARS-CoV-2 antibody comprises a K_D of less than 1.2 nM. In some instances, the SARS-CoV-2 antibody comprises a K_D of less than 2 nM. In some instances, the SARS-CoV-2 antibody comprises a K_D of less than 5 nM. In some instances, the SARS-CoV-2 antibody comprises a K_D of less than 10 nM. In some instances, the SARS-CoV-2 antibody comprises a K_D of less than 13.5 nM. In some instances, the SARS-CoV-2 antibody comprises a K_D of less than 15 nM. In some instances, the SARS-CoV-2 antibody comprises a K_D of less than 20 nM. In some instances, the SARS-CoV-2 antibody comprises a K_D of less than 25 nM. In some instances, the SARS-CoV-2 antibody comprises a K_D of less than 30 nM.

In some instances, the ACE2 antibody comprises a binding affinity (e.g., K_D) to ACE2 of less than 1 nM, less than 1.2 nM, less than 2 nM, less than 5 nM, less than 10 nM, less than 11 nm, less than 13.5 nM, less than 15 nM, less than 20 nM, less than 25 nM, or less than 30 nM. In some instances, the ACE2 antibody comprises a K_D of less than 1 nM. In some instances, the ACE2 antibody comprises a K_D of less than 1.2 nM. In some instances, the ACE2 antibody comprises a K_D of less than 2 nM. In some instances, the ACE2 antibody comprises a K_D of less than 5 nM. In some instances, the ACE2 antibody comprises a K_D of less than 10 nM. In some instances, the ACE2 antibody comprises a K_D of less than 13.5 nM. In some instances, the ACE2 antibody comprises a K_D of less than 15 nM. In some instances, the ACE2 antibody comprises a K_D of less than 20 nM. In some instances, the ACE2 antibody comprises a K_D of less than 25 nM. In some instances, the ACE2 antibody comprises a K_D of less than 30 nM.

In some instances, the SARS-CoV-2 or ACE2 immunoglobulin is an agonist. In some instances, the SARS-CoV-2 or ACE2 immunoglobulin is an antagonist. In some instances, the SARS-CoV-2 or ACE2 immunoglobulin is an allosteric modulator. In some instances, the allosteric modulator is a negative allosteric modulator. In some instances, the allosteric modulator is a positive allosteric modulator. In some instances, the SARS-CoV-2 or ACE2 immunoglobulin results in agonistic, antagonistic, or allosteric effects at a concentration of at least or about 1 nM, 2 nM, 4 nM, 6 nM, 8 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 120 nM, 140 nM, 160 nM, 180 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1000 nM, or more than 1000 nM. In some instances, the SARS-CoV-2 or ACE2 immunoglobulin is a negative allosteric modulator. In some instances, the SARS-CoV-2 or ACE2 immunoglobulin is a negative allosteric modulator at a concentration of at least or about 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1 nM, 2 nM, 4 nM, 6 nM, 8 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, or more than 100 nM. In some instances, the SARS-CoV-2 or ACE2 immunoglobulin is a negative allosteric modulator at a concentration in a range of about 0.001 to about 100, 0.01 to about 90, about 0.1 to about 80, 1 to about 50, about 10 to about 40 nM, or about 1 to about 10 nM. In some instances, the SARS-CoV-2 or ACE2 immunoglobulin comprises an EC50 or IC50 of at least or about 0.001, 0.0025, 0.005, 0.01, 0.025, 0.05, 0.06, 0.07, 0.08, 0.9, 0.1, 0.5, 1, 2, 3, 4, 5, 6, or more than 6 nM. In some instances, the SARS-CoV-2 or ACE2 immunoglobulin comprises an EC50 or IC50 of at least or about 1 nM, 2 nM, 4 nM, 6 nM, 8 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, or more than 100 nM.

In some instances, the affinity of the SARS-CoV-2 or ACE2 antibody generated by methods as described herein is at least or about 1.5×, 2.0×, 5×, 10×, 20×, 30×, 40×, 50×, 60×, 70×, 80×, 90×, 100×, 200×, or more than 200× improved binding affinity as compared to a comparator antibody. In some instances, the SARS-CoV-2 or ACE2 antibody generated by methods as described herein is at least or about 1.5×, 2.0×, 5×, 10×, 20×, 30×, 40×, 50×, 60×, 70×, 80×, 90×, 100×, 200×, or more than 200× improved function as compared to a comparator antibody. In some instances, the comparator antibody is an antibody with similar structure, sequence, or antigen target.

Provided herein are SARS-CoV-2 or ACE2 binding libraries comprising nucleic acids encoding for antibodies comprising SARS-CoV-2 or ACE2 binding domains comprise variation in domain type, domain length, or residue variation. In some instances, the domain is a region in the antibody comprising the SARS-CoV-2 or ACE2 binding domains. For example, the region is the VH, CDRH3, or VL domain. In some instances, the domain is the SARS-CoV-2 or ACE2 binding domain.

Methods described herein provide for synthesis of a SARS-CoV-2 or ACE21 binding library of nucleic acids each encoding for a predetermined variant of at least one predetermined reference nucleic acid sequence. In some cases, the predetermined reference sequence is a nucleic acid sequence encoding for a protein, and the variant library comprises sequences encoding for variation of at least a single codon such that a plurality of different variants of a single residue in the subsequent protein encoded by the synthesized nucleic acid are generated by standard translation processes. In some instances, the SARS-CoV-2 or ACE2 binding library comprises varied nucleic acids collectively encoding variations at multiple positions. In some instances, the variant library comprises sequences encoding for variation of at least a single codon of a VH or VL domain. In some instances, the variant library comprises sequences encoding for variation of at least a single codon in a SARS-CoV-2 or ACE2 binding domain. For example, at least one single codon of a SARS-CoV-2 or ACE2 binding domain is varied. In some instances, the variant library comprises sequences encoding for variation of multiple codons of a VH or VL domain. In some instances, the variant library comprises sequences encoding for variation of multiple codons in a SARS-CoV-2 or ACE2 binding domain. An exemplary number of codons for variation include, but are not limited to, at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275, 300, or more than 300 codons.

Methods described herein provide for synthesis of a SARS-CoV-2 or ACE2 binding library of nucleic acids each encoding for a predetermined variant of at least one predetermined reference nucleic acid sequence, wherein the SARS-CoV-2 or ACE2 binding library comprises sequences encoding for variation of length of a domain. In some instances, the domain is VH or VL domain. In some instances, the domain is the SARS-CoV-2 or ACE2 binding domain. In some instances, the library comprises sequences encoding for variation of length of at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275, 300, or more than 300 codons less as compared to a predetermined reference sequence. In some instances, the library comprises sequences encoding for variation of length of at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, or more than 300 codons more as compared to a predetermined reference sequence.

Provided herein are SARS-CoV-2 or ACE2 binding libraries comprising nucleic acids encoding for antibodies comprising SARS-CoV-2 or ACE2 binding domains, wherein the SARS-CoV-2 or ACE2 binding libraries are synthesized with various numbers of fragments. In some instances, the fragments comprise the VH or VL domain. In some instances, the SARS-CoV-2 or ACE2 binding libraries are synthesized with at least or about 2 fragments, 3 fragments, 4 fragments, 5 fragments, or more than 5 fragments. The length of each of the nucleic acid fragments or average length of the nucleic acids synthesized may be at least or about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, or more than 600 base pairs. In some instances, the length is about 50 to 600, 75 to 575, 100 to 550, 125 to 525, 150 to 500, 175 to 475, 200 to 450, 225 to 425, 250 to 400, 275 to 375, or 300 to 350 base pairs.

SARS-CoV-2 or ACE2 binding libraries comprising nucleic acids encoding for antibodies comprising SARS-CoV-2 or ACE2 binding domains as described herein comprise various lengths of amino acids when translated. In some instances, the length of each of the amino acid fragments or average length of the amino acid synthesized may be at least or about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, or more than 150 amino acids. In some instances, the length of the amino acid is about 15 to 150, 20 to 145, 25 to 140, 30 to 135, 35 to 130, 40 to 125, 45 to 120, 50 to 115, 55 to 110, 60 to 110, 65 to 105, 70 to 100, or 75 to 95 amino acids. In some instances, the length of the amino acid is about 22 to about 75 amino acids.

SARS-CoV-2 or ACE2 binding libraries comprising de novo synthesized variant sequences encoding for antibodies comprising SARS-CoV-2 or ACE2 binding domains comprise a number of variant sequences. In some instances, a number of variant sequences is de novo synthesized for a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VL, VH, or a combination thereof. In some instances, a number of variant sequences is de novo synthesized for framework element 1 (FW1), framework element 2 (FW2), framework element 3 (FW3), or framework element 4 (FW4). In some instances, a number of variant sequences are de novo synthesized for a SARS-CoV-2 or ACE2 binding domain. The number of variant sequences may be at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more than 500 sequences. In some instances, the number of variant sequences is about 10 to 300, 25 to 275, 50 to 250, 75 to 225, 100 to 200, or 125 to 150 sequences.

SARS-CoV-2 or ACE2 binding libraries comprising de novo synthesized variant sequences encoding for antibodies comprising SARS-CoV-2 or ACE2 binding domains comprise improved diversity. In some instances, variants include affinity maturation variants. Alternatively or in combination, variants include variants in other regions of the antibody including, but not limited to, CDRH1, CDRH2, CDRL1, CDRL2, and CDRL3. In some instances, the number of variants of the SARS-CoV-2 or ACE2 binding libraries is least or about 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴ or more than 10¹⁴ non-identical sequences.

Following synthesis of SARS-CoV-2 or ACE2 binding libraries comprising nucleic acids encoding antibodies comprising SARS-CoV-2 or ACE2 binding domains, libraries may be used for screening and analysis. For example, libraries are assayed for library displayability and panning. In some instances, displayability is assayed using a selectable tag. Exemplary tags include, but are not limited to, a radioactive label, a fluorescent label, an enzyme, a chemiluminescent tag, a colorimetric tag, an affinity tag or other labels or tags that are known in the art. In some instances, the tag is histidine, polyhistidine, myc, hemagglutinin (HA), or FLAG. For example, SARS-CoV-2 or ACE2 binding libraries comprise nucleic acids encoding antibodies comprising SARS-CoV-2 or ACE2 binding domains with multiple tags such as GFP, FLAG, and Lucy as well as a DNA barcode. In some instances, libraries are assayed by sequencing using various methods including, but not limited to, single-molecule real-time (SMRT) sequencing, Polony sequencing, sequencing by ligation, reversible terminator sequencing, proton detection sequencing, ion semiconductor sequencing, nanopore sequencing, electronic sequencing, pyrosequencing, Maxam-Gilbert sequencing, chain termination (e.g., Sanger) sequencing, +S sequencing, or sequencing by synthesis.

As used herein, the term antibody will be understood to include proteins having the characteristic two-armed, Y-shape of a typical antibody molecule as well as one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Exemplary antibodies include, but are not limited to, a monoclonal antibody, a polyclonal antibody, a bi-specific antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv) (including fragments in which the VL and VH are joined using recombinant methods by a synthetic or natural linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules, including single chain Fab and scFab), a single chain antibody, a Fab fragment (including monovalent fragments comprising the VL, VH, CL, and CH1 domains), a F(ab′)2 fragment (including bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region), a Fd fragment (including fragments comprising the VH and CH1 fragment), a Fv fragment (including fragments comprising the VL and VH domains of a single arm of an antibody), a single-domain antibody (dAb or sdAb) (including fragments comprising a VH domain), an isolated complementarity determining region (CDR), a diabody (including fragments comprising bivalent dimers such as two VL and VH domains bound to each other and recognizing two different antigens), a fragment comprised of only a single monomeric variable domain, disulfide-linked Fvs (sdFv), an intrabody, an anti-idiotypic (anti-Id) antibody, or ab antigen-binding fragments thereof. In some instances, the libraries disclosed herein comprise nucleic acids encoding for an antibody, wherein the antibody is a Fv antibody, including Fv antibodies comprised of the minimum antibody fragment which contains a complete antigen-recognition and antigen-binding site. In some embodiments, the Fv antibody consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association, and the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. In some embodiments, the six hypervariable regions confer antigen-binding specificity to the antibody. In some embodiments, a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen, including single domain antibodies isolated from camelid animals comprising one heavy chain variable domain such as VHH antibodies or nanobodies) has the ability to recognize and bind antigen. In some instances, the libraries disclosed herein comprise nucleic acids encoding for an antibody, wherein the antibody is a single-chain Fv or scFv, including antibody fragments comprising a VH, a VL, or both a VH and VL domain, wherein both domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains allowing the scFv to form the desired structure for antigen binding. In some instances, a scFv is linked to the Fc fragment or a VHH is linked to the Fc fragment (including minibodies). In some instances, the antibody comprises immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, e.g., molecules that contain an antigen binding site. Immunoglobulin molecules are of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG 2, IgG 3, IgG 4, IgA 1 and IgA 2) or subclass.

In some embodiments, the antibody is a multivalent antibody. In some embodiments, the antibody is a monovalent, bivalent, or multivalent antibody. In some instances, the antibody is monospecific, bispecific, or multispecific. In some embodiments, the antibody is monovalent monospecific, monovalent bispecific, monovalent multispecific, bivalent monospecific, bivalent bispecific, bivalent multispecific, multivalent monospecific, multivalent bispecific, multivalent multispecific. In some instances, the antibody is homodimeric, heterodimeric, or heterotrimeric.

In some embodiments, libraries comprise immunoglobulins that are adapted to the species of an intended therapeutic target. Generally, these methods include “mammalization” and comprises methods for transferring donor antigen-binding information to a less immunogenic mammal antibody acceptor to generate useful therapeutic treatments. In some instances, the mammal is mouse, rat, equine, sheep, cow, primate (e.g., chimpanzee, baboon, gorilla, orangutan, monkey), dog, cat, pig, donkey, rabbit, and human. In some instances, provided herein are libraries and methods for felinization and caninization of antibodies.

“Humanized” forms of non-human antibodies can be chimeric antibodies that contain minimal sequence derived from the non-human antibody. A humanized antibody is generally a human antibody (recipient antibody) in which residues from one or more CDRs are replaced by residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody having a desired specificity, affinity, or biological effect. In some instances, selected framework region residues of the recipient antibody are replaced by the corresponding framework region residues from the donor antibody. Humanized antibodies may also comprise residues that are not found in either the recipient antibody or the donor antibody. In some instances, these modifications are made to further refine antibody performance.

“Caninization” can comprise a method for transferring non-canine antigen-binding information from a donor antibody to a less immunogenic canine antibody acceptor to generate treatments useful as therapeutics in dogs. In some instances, caninized forms of non-canine antibodies provided herein are chimeric antibodies that contain minimal sequence derived from non-canine antibodies. In some instances, caninized antibodies are canine antibody sequences (“acceptor” or “recipient” antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-canine species (“donor” antibody) such as mouse, rat, rabbit, cat, dogs, goat, chicken, bovine, horse, llama, camel, dromedaries, sharks, non-human primates, human, humanized, recombinant sequence, or an engineered sequence having the desired properties. In some instances, framework region (FR) residues of the canine antibody are replaced by corresponding non-canine FR residues. In some instances, caninized antibodies include residues that are not found in the recipient antibody or in the donor antibody. In some instances, these modifications are made to further refine antibody performance. The caninized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc) of a canine antibody.

“Felinization” can comprise a method for transferring non-feline antigen-binding information from a donor antibody to a less immunogenic feline antibody acceptor to generate treatments useful as therapeutics in cats. In some instances, felinized forms of non-feline antibodies provided herein are chimeric antibodies that contain minimal sequence derived from non-feline antibodies. In some instances, felinized antibodies are feline antibody sequences (“acceptor” or “recipient” antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-feline species (“donor” antibody) such as mouse, rat, rabbit, cat, dogs, goat, chicken, bovine, horse, llama, camel, dromedaries, sharks, non-human primates, human, humanized, recombinant sequence, or an engineered sequence having the desired properties. In some instances, framework region (FR) residues of the feline antibody are replaced by corresponding non-feline FR residues. In some instances, felinized antibodies include residues that are not found in the recipient antibody or in the donor antibody. In some instances, these modifications are made to further refine antibody performance. The felinized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc) of a felinize antibody.

Methods as described herein may be used for optimization of libraries encoding a non-immunoglobulin. In some instances, the libraries comprise antibody mimetics. Exemplary antibody mimetics include, but are not limited to, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, atrimers, DARPins, fynomers, Kunitz domain-based proteins, monobodies, anticalins, knottins, armadillo repeat protein-based proteins, and bicyclic peptides.

Libraries described herein comprising nucleic acids encoding for an antibody comprise variations in at least one region of the antibody. Exemplary regions of the antibody for variation include, but are not limited to, a complementarity-determining region (CDR), a variable domain, or a constant domain. In some instances, the CDR is CDR1, CDR2, or CDR3. In some instances, the CDR is a heavy domain including, but not limited to, CDRH1, CDRH2, and CDRH3. In some instances, the CDR is a light domain including, but not limited to, CDRL1, CDRL2, and CDRL3. In some instances, the variable domain is variable domain, light chain (VL) or variable domain, heavy chain (VH). In some instances, the VL domain comprises kappa or lambda chains. In some instances, the constant domain is constant domain, light chain (CL) or constant domain, heavy chain (CH).

Methods described herein provide for synthesis of libraries comprising nucleic acids encoding an antibody, wherein each nucleic acid encodes for a predetermined variant of at least one predetermined reference nucleic acid sequence. In some cases, the predetermined reference sequence is a nucleic acid sequence encoding for a protein, and the variant library comprises sequences encoding for variation of at least a single codon such that a plurality of different variants of a single residue in the subsequent protein encoded by the synthesized nucleic acid are generated by standard translation processes. In some instances, the antibody library comprises varied nucleic acids collectively encoding variations at multiple positions. In some instances, the variant library comprises sequences encoding for variation of at least a single codon of a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VL, or VH domain. In some instances, the variant library comprises sequences encoding for variation of multiple codons of a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VL, or VH domain. In some instances, the variant library comprises sequences encoding for variation of multiple codons of framework element 1 (FW1), framework element 2 (FW2), framework element 3 (FW3), or framework element 4 (FW4). An exemplary number of codons for variation include, but are not limited to, at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275, 300, or more than 300 codons.

In some instances, the at least one region of the antibody for variation is from heavy chain V-gene family, heavy chain D-gene family, heavy chain J-gene family, light chain V-gene family, or light chain J-gene family. In some instances, the light chain V-gene family comprises immunoglobulin kappa (IGK) gene or immunoglobulin lambda (IGL). Exemplary regions of the antibody for variation include, but are not limited to, IGHV1-18, IGHV1-69, IGHV1-8, IGHV3-21, IGHV3-23, IGHV3-30/33m, IGHV3-28, IGHV1-69, IGHV3-74, IGHV4-39, IGHV4-59/61, IGKV1-39, IGKV1-9, IGKV2-28, IGKV3-11, IGKV3-15, IGKV3-20, IGKV4-1, IGLV1-51, IGLV2-14, IGLV1-40, and IGLV3-1. In some instances, the gene is IGHV1-69, IGHV3-30, IGHV3-23, IGHV3, IGHV1-46, IGHV3-7, IGHV1, or IGHV1-8. In some instances, the gene is IGHV1-69 and IGHV3-30. In some instances, the region of the antibody for variation is IGHJ3, IGHJ6, IGHJ, IGHJ4, IGHJ5, IGHJ2, or IGH1. In some instances, the region of the antibody for variation is IGHJ3, IGHJ6, IGHJ, or IGHJ4. In some instances, the at least one region of the antibody for variation is IGHV1-69, IGHV3-23, IGKV3-20, IGKV1-39, or combinations thereof. In some instances, the at least one region of the antibody for variation is IGHV1-69 and IGKV3-20, In some instances, the at least one region of the antibody for variation is IGHV1-69 and IGKV1-39. In some instances, the at least one region of the antibody for variation is IGHV3-23 and IGKV3-20. In some instances, the at least one region of the antibody for variation is IGHV3-23 and IGKV1-39.

Provided herein are libraries comprising nucleic acids encoding for antibodies, wherein the libraries are synthesized with various numbers of fragments. In some instances, the fragments comprise the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VL, or VH domain. In some instances, the fragments comprise framework element 1 (FW1), framework element 2 (FW2), framework element 3 (FW3), or framework element 4 (FW4). In some instances, the antibody libraries are synthesized with at least or about 2 fragments, 3 fragments, 4 fragments, 5 fragments, or more than 5 fragments. The length of each of the nucleic acid fragments or average length of the nucleic acids synthesized may be at least or about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, or more than 600 base pairs. In some instances, the length is about 50 to 600, 75 to 575, 100 to 550, 125 to 525, 150 to 500, 175 to 475, 200 to 450, 225 to 425, 250 to 400, 275 to 375, or 300 to 350 base pairs.

Libraries comprising nucleic acids encoding for antibodies as described herein comprise various lengths of amino acids when translated. In some instances, the length of each of the amino acid fragments or average length of the amino acid synthesized may be at least or about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, or more than 150 amino acids. In some instances, the length of the amino acid is about 15 to 150, 20 to 145, 25 to 140, 30 to 135, 35 to 130, 40 to 125, 45 to 120, 50 to 115, 55 to 110, 60 to 110, 65 to 105, 70 to 100, or 75 to 95 amino acids. In some instances, the length of the amino acid is about 22 amino acids to about 75 amino acids. In some instances, the antibodies comprise at least or about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more than 5000 amino acids.

A number of variant sequences for the at least one region of the antibody for variation are de novo synthesized using methods as described herein. In some instances, a number of variant sequences is de novo synthesized for CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VL, VH, or combinations thereof. In some instances, a number of variant sequences is de novo synthesized for framework element 1 (FW1), framework element 2 (FW2), framework element 3 (FW3), or framework element 4 (FW4). The number of variant sequences may be at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more than 500 sequences. In some instances, the number of variant sequences is at least or about 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, or more than 8000 sequences. In some instances, the number of variant sequences is about 10 to 500, 25 to 475, 50 to 450, 75 to 425, 100 to 400, 125 to 375, 150 to 350, 175 to 325, 200 to 300, 225 to 375, 250 to 350, or 275 to 325 sequences.

Variant sequences for the at least one region of the antibody, in some instances, vary in length or sequence. In some instances, the at least one region that is de novo synthesized is for CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VL, VH, or combinations thereof. In some instances, the at least one region that is de novo synthesized is for framework element 1 (FW1), framework element 2 (FW2), framework element 3 (FW3), or framework element 4 (FW4). In some instances, the variant sequence comprises at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more than 50 variant nucleotides or amino acids as compared to wild-type. In some instances, the variant sequence comprises at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 additional nucleotides or amino acids as compared to wild-type. In some instances, the variant sequence comprises at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 less nucleotides or amino acids as compared to wild-type. In some instances, the libraries comprise at least or about 10¹, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, or more than 10¹⁰ variants.

Following synthesis of antibody libraries, antibody libraries may be used for screening and analysis. For example, antibody libraries are assayed for library displayability and panning. In some instances, displayability is assayed using a selectable tag. Exemplary tags include, but are not limited to, a radioactive label, a fluorescent label, an enzyme, a chemiluminescent tag, a colorimetric tag, an affinity tag or other labels or tags that are known in the art. In some instances, the tag is histidine, polyhistidine, myc, hemagglutinin (HA), or FLAG. In some instances, antibody libraries are assayed by sequencing using various methods including, but not limited to, single-molecule real-time (SMRT) sequencing, Polony sequencing, sequencing by ligation, reversible terminator sequencing, proton detection sequencing, ion semiconductor sequencing, nanopore sequencing, electronic sequencing, pyrosequencing, Maxam-Gilbert sequencing, chain termination (e.g., Sanger) sequencing, +S sequencing, or sequencing by synthesis. In some instances, antibody libraries are displayed on the surface of a cell or phage. In some instances, antibody libraries are enriched for sequences with a desired activity using phage display.

In some instances, the antibody libraries are assayed for functional activity, structural stability (e.g., thermal stable or pH stable), expression, specificity, or a combination thereof. In some instances, the antibody libraries are assayed for antibody capable of folding. In some instances, a region of the antibody is assayed for functional activity, structural stability, expression, specificity, folding, or a combination thereof. For example, a VH region or VL region is assayed for functional activity, structural stability, expression, specificity, folding, or a combination thereof.

In some instances, the affinity of antibodies or IgGs generated by methods as described herein is at least or about 1.5×, 2.0×, 5×, 10×, 20×, 30×, 40×, 50×, 60×, 70×, 80×, 90×, 100×, 200×, or more than 200× improved binding affinity as compared to a comparator antibody. In some instances, the affinity of antibodies or IgGs generated by methods as described herein is at least or about 1.5×, 2.0×, 5×, 10×, 20×, 30×, 40×, 50×, 60×, 70×, 80×, 90×, 100×, 200×, or more than 200× improved function as compared to a comparator antibody. In some instances, the comparator antibody is an antibody with similar structure, sequence, or antigen target.

Expression Systems

Provided herein are libraries comprising nucleic acids encoding for antibody comprising binding domains, wherein the libraries have improved specificity, stability, expression, folding, or downstream activity. In some instances, libraries described herein are used for screening and analysis.

Provided herein are libraries comprising nucleic acids encoding for antibody comprising binding domains, wherein the nucleic acid libraries are used for screening and analysis. In some instances, screening and analysis comprises in vitro, in vivo, or ex vivo assays. Cells for screening include primary cells taken from living subjects or cell lines. Cells may be from prokaryotes (e.g., bacteria and fungi) or eukaryotes (e.g., animals and plants). Exemplary animal cells include, without limitation, those from a mouse, rabbit, primate, and insect. In some instances, cells for screening include a cell line including, but not limited to, Chinese Hamster Ovary (CHO) cell line, human embryonic kidney (HEK) cell line, or baby hamster kidney (BHK) cell line. In some instances, nucleic acid libraries described herein may also be delivered to a multicellular organism. Exemplary multicellular organisms include, without limitation, a plant, a mouse, rabbit, primate, and insect.

Nucleic acid libraries described herein may be screened for various pharmacological or pharmacokinetic properties. In some instances, the libraries are screened using in vitro assays, in vivo assays, or ex vivo assays. For example, in vitro pharmacological or pharmacokinetic properties that are screened include, but are not limited to, binding affinity, binding specificity, and binding avidity. Exemplary in vivo pharmacological or pharmacokinetic properties of libraries described herein that are screened include, but are not limited to, therapeutic efficacy, activity, preclinical toxicity properties, clinical efficacy properties, clinical toxicity properties, immunogenicity, potency, and clinical safety properties.

Provided herein are nucleic acid libraries, wherein the nucleic acid libraries may be expressed in a vector. Expression vectors for inserting nucleic acid libraries disclosed herein may comprise eukaryotic or prokaryotic expression vectors. Exemplary expression vectors include, without limitation, mammalian expression vectors: pSF-CMV-NEO-NH2-PPT-3×FLAG, pSF-CMV-NEO-COOH-3×FLAG, pSF-CMV-PURO-NH2-GST-TEV, pSF-OXB20-COOH-TEV-FLAG®-6His (“6His” disclosed as SEQ ID NO: 2672), pCEP4 pDEST27, pSF-CMV-Ub-KrYFP, pSF-CMV-FMDV-daGFP, pEF1a-mCherry-N1 Vector, pEF1a-tdTomato Vector, pSF-CMV-FMDV-Hygro, pSF-CMV-PGK-Puro, pMCP-tag(m), and pSF-CMV-PURO-NH2-CMYC; bacterial expression vectors: pSF-OXB20-BetaGal,pSF-OXB20-Fluc, pSF-OXB20, and pSF-Tac; plant expression vectors: pRI 101-AN DNA and pCambia2301; and yeast expression vectors: pTYB21 and pKLAC2, and insect vectors: pAc5.1/V5-His A and pDEST8. In some instances, the vector is pcDNA3 or pcDNA3.1.

Described herein are nucleic acid libraries that are expressed in a vector to generate a construct comprising an antibody. In some instances, a size of the construct varies. In some instances, the construct comprises at least or about 500, 600, 700, 800, 900, 1000, 1100, 1300, 1400, 1500, 1600, 1700, 1800, 2000, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 6000, 7000, 8000, 9000, 10000, or more than 10000 bases. In some instances, a the construct comprises a range of about 300 to 1,000, 300 to 2,000, 300 to 3,000, 300 to 4,000, 300 to 5,000, 300 to 6,000, 300 to 7,000, 300 to 8,000, 300 to 9,000, 300 to 10,000, 1,000 to 2,000, 1,000 to 3,000, 1,000 to 4,000, 1,000 to 5,000, 1,000 to 6,000, 1,000 to 7,000, 1,000 to 8,000, 1,000 to 9,000, 1,000 to 10,000, 2,000 to 3,000, 2,000 to 4,000, 2,000 to 5,000, 2,000 to 6,000, 2,000 to 7,000, 2,000 to 8,000, 2,000 to 9,000, 2,000 to 10,000, 3,000 to 4,000, 3,000 to 5,000, 3,000 to 6,000, 3,000 to 7,000, 3,000 to 8,000, 3,000 to 9,000, 3,000 to 10,000, 4,000 to 5,000, 4,000 to 6,000, 4,000 to 7,000, 4,000 to 8,000, 4,000 to 9,000, 4,000 to 10,000, 5,000 to 6,000, 5,000 to 7,000, 5,000 to 8,000, 5,000 to 9,000, 5,000 to 10,000, 6,000 to 7,000, 6,000 to 8,000, 6,000 to 9,000, 6,000 to 10,000, 7,000 to 8,000, 7,000 to 9,000, 7,000 to 10,000, 8,000 to 9,000, 8,000 to 10,000, or 9,000 to 10,000 bases.

Provided herein are libraries comprising nucleic acids encoding for antibodies, wherein the nucleic acid libraries are expressed in a cell. In some instances, the libraries are synthesized to express a reporter gene. Exemplary reporter genes include, but are not limited to, acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucuronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), cerulean fluorescent protein, citrine fluorescent protein, orange fluorescent protein, cherry fluorescent protein, turquoise fluorescent protein, blue fluorescent protein, horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), luciferase, and derivatives thereof. Methods to determine modulation of a reporter gene are well known in the art, and include, but are not limited to, fluorometric methods (e.g. fluorescence spectroscopy, Fluorescence Activated Cell Sorting (FACS), fluorescence microscopy), and antibiotic resistance determination.

Epitopes Bound by Therapeutically Useful SARS-CoV-2 or ACE2 Antibodies

Described herein is a unique epitope of SARS-CoV-2 or ACE2. The epitope described herein consists of stretches of amino acids that are present in the SARS-CoV-2 S protein receptor binding domain (RBD). In some embodiments, this binding comprises weak (Van der Waals attraction), medium (hydrogen binding), strong (salt bridge) interactions, or combinations thereof. In certain embodiments, a contact residue is a residue on SARS-CoV-2 that forms a hydrogen bond with a residue on an anti-SARS-CoV-2 antibody. In certain embodiments, a contact residue is a residue on SARS-CoV-2 that forms a salt bridge with a residue on an anti-SARS-CoV-2 antibody. In certain embodiments, a contact residue is a residue on SARS-CoV-2 that results in a Van der Waals attraction with and is within at least 5, 4, or 3 angstroms of a residue on an anti-SARS-CoV-2 antibody.

In certain embodiments, described herein is an isolated antibody that binds any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty of the following residues: R102, N125, F157, S172, F175, L176, R190, Y265, I326, R328, K378, V382, S383, P384, K417, T430, N450, L452, F456, I468, I472, G476, F486, N487, Y489, F490, T531, N532, L533, F543, L552, S555, F559, or F562 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an isolated antibody that binds all of the following residues: R102, N125, F157, S172, F175, L176, R190, Y265, I326, R328, K378, V382, S383, P384, K417, T430, N450, L452, F456, I468, I472, G476, F486, N487, Y489, F490, T531, N532, L533, F543, L552, S555, F559, or F562 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an isolated antibody that binds all of the following residues: V382, S383, P384, or T430 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an isolated antibody that binds all of the following residues K378 or P384 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an isolated antibody that binds all of the following residues: R102, N125, F157, S172, F175, L176, R190, or Y265 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an isolated antibody that binds all of the following residues: K417, F456, G476, F486, N487, or Y489 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an isolated antibody that binds all of the following residues: I326, R328, T531, N532, L533, F543, L552, S555, F559, or F562 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an isolated antibody that binds all of the following residues: N450, 1472, or F490 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an isolated antibody that binds all of the following residues: L452, 1468, or F490 of SARS-CoV-2 S protein RBD. In certain embodiments, the antibody only binds residues that participate with the antibody in strong or medium interactions. In certain embodiments, the antibody only binds residues that participate with the antibody in strong interactions.

In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721-779-1202, 1344-1883, and 2381-2596 that binds any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty of the following residues: R102, N125, F157, S172, F175, L176, R190, Y265, I326, R328, K378, V382, S383, P384, K417, T430, N450, L452, F456, I468, I472, G476, F486, N487, Y489, F490, T531, N532, L533, F543, L552, S555, F559, or F562 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721-779-1202, 1344-1883, and 2381-2596 that binds any one, two, three, or four of the following residues: V382, S383, P384, or T430 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721-779-1202, 1344-1883, and 2381-2596 that binds any one or two of the following residues following residues K378 or P384 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721-779-1202, 1344-1883, and 2381-2596 that binds any one, two, three, four, five, six, seven, or eight of the following residues: R102, N125, F157, S172, F175, L176, R190, or Y265 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721-779-1202, 1344-1883, and 2381-2596 that binds any one, two, three, four, five, or six of the following residues: K417, F456, G476, F486, N487, or Y489 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721-779-1202, 1344-1883, and 2381-2596 that binds any one, two, three, four, five, six, seven, eight, nine, or ten of the following residues: I326, R328, T531, N532, L533, F543, L552, S555, F559, or F562 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721-779-1202, 1344-1883, and 2381-2596 that binds any one, two, or three of the following residues: N450, I472, or F490 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721-779-1202, 1344-1883, and 2381-2596 that binds any one, two, or three of the following residues: L452, I468, or F490 of SARS-CoV-2 S protein RBD. In certain embodiments, the antibody only binds residues that participate with the antibody in strong or medium interactions. In certain embodiments, the antibody only binds residues that participate with the antibody in strong interactions. In certain embodiments, the antibody only binds residues that participate with the antibody in strong or medium interactions. In certain embodiments, the antibody only binds residues that participate with the antibody in strong interactions.

In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721-779-1202, 1344-1883, and 2381-2596 that binds to all of the following residues: R102, N125, F157, S172, F175, L176, R190, Y265, I326, R328, K378, V382, S383, P384, K417, T430, N450, L452, F456, I468, I472, G476, F486, N487, Y489, F490, T531, N532, L533, F543, L552, S555, F559, or F562 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721-779-1202, 1344-1883, and 2381-2596 that binds all of the following residues: V382, S383, P384, or T430 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721-779-1202, 1344-1883, and 2381-2596 that binds all of the following residues K378 or P384 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721-779-1202, 1344-1883, and 2381-2596 that binds all of the following residues: R102, N125, F157, S172, F175, L176, R190, or Y265 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721-779-1202, 1344-1883, and 2381-2596 that binds all of the following residues: K417, F456, G476, F486, N487, or Y489 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721-779-1202, 1344-1883, and 2381-2596 that binds all of the following residues: I326, R328, T531, N532, L533, F543, L552, S555, F559, or F562 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721-779-1202, 1344-1883, and 2381-2596 that binds all of the following residues: N450, I472, or F490 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721-779-1202, 1344-1883, and 2381-2596 that binds all of the following residues: L452, I468, or F490 of SARS-CoV-2 S protein RBD. In certain embodiments, the antibody only binds residues that participate with the antibody in strong or medium interactions. In certain embodiments, the antibody only binds residues that participate with the antibody in strong interactions. In certain embodiments, the antibody only binds residues that participate with the antibody in strong or medium interactions. In certain embodiments, the antibody only binds residues that participate with the antibody in strong interactions.

In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence that differ from the amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721-779-1202, 1344-1883, and 2381-2596 by 1, 2, 3, 4, or 5 amino acids and that binds any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty of the following residues: R102, N125, F157, S172, F175, L176, R190, Y265, I326, R328, K378, V382, S383, P384, K417, T430, N450, L452, F456, I468, I472, G476, F486, N487, Y489, F490, T531, N532, L533, F543, L552, S555, F559, or F562 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence that differ from the amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721-779-1202, 1344-1883, and 2381-2596 by 1, 2, 3, 4, or 5 amino acids and that binds any one, two, three, or four of the following residues: V382, S383, P384, or T430 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence that differ from the amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721-779-1202, 1344-1883, and 2381-2596 by 1, 2, 3, 4, or 5 amino acids and that binds any one or two of the following residues following residues K378 or P384 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence that differ from the amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721-779-1202, 1344-1883, and 2381-2596 by 1, 2, 3, 4, or 5 amino acids and that binds any one, two, three, four, five, six, seven, or eight of the following residues: R102, N125, F157, S172, F175, L176, R190, or Y265 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence that differ from the amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721-779-1202, 1344-1883, and 2381-2596 by 1, 2, 3, 4, or 5 amino acids and that binds any one, two, three, four, five, or six of the following residues: K417, F456, G476, F486, N487, or Y489 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence that differ from the amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721-779-1202, 1344-1883, and 2381-2596 by 1, 2, 3, 4, or 5 amino acids and that binds any one, two, three, four, five, six, seven, eight, nine, or ten of the following residues: I326, R328, T531, N532, L533, F543, L552, S555, F559, or F562 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence that differ from the amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721-779-1202, 1344-1883, and 2381-2596 by 1, 2, 3, 4, or 5 amino acids and that binds any one, two, or three of the following residues: N450, I472, or F490 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence that differ from the amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721, 779-1202, 1344-1883, and 2381-2596 by 1, 2, 3, 4, or 5 amino acids and that binds any one, two, or three of the following residues: L452, I468, or F490 of SARS-CoV-2 S protein RBD. In certain embodiments, the antibody only binds residues that participate with the antibody in strong or medium interactions. In certain embodiments, the antibody only binds residues that participate with the antibody in strong interactions. In certain embodiments, the antibody only binds residues that participate with the antibody in strong or medium interactions. In certain embodiments, the antibody only binds residues that participate with the antibody in strong interactions.

In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence that differ from the amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721, 779-1202, 1344-1883, and 2381-2596 by 1, 2, 3, 4, or 5 amino acids and that binds to all of the following residues: R102, N125, F157, S172, F175, L176, R190, Y265, I326, R328, K378, V382, S383, P384, K417, T430, N450, L452, F456, I468, I472, G476, F486, N487, Y489, F490, T531, N532, L533, F543, L552, S555, F559, or F562 of SARS-CoV-2 S protein RBD. In In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence that differ from the amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721, 779-1202, 1344-1883, and 2381-2596 by 1, 2, 3, 4, or 5 amino acids and that binds all of the following residues: V382, S383, P384, or T430 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence that differ from the amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721, 779-1202, 1344-1883, and 2381-2596 by 1, 2, 3, 4, or 5 amino acids and that binds all of the following residues K378 or P384 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence that differ from the amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721, 779-1202, 1344-1883, and 2381-2596 by 1, 2, 3, 4, or 5 amino acids and that binds all of the following residues: R102, N125, F157, S172, F175, L176, R190, or Y265 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence that differ from the amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721, 779-1202, 1344-1883, and 2381-2596 by 1, 2, 3, 4, or 5 amino acids and that binds all of the following residues: K417, F456, G476, F486, N487, or Y489 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence that differ from the amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721, 779-1202, 1344-1883, and 2381-2596 by 1, 2, 3, 4, or 5 amino acids and that binds all of the following residues: I326, R328, T531, N532, L533, F543, L552, S555, F559, or F562 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence that differ from the amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721, 779-1202, 1344-1883, and 2381-2596 by 1, 2, 3, 4, or 5 amino acids and that binds all of the following residues: N450, I472, or F490 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein is an antibody comprising CDRs with an amino acid sequence that differ from the amino acid sequence set forth in any one of SEQ ID NOs: 1-492, 547-721, 779-1202, 1344-1883, and 2381-2596 by 1, 2, 3, 4, or 5 amino acids and that binds all of the following residues: L452, I468, or F490 of SARS-CoV-2 S protein RBD. In certain embodiments, the antibody only binds residues that participate with the antibody in strong or medium interactions. In certain embodiments, the antibody only binds residues that participate with the antibody in strong interactions. In certain embodiments, the antibody only binds residues that participate with the antibody in strong or medium interactions. In certain embodiments, the antibody only binds residues that participate with the antibody in strong interactions.

In certain embodiments, described herein, is an antibody that specifically binds SARS-CoV-2 comprising a variable heavy chain amino acid sequence at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 493-519 and 721-749; and a variable light chain amino acid sequence at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 520-546 and 750-778 and binds any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty of the following residues: R102, N125, F157, S172, F175, L176, R190, Y265, I326, R328, K378, V382, S383, P384, K417, T430, N450, L452, F456, I468, I472, G476, F486, N487, Y489, F490, T531, N532, L533, F543, L552, S555, F559, or F562 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein, is an antibody that specifically binds SARS-CoV-2 comprising a variable heavy chain amino acid sequence at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 493-519 and 721-749; and a variable light chain amino acid sequence at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 520-546 and 750-778 and binds all of the following residues: R102, N125, F157, S172, F175, L176, R190, Y265, I326, R328, K378, V382, S383, P384, K417, T430, N450, L452, F456, I468, I472, G476, F486, N487, Y489, F490, T531, N532, L533, F543, L552, S555, F559, or F562 of SARS-CoV-2 S protein RBD. In certain embodiments, the antibody only binds residues that that participate with the antibody in strong or medium interactions. In certain embodiments, the antibody only binds residues that that participate with the antibody in strong interactions.

In certain embodiments, described herein, is an antibody that specifically binds SARS-CoV-2 comprising a variable heavy chain amino acid sequence at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 1884-1951, 1951-2063, 2302-2368, 2369-2380, 2597-2607, and 2608-2668 and binds any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty of the following residues: R102, N125, F157, S172, F175, L176, R190, Y265, I326, R328, K378, V382, S383, P384, K417, T430, N450, L452, F456, I468, I472, G476, F486, N487, Y489, F490, T531, N532, L533, F543, L552, S555, F559, or F562 of SARS-CoV-2 S protein RBD. In certain embodiments, described herein, is an antibody that specifically binds SARS-CoV-2 comprising a variable heavy chain amino acid sequence at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 1884-1951, 1951-2063, 2302-2368, 2369-2380, 2597-2607, and 2608-2668 and binds all of the following residues: R102, N125, F157, S172, F175, L176, R190, Y265, I326, R328, K378, V382, S383, P384, K417, T430, N450, L452, F456, I468, I472, G476, F486, N487, Y489, F490, T531, N532, L533, F543, L552, S555, F559, or F562 of SARS-CoV-2 S protein RBD. In certain embodiments, the antibody only binds residues that that participate with the antibody in strong or medium interactions. In certain embodiments, the antibody only binds residues that that participate with the antibody in strong interactions.

Diseases and Disorders

Provided herein are SARS-CoV-2 or ACE2 binding libraries comprising nucleic acids encoding for antibodies comprising SARS-CoV-2 or ACE2 binding domains may have therapeutic effects. In some instances, the SARS-CoV-2 or ACE2 binding libraries result in protein when translated that is used to treat a disease or disorder. In some instances, the protein is an immunoglobulin. In some instances, the protein is a peptidomimetic. In some instances, the disease or disorder is a viral infection caused by SARS-CoV-2. In some instances, the disease or disorder is a respiratory disease or disorder caused by SARS-CoV-2.

SARS-CoV-2 or ACE2 variant antibody libraries as described herein may be used to treat SARS-CoV-2. In some embodiments, the SARS-CoV-2 or ACE2 variant antibody libraries are used to treat or prevent symptoms of SARS-CoV-2. These symptoms include, but are not limited to, fever, chills, cough, fatigue, headaches, loss of taste, loss of smell, nausea, vomiting, muscle weakness, sleep difficulties, anxiety, and depression. In some embodiments, the SARS-CoV-2 or ACE2 variant antibody libraries are used to treat a subject who has symptoms for an extended period of time. In some embodiments, the subject has symptoms for an extended period of time after testing negative for SARS-CoV-2. In some embodiments, the subject has symptoms for an extended period of time including at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, or more than 1 year.

In some instances, the subject is a mammal. In some instances, the subject is a mouse, rabbit, dog, or human. Subjects treated by methods described herein may be infants, adults, or children. Pharmaceutical compositions comprising antibodies or antibody fragments as described herein may be administered intravenously or subcutaneously. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a CDRH1 sequence of any one of SEQ ID NOs: 1-50, 779-919, 1344-1523, and 2381-2452. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a CDRH2 sequence of any one of SEQ ID NOs: 51-100, 920-1061, 1524-1703, and 2453-2524 In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a CDRH3 sequence of any one of SEQ ID NOs: 101-150, 1062-1202, 1704-1883, and 2525-2596. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a variable domain, heavy chain region (VH) and a variable domain, light chain region (VL), wherein VH comprises complementarity determining regions CDRH1, CDRH2, and CDRH3, wherein VL comprises complementarity determining regions CDRL1, CDRL2, and CDRL3, and wherein (a) an amino acid sequence of CDRH1 is as set forth in any one of SEQ ID NOs: 151-165, 241-255, 331-357, and 547-575; (b) an amino acid sequence of CDRH2 is as set forth in any one of SEQ ID NOs: 166-180, 256-270, 358-384, and 576-604; (c) an amino acid sequence of CDRH3 is as set forth in any one of SEQ ID NOs: 181-195, 271-285, 385-411, and 605-633; (d) an amino acid sequence of CDRL1 is as set forth in any one of SEQ ID NOs: 196-210, 286-300, 412-438, and 634-662; (e) an amino acid sequence of CDRL2 is as set forth in any one of SEQ ID NOs: 211-225, 301-315, 439-465, and 663-691; and (f) an amino acid sequence of CDRL3 is as set forth in any one of SEQ ID NOs: 226-240, 316-330, 466-492, and 692-720. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a VH comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 493-519 and 721-749, and VL comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 520-546 and 750-778. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a heavy chain variable domain comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 1918-2058, 2599-2778, and 3095-3173.

SARS-CoV-2 or ACE2 antibodies as described herein may confer immunity after exposure to SARS-CoV-2 or ACE2 antibodies. In some embodiments, the SARS-CoV-2 or ACE2 antibodies described herein are used for passive immunization of a subject. In some instances, the subject is actively immunized after exposure to SARS-CoV-2 or ACE2 antibodies followed by exposure to SARS-CoV-2. In some embodiments, SARS-CoV-2 or ACE2 antibodies are derived from a subject who has recovered from SARS-CoV-2.

In some embodiments, the immunity occurs at least about 30 minutes, 1 hour, 5 hours, 10 hours, 16 hours, 20 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, or more than 2 weeks after exposure to SARS-CoV-2 or ACE2 antibodies. In some instances, the immunity lasts for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, or more than 5 years after exposure to SARS-CoV-2 or ACE2 antibodies.

In some embodiments, the subject receives the SARS-CoV-2 or ACE2 antibodies prior to exposure to SARS-CoV-2. In some embodiments, the subject receives the SARS-CoV-2 or ACE2 antibodies at least about 30 minutes, 1 hour, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, or more than 5 years prior to exposure to SARS-CoV-2. In some embodiments, the subject receives the SARS-CoV-2 or ACE2 antibodies after exposure to SARS-CoV-2. In some embodiments, the subject receives the SARS-CoV-2 or ACE2 antibodies at least about 30 minutes, 1 hour, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, or more than 5 years after exposure to SARS-CoV-2.

SARS-CoV-2 or ACE2 antibodies described herein may be administered through various routes. The administration may, depending on the composition being administered, for example, be oral, pulmonary, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal.

Described herein are compositions or pharmaceutical compositions comprising SARS-CoV-2 or ACE2 antibodies or antibody fragment thereof that comprise various dosages of the antibodies or antibody fragment. In some instances, the dosage is ranging from about 1 to 25 mg/kg, from about 1 to 50 mg/kg, from about 1 to 80 mg/kg, from about 1 to about 100 mg/kg, from about 5 to about 100 mg/kg, from about 5 to about 80 mg/kg, from about 5 to about 60 mg/kg, from about 5 to about 50 mg/kg or from about 5 to about 500 mg/kg which can be administered in single or multiple doses. In some instances, the dosage is administered in an amount of about 0.01 mg/kg, about 0.05 mg/kg, about 0.10 mg/kg, about 0.25 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg, about 105 mg/kg, about 110 mg/kg, about 115 mg/kg, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, about 200, about 205, about 210, about 215, about 220, about 225, about 230, about 240, about 250, about 260, about 270, about 275, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360 mg/kg, about 370 mg/kg, about 380 mg/kg, about 390 mg/kg, about 400 mg/kg, 410 mg/kg, about 420 mg/kg, about 430 mg/kg, about 440 mg/kg, about 450 mg/kg, about 460 mg/kg, about 470 mg/kg, about 480 mg/kg, about 490 mg/kg, or about 500 mg/kg.

SARS-CoV-2 or ACE2 antibodies or antibody fragment thereof described herein, in some embodiments, improve disease severity. In some embodiments, the SARS-CoV-2 or ACE2 antibodies or antibody fragment thereof improve disease severity at a dose level of about 0.01 mg/kg, about 0.05 mg/kg, about 0.10 mg/kg, about 0.25 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, or about 20 mg/kg. In some embodiments, the SARS-CoV-2 or ACE2 antibodies or antibody fragment thereof improve disease severity at a dose level of about 1 mg/kg, about 5 mg/kg, or about 10 mg/kg. In some embodiments, disease severity is determined by percent weight loss. In some embodiments, the SARS-CoV-2 or ACE2 antibodies or antibody fragment thereof protects against weight loss at a dose level of about 0.01 mg/kg, about 0.05 mg/kg, about 0.10 mg/kg, about 0.25 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, or about 20 mg/kg. In some embodiments, the SARS-CoV-2 or ACE2 antibodies or antibody fragment thereof protects against weight loss at a dose level of about 1 mg/kg, about 5 mg/kg, or about 10 mg/kg. In some embodiments, SARS-CoV-2 or ACE2 antibodies or antibody fragment thereof

Variant Libraries

Codon Variation

Variant nucleic acid libraries described herein may comprise a plurality of nucleic acids, wherein each nucleic acid encodes for a variant codon sequence compared to a reference nucleic acid sequence. In some instances, each nucleic acid of a first nucleic acid population contains a variant at a single variant site. In some instances, the first nucleic acid population contains a plurality of variants at a single variant site such that the first nucleic acid population contains more than one variant at the same variant site. The first nucleic acid population may comprise nucleic acids collectively encoding multiple codon variants at the same variant site. The first nucleic acid population may comprise nucleic acids collectively encoding up to 19 or more codons at the same position. The first nucleic acid population may comprise nucleic acids collectively encoding up to 60 variant triplets at the same position, or the first nucleic acid population may comprise nucleic acids collectively encoding up to 61 different triplets of codons at the same position. Each variant may encode for a codon that results in a different amino acid during translation. Table 1 provides a listing of each codon possible (and the representative amino acid) for a variant site.

TABLE 1
List of codons and amino acids
	One	Three
	letter	letter
Amino Acids	code	code	Codons
Alanine	A	Ala	GCA GCC GCG GCT
Cysteine	C	Cys	TGC TGT
Aspartic acid	D	Asp	GAC GAT
Glutamic acid	E	Glu	GAA GAG
Phenylalanine	F	Phe	TTC TTT
Glycine	G	Gly	GGA GGC GGG GGT
Histidine	H	His	CAC CAT
Isoleucine	I	Iso	ATA ATC ATT
Lysine	K	Lys	AAA AAG
Leucine	L	Leu	TTA TTG CTA CTC CTG
			CTT
Methionine	M	Met	ATG
Asparagine	N	Asn	AAC AAT
Proline	P	Pro	CCA CCC CCG CCT
Glutamine	Q	Gln	CAA CAG
Arginine	R	Arg	AGA AGG CGA CGC CGG
			CGT
Serine	S	Ser	AGC AGT TCA TCC TCG
			TCT
Threonine	T	Thr	ACA ACC ACG ACT
Valine	V	Val	GTA GTC GTG GTT
Tryptophan	W	Trp	TGG
Tyrosine	Y	Tyr	TAC TAT

A nucleic acid population may comprise varied nucleic acids collectively encoding up to 20 codon variations at multiple positions. In such cases, each nucleic acid in the population comprises variation for codons at more than one position in the same nucleic acid. In some instances, each nucleic acid in the population comprises variation for codons at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more codons in a single nucleic acid. In some instances, each variant long nucleic acid comprises variation for codons at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more codons in a single long nucleic acid. In some instances, the variant nucleic acid population comprises variation for codons at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more codons in a single nucleic acid. In some instances, the variant nucleic acid population comprises variation for codons in at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more codons in a single long nucleic acid.

Highly Parallel Nucleic Acid Synthesis

Provided herein is a platform approach utilizing miniaturization, parallelization, and vertical integration of the end-to-end process from polynucleotide synthesis to gene assembly within nanowells on silicon to create a revolutionary synthesis platform. Devices described herein provide, with the same footprint as a 96-well plate, a silicon synthesis platform is capable of increasing throughput by a factor of up to 1,000 or more compared to traditional synthesis methods, with production of up to approximately 1,000,000 or more polynucleotides, or 10,000 or more genes in a single highly-parallelized run.

With the advent of next-generation sequencing, high resolution genomic data has become an important factor for studies that delve into the biological roles of various genes in both normal biology and disease pathogenesis. At the core of this research is the central dogma of molecular biology and the concept of “residue-by-residue transfer of sequential information.” Genomic information encoded in the DNA is transcribed into a message that is then translated into the protein that is the active product within a given biological pathway.

Another exciting area of study is on the discovery, development and manufacturing of therapeutic molecules focused on a highly-specific cellular target. High diversity DNA sequence libraries are at the core of development pipelines for targeted therapeutics. Gene variants are used to express proteins in a design, build, and test protein engineering cycle that ideally culminates in an optimized gene for high expression of a protein with high affinity for its therapeutic target. As an example, consider the binding pocket of a receptor. The ability to test all sequence permutations of all residues within the binding pocket simultaneously will allow for a thorough exploration, increasing chances of success. Saturation mutagenesis, in which a researcher attempts to generate all possible mutations or variants at a specific site within the receptor, represents one approach to this development challenge. Though costly and time and labor-intensive, it enables each variant to be introduced into each position. In contrast, combinatorial mutagenesis, where a few selected positions or short stretch of DNA may be modified extensively, generates an incomplete repertoire of variants with biased representation.

To accelerate the drug development pipeline, a library with the desired variants available at the intended frequency in the right position available for testing—in other words, a precision library, enables reduced costs as well as turnaround time for screening. Provided herein are methods for synthesizing nucleic acid synthetic variant libraries which provide for precise introduction of each intended variant at the desired frequency. To the end user, this translates to the ability to not only thoroughly sample sequence space but also be able to query these hypotheses in an efficient manner, reducing cost and screening time. Genome-wide editing can elucidate important pathways, libraries where each variant and sequence permutation can be tested for optimal functionality, and thousands of genes can be used to reconstruct entire pathways and genomes to re-engineer biological systems for drug discovery.

In a first example, a drug itself can be optimized using methods described herein. For example, to improve a specified function of an antibody, a variant polynucleotide library encoding for a portion of the antibody is designed and synthesized. A variant nucleic acid library for the antibody can then be generated by processes described herein (e.g., PCR mutagenesis followed by insertion into a vector). The antibody is then expressed in a production cell line and screened for enhanced activity. Example screens include examining modulation in binding affinity to an antigen, stability, or effector function (e.g., ADCC, complement, or apoptosis). Exemplary regions to optimize the antibody include, without limitation, the Fc region, Fab region, variable region of the Fab region, constant region of the Fab region, variable domain of the heavy chain or light chain (V_H or V_L), and specific complementarity-determining regions (CDRs) of V_H or V_L.

Nucleic acid libraries synthesized by methods described herein may be expressed in various cells associated with a disease state. Cells associated with a disease state include cell lines, tissue samples, primary cells from a subject, cultured cells expanded from a subject, or cells in a model system. Exemplary model systems include, without limitation, plant and animal models of a disease state.

To identify a variant molecule associated with prevention, reduction or treatment of a disease state, a variant nucleic acid library described herein is expressed in a cell associated with a disease state, or one in which a cell a disease state can be induced. In some instances, an agent is used to induce a disease state in cells. Exemplary tools for disease state induction include, without limitation, a Cre/Lox recombination system, LPS inflammation induction, and streptozotocin to induce hypoglycemia. The cells associated with a disease state may be cells from a model system or cultured cells, as well as cells from a subject having a particular disease condition. Exemplary disease conditions include a bacterial, fungal, viral, autoimmune, or proliferative disorder (e.g., cancer). In some instances, the variant nucleic acid library is expressed in the model system, cell line, or primary cells derived from a subject, and screened for changes in at least one cellular activity. Exemplary cellular activities include, without limitation, proliferation, cycle progression, cell death, adhesion, migration, reproduction, cell signaling, energy production, oxygen utilization, metabolic activity, and aging, response to free radical damage, or any combination thereof

Substrates

Devices used as a surface for polynucleotide synthesis may be in the form of substrates which include, without limitation, homogenous array surfaces, patterned array surfaces, channels, beads, gels, and the like. Provided herein are substrates comprising a plurality of clusters, wherein each cluster comprises a plurality of loci that support the attachment and synthesis of polynucleotides. In some instances, substrates comprise a homogenous array surface. For example, the homogenous array surface is a homogenous plate. The term “locus” as used herein refers to a discrete region on a structure which provides support for polynucleotides encoding for a single predetermined sequence to extend from the surface. In some instances, a locus is on a two-dimensional surface, e.g., a substantially planar surface. In some instances, a locus is on a three-dimensional surface, e.g., a well, microwell, channel, or post. In some instances, a surface of a locus comprises a material that is actively functionalized to attach to at least one nucleotide for polynucleotide synthesis, or preferably, a population of identical nucleotides for synthesis of a population of polynucleotides. In some instances, polynucleotide refers to a population of polynucleotides encoding for the same nucleic acid sequence. In some cases, a surface of a substrate is inclusive of one or a plurality of surfaces of a substrate. The average error rates for polynucleotides synthesized within a library described here using the systems and methods provided are often less than 1 in 1000, less than about 1 in 2000, less than about 1 in 3000 or less often without error correction.

Provided herein are surfaces that support the parallel synthesis of a plurality of polynucleotides having different predetermined sequences at addressable locations on a common support. In some instances, a substrate provides support for the synthesis of more than 50, 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2,000; 5,000; 10,000; 20,000; 50,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; 10,000,000 or more non-identical polynucleotides. In some cases, the surfaces provide support for the synthesis of more than 50, 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2,000; 5,000; 10,000; 20,000; 50,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; 10,000,000 or more polynucleotides encoding for distinct sequences. In some instances, at least a portion of the polynucleotides have an identical sequence or are configured to be synthesized with an identical sequence. In some instances, the substrate provides a surface environment for the growth of polynucleotides having at least 80, 90, 100, 120, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 bases or more.

Provided herein are methods for polynucleotide synthesis on distinct loci of a substrate, wherein each locus supports the synthesis of a population of polynucleotides. In some cases, each locus supports the synthesis of a population of polynucleotides having a different sequence than a population of polynucleotides grown on another locus. In some instances, each polynucleotide sequence is synthesized with 1, 2, 3, 4, 5, 6, 7, 8, 9 or more redundancy across different loci within the same cluster of loci on a surface for polynucleotide synthesis. In some instances, the loci of a substrate are located within a plurality of clusters. In some instances, a substrate comprises at least 10, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 20000, 30000, 40000, 50000 or more clusters. In some instances, a substrate comprises more than 2,000; 5,000; 10,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,100,000; 1,200,000; 1,300,000; 1,400,000; 1,500,000; 1,600,000; 1,700,000; 1,800,000; 1,900,000; 2,000,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; or 10,000,000 or more distinct loci. In some instances, a substrate comprises about 10,000 distinct loci. The number of loci within a single cluster is varied in different instances. In some cases, each cluster includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 150, 200, 300, 400, 500 or more loci. In some instances, each cluster includes about 50-500 loci. In some instances, each cluster includes about 100-200 loci. In some instances, each cluster includes about 100-150 loci. In some instances, each cluster includes about 109, 121, 130 or 137 loci. In some instances, each cluster includes about 19, 20, 61, 64 or more loci. Alternatively or in combination, polynucleotide synthesis occurs on a homogenous array surface.

In some instances, the number of distinct polynucleotides synthesized on a substrate is dependent on the number of distinct loci available in the substrate. In some instances, the density of loci within a cluster or surface of a substrate is at least or about 1, 10, 25, 50, 65, 75, 100, 130, 150, 175, 200, 300, 400, 500, 1,000 or more loci per mm². In some cases, a substrate comprises 10-500, 25-400, 50-500, 100-500, 150-500, 10-250, 50-250, 10-200, or 50-200 mm². In some instances, the distance between the centers of two adjacent loci within a cluster or surface is from about 10-500, from about 10-200, or from about 10-100 um. In some instances, the distance between two centers of adjacent loci is greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 um. In some instances, the distance between the centers of two adjacent loci is less than about 200, 150, 100, 80, 70, 60, 50, 40, 30, 20 or 10 um. In some instances, each locus has a width of about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 um. In some cases, each locus has a width of about 0.5-100, 0.5-50, 10-75, or 0.5-50 um.

In some instances, the density of clusters within a substrate is at least or about 1 cluster per 100 mm², 1 cluster per 10 mm², 1 cluster per 5 mm², 1 cluster per 4 mm², 1 cluster per 3 mm², 1 cluster per 2 mm², 1 cluster per 1 mm², 2 clusters per 1 mm², 3 clusters per 1 mm², 4 clusters per 1 mm², 5 clusters per 1 mm², 10 clusters per 1 mm², 50 clusters per 1 mm² or more. In some instances, a substrate comprises from about 1 cluster per 10 mm² to about 10 clusters per 1 mm². In some instances, the distance between the centers of two adjacent clusters is at least or about 50, 100, 200, 500, 1000, 2000, or 5000 um. In some cases, the distance between the centers of two adjacent clusters is between about 50-100, 50-200, 50-300, 50-500, and 100-2000 um. In some cases, the distance between the centers of two adjacent clusters is between about 0.05-50, 0.05-10, 0.05-5, 0.05-4, 0.05-3, 0.05-2, 0.1-10, 0.2-10, 0.3-10, 0.4-10, 0.5-10, 0.5-5, or 0.5-2 mm. In some cases, each cluster has a cross section of about 0.5 to about 2, about 0.5 to about 1, or about 1 to about 2 mm. In some cases, each cluster has a cross section of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 mm. In some cases, each cluster has an interior cross section of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.15, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 mm.

In some instances, a substrate is about the size of a standard 96 well plate, for example between about 100 and about 200 mm by between about 50 and about 150 mm. In some instances, a substrate has a diameter less than or equal to about 1000, 500, 450, 400, 300, 250, 200, 150, 100 or 50 mm. In some instances, the diameter of a substrate is between about 25-1000, 25-800, 25-600, 25-500, 25-400, 25-300, or 25-200 mm. In some instances, a substrate has a planar surface area of at least about 100; 200; 500; 1,000; 2,000; 5,000; 10,000; 12,000; 15,000; 20,000; 30,000; 40,000; 50,000 mm² or more. In some instances, the thickness of a substrate is between about 50-2000, 50-1000, 100-1000, 200-1000, or 250-1000 mm.

Surface Materials

Substrates, devices, and reactors provided herein are fabricated from any variety of materials suitable for the methods, compositions, and systems described herein. In certain instances, substrate materials are fabricated to exhibit a low level of nucleotide binding. In some instances, substrate materials are modified to generate distinct surfaces that exhibit a high level of nucleotide binding. In some instances, substrate materials are transparent to visible and/or UV light. In some instances, substrate materials are sufficiently conductive, e.g., are able to form uniform electric fields across all or a portion of a substrate. In some instances, conductive materials are connected to an electric ground. In some instances, the substrate is heat conductive or insulated. In some instances, the materials are chemical resistant and heat resistant to support chemical or biochemical reactions, for example polynucleotide synthesis reaction processes. In some instances, a substrate comprises flexible materials. For flexible materials, materials can include, without limitation: nylon, both modified and unmodified, nitrocellulose, polypropylene, and the like. In some instances, a substrate comprises rigid materials. For rigid materials, materials can include, without limitation: glass; fuse silica; silicon, plastics (for example polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, and blends thereof, and the like); metals (for example, gold, platinum, and the like). The substrate, solid support or reactors can be fabricated from a material selected from the group consisting of silicon, polystyrene, agarose, dextran, cellulosic polymers, polyacrylamides, polydimethylsiloxane (PDMS), and glass. The substrates/solid supports or the microstructures, reactors therein may be manufactured with a combination of materials listed herein or any other suitable material known in the art.

Surface Architecture

Provided herein are substrates for the methods, compositions, and systems described herein, wherein the substrates have a surface architecture suitable for the methods, compositions, and systems described herein. In some instances, a substrate comprises raised and/or lowered features. One benefit of having such features is an increase in surface area to support polynucleotide synthesis. In some instances, a substrate having raised and/or lowered features is referred to as a three-dimensional substrate. In some cases, a three-dimensional substrate comprises one or more channels. In some cases, one or more loci comprise a channel. In some cases, the channels are accessible to reagent deposition via a deposition device such as a material deposition device. In some cases, reagents and/or fluids collect in a larger well in fluid communication one or more channels. For example, a substrate comprises a plurality of channels corresponding to a plurality of loci with a cluster, and the plurality of channels are in fluid communication with one well of the cluster. In some methods, a library of polynucleotides is synthesized in a plurality of loci of a cluster.

Provided herein are substrates for the methods, compositions, and systems described herein, wherein the substrates are configured for polynucleotide synthesis. In some instances, the structure is configured to allow for controlled flow and mass transfer paths for polynucleotide synthesis on a surface. In some instances, the configuration of a substrate allows for the controlled and even distribution of mass transfer paths, chemical exposure times, and/or wash efficacy during polynucleotide synthesis. In some instances, the configuration of a substrate allows for increased sweep efficiency, for example by providing sufficient volume for a growing polynucleotide such that the excluded volume by the growing polynucleotide does not take up more than 50, 45, 40, 35, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1%, or less of the initially available volume that is available or suitable for growing the polynucleotide. In some instances, a three-dimensional structure allows for managed flow of fluid to allow for the rapid exchange of chemical exposure.

Provided herein are substrates for the methods, compositions, and systems described herein, wherein the substrates comprise structures suitable for the methods, compositions, and systems described herein. In some instances, segregation is achieved by physical structure. In some instances, segregation is achieved by differential functionalization of the surface generating active and passive regions for polynucleotide synthesis. In some instances, differential functionalization is achieved by alternating the hydrophobicity across the substrate surface, thereby creating water contact angle effects that cause beading or wetting of the deposited reagents. Employing larger structures can decrease splashing and cross-contamination of distinct polynucleotide synthesis locations with reagents of the neighboring spots. In some cases, a device, such as a material deposition device, is used to deposit reagents to distinct polynucleotide synthesis locations. Substrates having three-dimensional features are configured in a manner that allows for the synthesis of a large number of polynucleotides (e.g., more than about 10,000) with a low error rate (e.g., less than about 1:500, 1:1000, 1:1500, 1:2,000, 1:3,000, 1:5,000, or 1:10,000). In some cases, a substrate comprises features with a density of about or greater than about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400 or 500 features per mm².

A well of a substrate may have the same or different width, height, and/or volume as another well of the substrate. A channel of a substrate may have the same or different width, height, and/or volume as another channel of the substrate. In some instances, the diameter of a cluster or the diameter of a well comprising a cluster, or both, is between about 0.05-50, 0.05-10, 0.05-5, 0.05-4, 0.05-3, 0.05-2, 0.05-1, 0.05-0.5, 0.05-0.1, 0.1-10, 0.2-10, 0.3-10, 0.4-10, 0.5-10, 0.5-5, or 0.5-2 mm. In some instances, the diameter of a cluster or well or both is less than or about 5, 4, 3, 2, 1, 0.5, 0.1, 0.09, 0.08, 0.07, 0.06, or 0.05 mm. In some instances, the diameter of a cluster or well or both is between about 1.0 and 1.3 mm. In some instances, the diameter of a cluster or well, or both is about 1.150 mm. In some instances, the diameter of a cluster or well, or both is about 0.08 mm. The diameter of a cluster refers to clusters within a two-dimensional or three-dimensional substrate.

In some instances, the height of a well is from about 20-1000, 50-1000, 100-1000, 200-1000, 300-1000, 400-1000, or 500-1000 um. In some cases, the height of a well is less than about 1000, 900, 800, 700, or 600 um.

In some instances, a substrate comprises a plurality of channels corresponding to a plurality of loci within a cluster, wherein the height or depth of a channel is 5-500, 5-400, 5-300, 5-200, 5-100, 5-50, or 10-50 um. In some cases, the height of a channel is less than 100, 80, 60, 40, or 20 um.

In some instances, the diameter of a channel, locus (e.g., in a substantially planar substrate) or both channel and locus (e.g., in a three-dimensional substrate wherein a locus corresponds to a channel) is from about 1-1000, 1-500, 1-200, 1-100, 5-100, or 10-100 um, for example, about 90, 80, 70, 60, 50, 40, 30, 20 or 10 um. In some instances, the diameter of a channel, locus, or both channel and locus is less than about 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 um. In some instances, the distance between the center of two adjacent channels, loci, or channels and loci is from about 1-500, 1-200, 1-100, 5-200, 5-100, 5-50, or 5-30, for example, about 20 um.

Surface Modifications

Provided herein are methods for polynucleotide synthesis on a surface, wherein the surface comprises various surface modifications. In some instances, the surface modifications are employed for the chemical and/or physical alteration of a surface by an additive or subtractive process to change one or more chemical and/or physical properties of a substrate surface or a selected site or region of a substrate surface. For example, surface modifications include, without limitation, (1) changing the wetting properties of a surface, (2) functionalizing a surface, i.e., providing, modifying or substituting surface functional groups, (3) defunctionalizing a surface, i.e., removing surface functional groups, (4) otherwise altering the chemical composition of a surface, e.g., through etching, (5) increasing or decreasing surface roughness, (6) providing a coating on a surface, e.g., a coating that exhibits wetting properties that are different from the wetting properties of the surface, and/or (7) depositing particulates on a surface.

In some cases, the addition of a chemical layer on top of a surface (referred to as adhesion promoter) facilitates structured patterning of loci on a surface of a substrate. Exemplary surfaces for application of adhesion promotion include, without limitation, glass, silicon, silicon dioxide and silicon nitride. In some cases, the adhesion promoter is a chemical with a high surface energy. In some instances, a second chemical layer is deposited on a surface of a substrate. In some cases, the second chemical layer has a low surface energy. In some cases, surface energy of a chemical layer coated on a surface supports localization of droplets on the surface. Depending on the patterning arrangement selected, the proximity of loci and/or area of fluid contact at the loci are alterable.

In some instances, a substrate surface, or resolved loci, onto which nucleic acids or other moieties are deposited, e.g., for polynucleotide synthesis, are smooth or substantially planar (e.g., two-dimensional) or have irregularities, such as raised or lowered features (e.g., three-dimensional features). In some instances, a substrate surface is modified with one or more different layers of compounds. Such modification layers of interest include, without limitation, inorganic and organic layers such as metals, metal oxides, polymers, small organic molecules and the like.

In some instances, resolved loci of a substrate are functionalized with one or more moieties that increase and/or decrease surface energy. In some cases, a moiety is chemically inert. In some cases, a moiety is configured to support a desired chemical reaction, for example, one or more processes in a polynucleotide synthesis reaction. The surface energy, or hydrophobicity, of a surface is a factor for determining the affinity of a nucleotide to attach onto the surface. In some instances, a method for substrate functionalization comprises: (a) providing a substrate having a surface that comprises silicon dioxide; and (b) silanizing the surface using, a suitable silanizing agent described herein or otherwise known in the art, for example, an organofunctional alkoxysilane molecule. Methods and functionalizing agents are described in U.S. Pat. No. 5,474,796, which is herein incorporated by reference in its entirety.

In some instances, a substrate surface is functionalized by contact with a derivatizing composition that contains a mixture of silanes, under reaction conditions effective to couple the silanes to the substrate surface, typically via reactive hydrophilic moieties present on the substrate surface. Silanization generally covers a surface through self-assembly with organofunctional alkoxysilane molecules. A variety of siloxane functionalizing reagents can further be used as currently known in the art, e.g., for lowering or increasing surface energy. The organofunctional alkoxysilanes are classified according to their organic functions.

Polynucleotide Synthesis

Methods of the current disclosure for polynucleotide synthesis may include processes involving phosphoramidite chemistry. In some instances, polynucleotide synthesis comprises coupling a base with phosphoramidite. Polynucleotide synthesis may comprise coupling a base by deposition of phosphoramidite under coupling conditions, wherein the same base is optionally deposited with phosphoramidite more than once, i.e., double coupling. Polynucleotide synthesis may comprise capping of unreacted sites. In some instances, capping is optional. Polynucleotide synthesis may also comprise oxidation or an oxidation step or oxidation steps. Polynucleotide synthesis may comprise deblocking, detritylation, and sulfurization. In some instances, polynucleotide synthesis comprises either oxidation or sulfurization. In some instances, between one or each step during a polynucleotide synthesis reaction, the device is washed, for example, using tetrazole or acetonitrile. Time frames for any one step in a phosphoramidite synthesis method may be less than about 2 min, 1 min, 50 sec, 40 sec, 30 sec, 20 sec and 10 sec.

Polynucleotide synthesis using a phosphoramidite method may comprise a subsequent addition of a phosphoramidite building block (e.g., nucleoside phosphoramidite) to a growing polynucleotide chain for the formation of a phosphite triester linkage. Phosphoramidite polynucleotide synthesis proceeds in the 3′ to 5′ direction. Phosphoramidite polynucleotide synthesis allows for the controlled addition of one nucleotide to a growing nucleic acid chain per synthesis cycle. In some instances, each synthesis cycle comprises a coupling step. Phosphoramidite coupling involves the formation of a phosphite triester linkage between an activated nucleoside phosphoramidite and a nucleoside bound to the substrate, for example, via a linker. In some instances, the nucleoside phosphoramidite is provided to the device activated. In some instances, the nucleoside phosphoramidite is provided to the device with an activator. In some instances, nucleoside phosphoramidites are provided to the device in a 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100-fold excess or more over the substrate-bound nucleosides. In some instances, the addition of nucleoside phosphoramidite is performed in an anhydrous environment, for example, in anhydrous acetonitrile. Following addition of a nucleoside phosphoramidite, the device is optionally washed. In some instances, the coupling step is repeated one or more additional times, optionally with a wash step between nucleoside phosphoramidite additions to the substrate. In some instances, a polynucleotide synthesis method used herein comprises 1, 2, 3 or more sequential coupling steps. Prior to coupling, in many cases, the nucleoside bound to the device is de-protected by removal of a protecting group, where the protecting group functions to prevent polymerization. A common protecting group is 4,4′-dimethoxytrityl (DMT).

Following coupling, phosphoramidite polynucleotide synthesis methods optionally comprise a capping step. In a capping step, the growing polynucleotide is treated with a capping agent. A capping step is useful to block unreacted substrate-bound 5′-OH groups after coupling from further chain elongation, preventing the formation of polynucleotides with internal base deletions. Further, phosphoramidites activated with 1H-tetrazole may react, to a small extent, with the O6 position of guanosine. Without being bound by theory, upon oxidation with I₂/water, this side product, possibly via O6-N7 migration, may undergo depurination. The apurinic sites may end up being cleaved in the course of the final deprotection of the polynucleotide thus reducing the yield of the full-length product. The O6 modifications may be removed by treatment with the capping reagent prior to oxidation with I₂/water. In some instances, inclusion of a capping step during polynucleotide synthesis decreases the error rate as compared to synthesis without capping. As an example, the capping step comprises treating the substrate-bound polynucleotide with a mixture of acetic anhydride and 1-methylimidazole. Following a capping step, the device is optionally washed.

In some instances, following addition of a nucleoside phosphoramidite, and optionally after capping and one or more wash steps, the device bound growing nucleic acid is oxidized. The oxidation step comprises the phosphite triester is oxidized into a tetracoordinated phosphate triester, a protected precursor of the naturally occurring phosphate diester internucleoside linkage. In some instances, oxidation of the growing polynucleotide is achieved by treatment with iodine and water, optionally in the presence of a weak base (e.g., pyridine, lutidine, collidine). Oxidation may be carried out under anhydrous conditions using, e.g. tert-Butyl hydroperoxide or (1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO). In some methods, a capping step is performed following oxidation. A second capping step allows for device drying, as residual water from oxidation that may persist can inhibit subsequent coupling. Following oxidation, the device and growing polynucleotide is optionally washed. In some instances, the step of oxidation is substituted with a sulfurization step to obtain polynucleotide phosphorothioates, wherein any capping steps can be performed after the sulfurization. Many reagents are capable of the efficient sulfur transfer, including but not limited to 3-(Dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-3-thione, DDTT, 3H-1,2-benzodithiol-3-one 1,1-dioxide, also known as Beaucage reagent, and N,N,N′N′-Tetraethylthiuram disulfide (TETD).

In order for a subsequent cycle of nucleoside incorporation to occur through coupling, the protected 5′ end of the device bound growing polynucleotide is removed so that the primary hydroxyl group is reactive with a next nucleoside phosphoramidite. In some instances, the protecting group is DMT and deblocking occurs with trichloroacetic acid in dichloromethane. Conducting detritylation for an extended time or with stronger than recommended solutions of acids may lead to increased depurination of solid support-bound polynucleotide and thus reduces the yield of the desired full-length product. Methods and compositions of the disclosure described herein provide for controlled deblocking conditions limiting undesired depurination reactions. In some instances, the device bound polynucleotide is washed after deblocking. In some instances, efficient washing after deblocking contributes to synthesized polynucleotides having a low error rate.

Methods for the synthesis of polynucleotides typically involve an iterating sequence of the following steps: application of a protected monomer to an actively functionalized surface (e.g., locus) to link with either the activated surface, a linker or with a previously deprotected monomer; deprotection of the applied monomer so that it is reactive with a subsequently applied protected monomer; and application of another protected monomer for linking. One or more intermediate steps include oxidation or sulfurization. In some instances, one or more wash steps precede or follow one or all of the steps.

Methods for phosphoramidite-based polynucleotide synthesis comprise a series of chemical steps. In some instances, one or more steps of a synthesis method involve reagent cycling, where one or more steps of the method comprise application to the device of a reagent useful for the step. For example, reagents are cycled by a series of liquid deposition and vacuum drying steps. For substrates comprising three-dimensional features such as wells, microwells, channels and the like, reagents are optionally passed through one or more regions of the device via the wells and/or channels.

Methods and systems described herein relate to polynucleotide synthesis devices for the synthesis of polynucleotides. The synthesis may be in parallel. For example, at least or about at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 10000, 50000, 75000, 100000 or more polynucleotides can be synthesized in parallel. The total number polynucleotides that may be synthesized in parallel may be from 2-100000, 3-50000, 4-10000, 5-1000, 6-900, 7-850, 8-800, 9-750, 10-700, 11-650, 12-600, 13-550, 14-500, 15-450, 16-400, 17-350, 18-300, 19-250, 20-200, 21-150,22-100, 23-50, 24-45, 25-40, 30-35. Those of skill in the art appreciate that the total number of polynucleotides synthesized in parallel may fall within any range bound by any of these values, for example 25-100. The total number of polynucleotides synthesized in parallel may fall within any range defined by any of the values serving as endpoints of the range. Total molar mass of polynucleotides synthesized within the device or the molar mass of each of the polynucleotides may be at least or at least about 10, 20, 30, 40, 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 25000, 50000, 75000, 100000 picomoles, or more. The length of each of the polynucleotides or average length of the polynucleotides within the device may be at least or about at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 300, 400, 500 nucleotides, or more. The length of each of the polynucleotides or average length of the polynucleotides within the device may be at most or about at most 500, 400, 300, 200, 150, 100, 50, 45, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 nucleotides, or less. The length of each of the polynucleotides or average length of the polynucleotides within the device may fall from 10-500, 9-400, 11-300, 12-200, 13-150, 14-100, 15-50, 16-45, 17-40, 18-35, 19-25. Those of skill in the art appreciate that the length of each of the polynucleotides or average length of the polynucleotides within the device may fall within any range bound by any of these values, for example 100-300. The length of each of the polynucleotides or average length of the polynucleotides within the device may fall within any range defined by any of the values serving as endpoints of the range.

Methods for polynucleotide synthesis on a surface provided herein allow for synthesis at a fast rate. As an example, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 125, 150, 175, 200 nucleotides per hour, or more are synthesized. Nucleotides include adenine, guanine, thymine, cytosine, uridine building blocks, or analogs/modified versions thereof. In some instances, libraries of polynucleotides are synthesized in parallel on substrate. For example, a device comprising about or at least about 100; 1,000; 10,000; 30,000; 75,000; 100,000; 1,000,000; 2,000,000; 3,000,000; 4,000,000; or 5,000,000 resolved loci is able to support the synthesis of at least the same number of distinct polynucleotides, wherein polynucleotide encoding a distinct sequence is synthesized on a resolved locus. In some instances, a library of polynucleotides is synthesized on a device with low error rates described herein in less than about three months, two months, one month, three weeks, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days, 24 hours or less. In some instances, larger nucleic acids assembled from a polynucleotide library synthesized with low error rate using the substrates and methods described herein are prepared in less than about three months, two months, one month, three weeks, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days, 24 hours or less.

In some instances, methods described herein provide for generation of a library of nucleic acids comprising variant nucleic acids differing at a plurality of codon sites. In some instances, a nucleic acid may have 1 site, 2 sites, 3 sites, 4 sites, 5 sites, 6 sites, 7 sites, 8 sites, 9 sites, 10 sites, 11 sites, 12 sites, 13 sites, 14 sites, 15 sites, 16 sites, 17 sites 18 sites, 19 sites, 20 sites, 30 sites, 40 sites, 50 sites, or more of variant codon sites.

In some instances, the one or more sites of variant codon sites may be adjacent. In some instances, the one or more sites of variant codon sites may not be adjacent and separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more codons.

In some instances, a nucleic acid may comprise multiple sites of variant codon sites, wherein all the variant codon sites are adjacent to one another, forming a stretch of variant codon sites. In some instances, a nucleic acid may comprise multiple sites of variant codon sites, wherein none the variant codon sites are adjacent to one another. In some instances, a nucleic acid may comprise multiple sites of variant codon sites, wherein some the variant codon sites are adjacent to one another, forming a stretch of variant codon sites, and some of the variant codon sites are not adjacent to one another.

Referring to the Figures, FIG. 2 illustrates an exemplary process workflow for synthesis of nucleic acids (e.g., genes) from shorter nucleic acids. The workflow is divided generally into phases: (1) de novo synthesis of a single stranded nucleic acid library, (2) joining nucleic acids to form larger fragments, (3) error correction, (4) quality control, and (5) shipment. Prior to de novo synthesis, an intended nucleic acid sequence or group of nucleic acid sequences is preselected. For example, a group of genes is preselected for generation.

Once large nucleic acids for generation are selected, a predetermined library of nucleic acids is designed for de novo synthesis. Various suitable methods are known for generating high density polynucleotide arrays. In the workflow example, a device surface layer is provided. In the example, chemistry of the surface is altered in order to improve the polynucleotide synthesis process. Areas of low surface energy are generated to repel liquid while areas of high surface energy are generated to attract liquids. The surface itself may be in the form of a planar surface or contain variations in shape, such as protrusions or microwells which increase surface area. In the workflow example, high surface energy molecules selected serve a dual function of supporting DNA chemistry, as disclosed in International Patent Application Publication WO/2015/021080, which is herein incorporated by reference in its entirety.

In situ preparation of polynucleotide arrays is generated on a solid support and utilizes single nucleotide extension process to extend multiple oligomers in parallel. A deposition device, such as a material deposition device 201, is designed to release reagents in a step wise fashion such that multiple polynucleotides extend, in parallel, one residue at a time to generate oligomers with a predetermined nucleic acid sequence 202. In some instances, polynucleotides are cleaved from the surface at this stage. Cleavage includes gas cleavage, e.g., with ammonia or methylamine.

The generated polynucleotide libraries are placed in a reaction chamber. In this exemplary workflow, the reaction chamber (also referred to as “nanoreactor”) is a silicon coated well, containing PCR reagents and lowered onto the polynucleotide library 203. Prior to or after the sealing 204 of the polynucleotides, a reagent is added to release the polynucleotides from the substrate. In the exemplary workflow, the polynucleotides are released subsequent to sealing of the nanoreactor 205. Once released, fragments of single stranded polynucleotides hybridize in order to span an entire long range sequence of DNA. Partial hybridization 205 is possible because each synthesized polynucleotide is designed to have a small portion overlapping with at least one other polynucleotide in the pool.

After hybridization, a PCA reaction is commenced. During the polymerase cycles, the polynucleotides anneal to complementary fragments and gaps are filled in by a polymerase. Each cycle increases the length of various fragments randomly depending on which polynucleotides find each other. Complementarity amongst the fragments allows for forming a complete large span of double stranded DNA 206.

After PCA is complete, the nanoreactor is separated from the device 207 and positioned for interaction with a device having primers for PCR 208. After sealing, the nanoreactor is subject to PCR 209 and the larger nucleic acids are amplified. After PCR 210, the nanochamber is opened 211, error correction reagents are added 212, the chamber is sealed 213 and an error correction reaction occurs to remove mismatched base pairs and/or strands with poor complementarity from the double stranded PCR amplification products 214. The nanoreactor is opened and separated 215. Error corrected product is next subject to additional processing steps, such as PCR and molecular bar coding, and then packaged 222 for shipment 223.

In some instances, quality control measures are taken. After error correction, quality control steps include for example interaction with a wafer having sequencing primers for amplification of the error corrected product 216, sealing the wafer to a chamber containing error corrected amplification product 217, and performing an additional round of amplification 218. The nanoreactor is opened 219 and the products are pooled 220 and sequenced 221. After an acceptable quality control determination is made, the packaged product 222 is approved for shipment 223.

In some instances, a nucleic acid generate by a workflow such as that in FIG. 2 is subject to mutagenesis using overlapping primers disclosed herein. In some instances, a library of primers are generated by in situ preparation on a solid support and utilize single nucleotide extension process to extend multiple oligomers in parallel. A deposition device, such as a material deposition device, is designed to release reagents in a step wise fashion such that multiple polynucleotides extend, in parallel, one residue at a time to generate oligomers with a predetermined nucleic acid sequence 202.

Computer Systems

Any of the systems described herein, may be operably linked to a computer and may be automated through a computer either locally or remotely. In various instances, the methods and systems of the disclosure may further comprise software programs on computer systems and use thereof. Accordingly, computerized control for the synchronization of the dispense/vacuum/refill functions such as orchestrating and synchronizing the material deposition device movement, dispense action and vacuum actuation are within the bounds of the disclosure. The computer systems may be programmed to interface between the user specified base sequence and the position of a material deposition device to deliver the correct reagents to specified regions of the substrate.

The computer system 300 illustrated in FIG. 3 may be understood as a logical apparatus that can read instructions from media 311 and/or a network port 305, which can optionally be connected to server 309 having fixed media 312. The system, such as shown in FIG. 3 can include a CPU 301, disk drives 303, optional input devices such as keyboard 315 and/or mouse 316 and optional monitor 307. Data communication can be achieved through the indicated communication medium to a server at a local or a remote location. The communication medium can include any means of transmitting and/or receiving data. For example, the communication medium can be a network connection, a wireless connection or an internet connection. Such a connection can provide for communication over the World Wide Web. It is envisioned that data relating to the present disclosure can be transmitted over such networks or connections for reception and/or review by a party 322 as illustrated in FIG. 3.

FIG. 4 is a block diagram illustrating a first example architecture of a computer system 400 that can be used in connection with example instances of the present disclosure. As depicted in FIG. 4, the example computer system can include a processor 402 for processing instructions. Non-limiting examples of processors include: Intel Xeon™ processor, AMD Opteron™ processor, Samsung 32-bit RISC ARM 1176JZ(F)-S v1.0™ processor, ARM Cortex-A8 Samsung S5PC100™ processor, ARM Cortex-A8 Apple A4™ processor, Marvell PXA 930™ processor, or a functionally-equivalent processor. Multiple threads of execution can be used for parallel processing. In some instances, multiple processors or processors with multiple cores can also be used, whether in a single computer system, in a cluster, or distributed across systems over a network comprising a plurality of computers, cell phones, and/or personal data assistant devices.

As illustrated in FIG. 4, a high speed cache 404 can be connected to, or incorporated in, the processor 402 to provide a high speed memory for instructions or data that have been recently, or are frequently, used by processor 402. The processor 402 is connected to a north bridge 406 by a processor bus 408. The north bridge 406 is connected to random access memory (RAM) 410 by a memory bus 412 and manages access to the RAM 410 by the processor 402. The north bridge 406 is also connected to a south bridge 414 by a chipset bus 416. The south bridge 414 is, in turn, connected to a peripheral bus 418. The peripheral bus can be, for example, PCI, PCI-X, PCI Express, or other peripheral bus. The north bridge and south bridge are often referred to as a processor chipset and manage data transfer between the processor, RAM, and peripheral components on the peripheral bus 418. In some alternative architectures, the functionality of the north bridge can be incorporated into the processor instead of using a separate north bridge chip. In some instances, system 400 can include an accelerator card 422 attached to the peripheral bus 418. The accelerator can include field programmable gate arrays (FPGAs) or other hardware for accelerating certain processing. For example, an accelerator can be used for adaptive data restructuring or to evaluate algebraic expressions used in extended set processing.

Software and data are stored in external storage 424 and can be loaded into RAM 410 and/or cache 404 for use by the processor. The system 400 includes an operating system for managing system resources; non-limiting examples of operating systems include: Linux, Windows™, MACOS™, BlackBerry OS™, iOS™, and other functionally equivalent operating systems, as well as application software running on top of the operating system for managing data storage and optimization in accordance with example instances of the present disclosure. In this example, system 400 also includes network interface cards (NICs) 420 and 421 connected to the peripheral bus for providing network interfaces to external storage, such as Network Attached Storage (NAS) and other computer systems that can be used for distributed parallel processing.

FIG. 5 is a diagram showing a network 500 with a plurality of computer systems 502a, and 502b, a plurality of cell phones and personal data assistants 502c, and Network Attached Storage (NAS) 504a, and 504b. In example instances, systems 502a, 502b, and 502c can manage data storage and optimize data access for data stored in Network Attached Storage (NAS) 504a and 504b. A mathematical model can be used for the data and be evaluated using distributed parallel processing across computer systems 502a, and 502b, and cell phone and personal data assistant systems 502c. Computer systems 502a, and 502b, and cell phone and personal data assistant systems 502c can also provide parallel processing for adaptive data restructuring of the data stored in Network Attached Storage (NAS) 504a and 504b. FIG. 5 illustrates an example only, and a wide variety of other computer architectures and systems can be used in conjunction with the various instances of the present disclosure. For example, a blade server can be used to provide parallel processing. Processor blades can be connected through a back plane to provide parallel processing. Storage can also be connected to the back plane or as Network Attached Storage (NAS) through a separate network interface. In some example instances, processors can maintain separate memory spaces and transmit data through network interfaces, back plane or other connectors for parallel processing by other processors. In other instances, some or all of the processors can use a shared virtual address memory space.

FIG. 6 is a block diagram of a multiprocessor computer system using a shared virtual address memory space in accordance with an example instance. The system includes a plurality of processors 602a-f that can access a shared memory subsystem 604. The system incorporates a plurality of programmable hardware memory algorithm processors (MAPs) 606a-f in the memory subsystem 604. Each MAP 606a-f can comprise a memory 608a-f and one or more field programmable gate arrays (FPGAs) 610a-f. The MAP provides a configurable functional unit and particular algorithms, or portions of algorithms can be provided to the FPGAs 610a-f for processing in close coordination with a respective processor. For example, the MAPs can be used to evaluate algebraic expressions regarding the data model and to perform adaptive data restructuring in example instances. In this example, each MAP is globally accessible by all of the processors for these purposes. In one configuration, each MAP can use Direct Memory Access (DMA) to access an associated memory 608a-f, allowing it to execute tasks independently of, and asynchronously from the respective microprocessor 602a-f In this configuration, a MAP can feed results directly to another MAP for pipelining and parallel execution of algorithms.

The above computer architectures and systems are examples only, and a wide variety of other computer, cell phone, and personal data assistant architectures and systems can be used in connection with example instances, including systems using any combination of general processors, co-processors, FPGAs and other programmable logic devices, system on chips (SOCs), application specific integrated circuits (ASICs), and other processing and logic elements. In some instances, all or part of the computer system can be implemented in software or hardware. Any variety of data storage media can be used in connection with example instances, including random access memory, hard drives, flash memory, tape drives, disk arrays, Network Attached Storage (NAS) and other local or distributed data storage devices and systems.

In example instances, the computer system can be implemented using software modules executing on any of the above or other computer architectures and systems. In other instances, the functions of the system can be implemented partially or completely in firmware, programmable logic devices such as field programmable gate arrays (FPGAs) as referenced in FIG. 4, system on chips (SOCs), application specific integrated circuits (ASICs), or other processing and logic elements. For example, the Set Processor and Optimizer can be implemented with hardware acceleration through the use of a hardware accelerator card, such as accelerator card 422 illustrated in FIG. 4.

The following examples are set forth to illustrate more clearly the principle and practice of embodiments disclosed herein to those skilled in the art and are not to be construed as limiting the scope of any claimed embodiments. Unless otherwise stated, all parts and percentages are on a weight basis.

EXAMPLES

The following examples are given for the purpose of illustrating various embodiments of the disclosure and are not meant to limit the present disclosure in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure. Changes therein and other uses which are encompassed within the spirit of the disclosure as defined by the scope of the claims will occur to those skilled in the art.

Example 1: Functionalization of a Device Surface

A device was functionalized to support the attachment and synthesis of a library of polynucleotides. The device surface was first wet cleaned using a piranha solution comprising 90% H₂SO₄ and 10% H₂O₂ for 20 minutes. The device was rinsed in several beakers with DI water, held under a DI water gooseneck faucet for 5 min, and dried with N2. The device was subsequently soaked in NH₄OH (1:100; 3 mL:300 mL) for 5 min, rinsed with DI water using a handgun, soaked in three successive beakers with DI water for 1 min each, and then rinsed again with DI water using the handgun. The device was then plasma cleaned by exposing the device surface to O₂. A SAMCO PC-300 instrument was used to plasma etch O₂ at 250 watts for 1 min in downstream mode.

The cleaned device surface was actively functionalized with a solution comprising N-(3-triethoxysilylpropyl)-4-hydroxybutyramide using a YES-1224P vapor deposition oven system with the following parameters: 0.5 to 1 torr, 60 min, 70° C., 135° C. vaporizer. The device surface was resist coated using a Brewer Science 200× spin coater. SPR™ 3612 photoresist was spin coated on the device at 2500 rpm for 40 sec. The device was pre-baked for 30 min at 90° C. on a Brewer hot plate. The device was subjected to photolithography using a Karl Suss MA6 mask aligner instrument. The device was exposed for 2.2 sec and developed for 1 min in MSF 26A. Remaining developer was rinsed with the handgun and the device soaked in water for 5 min. The device was baked for 30 min at 100° C. in the oven, followed by visual inspection for lithography defects using a Nikon L200. A descum process was used to remove residual resist using the SAMCO PC-300 instrument to O₂ plasma etch at 250 watts for 1 min.

The device surface was passively functionalized with a 100 μL solution of perfluorooctyltrichlorosilane mixed with 10 μL light mineral oil. The device was placed in a chamber, pumped for 10 min, and then the valve was closed to the pump and left to stand for 10 min. The chamber was vented to air. The device was resist stripped by performing two soaks for 5 min in 500 mL NMP at 70° C. with ultrasonication at maximum power (9 on Crest system). The device was then soaked for 5 min in 500 mL isopropanol at room temperature with ultrasonication at maximum power. The device was dipped in 300 mL of 200 proof ethanol and blown dry with N2. The functionalized surface was activated to serve as a support for polynucleotide synthesis.

Example 2: Synthesis of a 50-Mer Sequence on an Oligonucleotide Synthesis Device

A two-dimensional oligonucleotide synthesis device was assembled into a flowcell, which was connected to a flowcell (Applied Biosystems (ABI394 DNA Synthesizer”). The two-dimensional oligonucleotide synthesis device was uniformly functionalized with N-(3-TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE (Gelest) was used to synthesize an exemplary polynucleotide of 50 bp (“50-mer polynucleotide”) using polynucleotide synthesis methods described herein.

The sequence of the 50-mer was as described. 5′AGACAATCAACCATTTGGGGTGGACAGCCTTGACCTCTAGACTTCGGCAT ##TTTTT TTTTT3′ (SEQ ID NO: 2673), where # denotes Thymidine-succinyl hexamide CED phosphoramidite (CLP-2244 from ChemGenes), which is a cleavable linker enabling the release of oligos from the surface during deprotection.

The synthesis was done using standard DNA synthesis chemistry (coupling, capping, oxidation, and deblocking) according to the protocol in Table 2 and an ABI synthesizer.

TABLE 2
Synthesis protocols
General DNA Synthesis	Table 2
Process Name	Process Step	Time (sec)
WASH (Acetonitrile	Acetonitrile System Flush	4
Wash Flow)	Acetonitrile to Flowcell	23
	N2 System Flush	4
	Acetonitrile System Flush	4
DNA BASE ADDITION	Activator Manifold Flush	2
(Phosphoramidite +	Activator to Flowcell	6
Activator Flow)	Activator +	6
	Phosphoramidite to
	Flowcell
	Activator to Flowcell	0.5
	Activator +	5
	Phosphoramidite to
	Flowcell
	Activator to Flowcell	0.5
	Activator +	5
	Phosphoramidite to
	Flowcell
	Activator to Flowcell	0.5
	Activator +	5
	Phosphoramidite to
	Flowcell
	Incubate for 25sec	25
WASH (Acetonitrile	Acetonitrile System Flush	4
Wash Flow)	Acetonitrile to Flowcell	15
	N2 System Flush	4
	Acetonitrile System Flush	4
DNA BASE ADDITION	Activator Manifold Flush	2
(Phosphoramidite +	Activator to Flowcell	5
Activator Flow)	Activator +	18
	Phosphoramidite to
	Flowcell
	Incubate for 25sec	25
WASH (Acetonitrile	Acetonitrile System Flush	4
Wash Flow)	Acetonitrile to Flowcell	15
	N2 System Flush	4
	Acetonitrile System Flush	4
CAPPING (CapA + B,	CapA+B to Flowcell	15
1:1, Flow)
WASH (Acetonitrile	Acetonitrile System Flush	4
Wash Flow)	Acetonitrile to Flowcell	15
	Acetonitrile System Flush	4
OXIDATION (Oxidizer	Oxidizer to Flowcell	18
Flow)
WASH (Acetonitrile	Acetonitrile System Flush	4
Wash Flow)	N2 System Flush	4
	Acetonitrile System Flush	4
	Acetonitrile to Flowcell	15
	Acetonitrile System Flush	4
	Acetonitrile to Flowcell	15
	N2 System Flush	4
	Acetonitrile System Flush	4
	Acetonitrile to Flowcell	23
	N2 System Flush	4
	Acetonitrile System Flush	4
DEBLOCKING	Deblock to Flowcell	36
(Deblock Flow)
WASH (Acetonitrile	Acetonitrile System Flush	4
Wash Flow)	N2 System Flush	4
	Acetonitrile System Flush	4
	Acetonitrile to Flowcell	18
	N2 System Flush	4.13
	Acetonitrile System Flush	4.13
	Acetonitrile to Flowcell	15

The phosphoramidite/activator combination was delivered similar to the delivery of bulk reagents through the flowcell. No drying steps were performed as the environment stays “wet” with reagent the entire time.

The flow restrictor was removed from the ABI 394 synthesizer to enable faster flow. Without flow restrictor, flow rates for amidites (0.1M in ACN), Activator, (0.25M Benzoylthiotetrazole (“BTT”; 30-3070-xx from GlenResearch) in ACN), and Ox (0.02M 12 in 20% pyridine, 10% water, and 70% THF) were roughly ˜100 uL/sec, for acetonitrile (“ACN”) and capping reagents (1:1 mix of CapA and CapB, wherein CapA is acetic anhydride in THF/Pyridine and CapB is 16% 1-methylimidizole in THF), roughly ˜200 uL/sec, and for Deblock (3% dichloroacetic acid in toluene), roughly ˜300 uL/sec (compared to ˜50 uL/sec for all reagents with flow restrictor). The time to completely push out Oxidizer was observed, the timing for chemical flow times was adjusted accordingly and an extra ACN wash was introduced between different chemicals. After polynucleotide synthesis, the chip was deprotected in gaseous ammonia overnight at 75 psi. Five drops of water were applied to the surface to recover polynucleotides. The recovered polynucleotides were then analyzed on a BioAnalyzer small RNA chip.

Example 3: Synthesis of a 100-Mer Sequence on an Oligonucleotide Synthesis Device

The same process as described in Example 2 for the synthesis of the 50-mer sequence was used for the synthesis of a 100-mer polynucleotide (“100-mer polynucleotide”; 5′ CGGGATCCTTATCGTCATCGTCGTACAGATCCCGACCCATTTGCTGTCCACCAGTCA TGCTAGCCATACCATGATGATGATGATGATGAGAACCCCGCAT ##TTTTTTTTTT3′ (SEQ ID NO: 2674), where # denotes Thymidine-succinyl hexamide CED phosphoramidite (CLP-2244 from ChemGenes) on two different silicon chips, the first one uniformly functionalized with N-(3-TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE and the second one functionalized with 5/95 mix of 11-acetoxyundecyltriethoxysilane and n-decyltriethoxysilane, and the polynucleotides extracted from the surface were analyzed on a BioAnalyzer instrument.

All ten samples from the two chips were further PCR amplified using a forward (5′ATGCGGGGTTCTCATCATC3′ (SEQ ID NO: 2675)) and a reverse (5′CGGGATCCTTATCGTCATCG3′ (SEQ ID NO: 2676)) primer in a 50 uL PCR mix (25 uL NEB Q5 mastermix, 2.5 uL 10 uM Forward primer, 2.5 uL 10 uM Reverse primer, 1 uL polynucleotide extracted from the surface, and water up to 50 uL) using the following thermalcycling program:

98° C., 30 sec

98° C., 10 sec; 63° C., 10 sec; 72° C., 10 sec; repeat 12 cycles

72° C., 2 min

The PCR products were also run on a BioAnalyzer, demonstrating sharp peaks at the 100-mer position. Next, the PCR amplified samples were cloned, and Sanger sequenced. Table 3 summarizes the results from the Sanger sequencing for samples taken from spots 1-5 from chip 1 and for samples taken from spots 6-10 from chip 2.

TABLE 3
Sequencing results
	Spot	Error rate	Cycle efficiency
	1	1/763	bp	99.87%
	2	1/824	bp	99.88%
	3	1/780	bp	99.87%
	4	1/429	bp	99.77%
	5	1/1525	bp	99.93%
	6	1/1615	bp	99.94%
	7	1/531	bp	99.81%
	8	1/1769	bp	99.94%
	9	1/854	bp	99.88%
	10	1/1451	bp	99.93%

Thus, the high quality and uniformity of the synthesized polynucleotides were repeated on two chips with different surface chemistries. Overall, 89% of the 100-mers that were sequenced were perfect sequences with no errors, corresponding to 233 out of 262.

Table 4 summarizes error characteristics for the sequences obtained from the polynucleotides samples from spots 1-10.

TABLE 4
Error characteristics
Sample	OSA_0	OSA_0	OSA_0	OSA_0	OSA_0	OSA_0	OSA_0	OSA_0	OSA_0	OSA_0
ID/Spot	046/1	047/2	048/3	049/4	050/5	051/6	052/7	053/8	054/9	055/10
no.
Total	32	32	32	32	32	32	32	32	32	32
Sequences
Sequencing	25 of	27 of	26 of	21 of	25 of	29 of	27 of	29 of	28 of	25 of 28
Quality	28	27	30	23	26	30	31	31	29
Oligo	23 of	25 of	22 of	18 of	24 of	25 of	22 of	28 of	26 of	20 of 25
Quality	25	27	26	21	25	29	27	29	28
ROI	2500	2698	2561	2122	2499	2666	2625	2899	2798	2348
Match
Count
ROI	2	2	1	3	1	0	2	1	2	1
Mutation
ROI Multi	0	0	0	0	0	0	0	0	0	0
Base
Deletion
ROI	1	0	0	0	0	0	0	0	0	0
Small
Insertion
ROI	0	0	0	0	0	0	0	0	0	0
Single
Base
Deletion
Large	0	0	1	0	0	1	1	0	0	0
Deletion
Count
Mutation:	2	2	1	2	1	0	2	1	2	1
G > A
Mutation:	0	0	0	1	0	0	0	0	0	0
T > C
ROI Error	3	2	2	3	1	1	3	1	2	1
Count
ROI Error	Err: ~1	Err: ~1	Err: ~1	Err: ~1	Err: ~1	Err: ~1	Err: ~1	Err: ~1	Err: ~1	Err: ~1
Rate	in 834	in 1350	in 1282	in 708	in 2500	in 2667	in 876	in 2900	in 1400	in 2349
ROI	MP	MP	MP	MP	MP	MP	MP	MP	MP	MP Err:
Minus	Err: ~1	Err: ~1	Err: ~1	Err: ~1	Err: ~1	Err: ~1	Err: ~1	Err: ~1	Err: ~1	~1 in
Primer	in 763	in 824	in 780	in 429	in 1525	in 1615	in 531	in 1769	in 854	1451
Error Rate

Example 4: Panning and Screening for Identification of Antibodies for SARS-CoV-2 and ACE2

This example describes identification of antibodies for SARS-CoV-2 and ACE2.

Design, Construction, and Screening of Anti-S1 Antibody Phage Libraries

Four phage antibody libraries were generated for screening against the SARS-CoV-2 S1 (GenBank QHD43416.1, residues 16-685). The CDR diversity of the libraries based on the repertoires from human and/or llama CDR sequences as described below, which were subsequently synthesized and assembled into antibody hypervariable regions for phage display were maximized. The four such libraries included the following:

• • (1) a short-chain variable fragment (scFv) library constructed using CDRs identified in the memory B cells of a convalescent COVID-19 donor (“COVID-19 scFv”, Antibody 1)
  • (2) an antigen-binding fragment (Fab) library constructed using CDRs from human naïve and memory B cells (“Hyperimmune Fab”, Antibody 2)
  • (3) a humanized llama nanobody library with shuffled, llama-based CDR diversity (“VHH hShuffle”, Antibody 5)
  • (4) a humanized llama nanobody library constructed using natural llama CDR1/2 sequences and human CDR3s identified from human naïve and memory B cells (“VHH Hyperimmune”, Antibody 6)

The antibodies are listed in Example 11 in Tables 8-30. Each library possessed a CDR diversity of >10¹⁰. Antibodies were selected for SARS-CoV-2 S1 binding using a bead-based biopanning strategy. For each library, phages were selected over four rounds of panning to identify putative high-affinity S1-binding antibodies. After panning, enzyme-linked immunosorbent assay (ELISA) was employed to assess the binding of phage-displayed antibodies to S1 protein. Antibody candidates from each library that elicited a greater than 10-fold enrichment over a bovine serum albumin control protein were selected as initial leads. From the COVID-19 scFv (Antibody 1), Hyperimmune Fab (Antibody 2), VHH hShuffle (Antibody 5), VHH Hyperimmune (Antibody 6) libraries, 41, 14, 68, and 112 unique clones, respectively were identified.

Following phage display ELISA screening, S1-binding antibody candidates were reformatted to human IgG1 (COVID-19 scFv, Hyperimmune Fab) or a VHH-Fc fusion containing the Fc region of human IgG1 (VHH hShuffle, VHH Hyperimmune hShuffle) for further characterization and development.

Biophysical Characterization and Competition Binning of Antibody Candidates

The phage display workflow identified 235 S1-binding leads across the four phage libraries that were screened. Given the diverse sources of CDR repertoires that were used to design these libraries, the sequence diversity of the hypervariable region by Sanger DNA sequencing was identified for each candidate and aligning the resulting hypervariable sequences across candidates from all four libraries. Antibody candidates from libraries containing overlapping CDR diversity were more closely related than those that drew from entirely distinct CDR sources. For example, many Antibody 5 and Antibody 6 candidates—both of which contained natural llama CDR1/2s—were interspersed in closely related sequence families.

The binding affinity and specificity of the S1 antibody candidates using surface plasmon resonance (SPR) and S1 RBD-ACE2 competition assays, respectively were determined. Multiple S1 antibody candidates with nanomolar affinities against SARS-CoV-2 S1, including 1-35 (K_D=83 nM), 2-2 (K_D=21 nM), 2-6 (K_D=25 nM), 5-1 (K_D=6.6 nM), 6-3 (K_D=32 nM), and 6-63 (K_D=46 nM) were identified. Additionally, all but one of these candidates bound to the prefusion-stabilized SARS-CoV-2 S trimer with picomolar affinities (data not shown). The cross-binding of antibody candidates to the S1 domain of SARS-CoV S protein was also assayed. All antibody candidates specifically bound SARS-CoV-2 S1 except for 2-5 and 2-2, which both cross-bound with SARS-CoV spike protein. The binding of S1 antibody candidates to ACE2 in an ELISA and flow cytometric competition binding assays were also determined. For the flow cytometry assay, each mAb candidate was incubated with recombinant SARS-CoV-2 S1 RBD and Vero E6 cells, which are susceptible to SARS-CoV and SARS-CoV-2 infection via ACE2. Many high-affinity anti-S1 mAbs effectively blocked the interaction between SARS-CoV-2 S1 RBD and ACE2 on Vero E6 cells as measured by flow cytometry, including 2-5, 2-2, 5-1, and 6-63. Nonetheless, some high-affinity, S1-binding candidates such as 6-42 and 1-12 failed to block this interaction. Notably, 1-12 did compete with ACE2 in the less physiologically relevant ELISA assay.

The cross-competition of the S1 antibody candidates and existing SARS-CoV-2 antibodies, including CR3022 and SAD-S35 (Acro Biosystems), with S1 using high-throughput surface plasmon resonance (HT-SPR) were investigated. This assay revealed four competition bins: namely, two bins that overlapped serially, and two additional, independent bins. The first bin (bin 1) included numerous VHH (Antibody 5) candidates and SAD-535. CR3022 competed with a few Antibody 2 candidates in bin 2. 2-2 bridged bins 1 and 2, forming a bin with CR3022 and SAD-535. The remaining bins, 3 and 4, exhibited no overlap. Bin 3 included 2-6, 1-35, 1-16, and 1-32. Bin 4 only contained 6-24, suggesting that it binds a unique epitope not targeted by the other candidates. The bins identified here may not reflect epitope bins per se, as other factors such as steric hindrance can allow antibodies with distinct epitopes to compete with one another for S1 binding.

Example 5. Epitope Mapping

Having identified several neutralizing mAbs, their binding epitopes were investigated.

Based on the aggregate data from the Vero E6 flow cytometry assay, competition binning analysis, and neutralization assays, it was hypothesized that many of the candidates bound divergent sites on SARS-CoV-2 S1. To test this, a shotgun mutagenesis library of SARS-CoV-2 S protein RBD mutants were generated and screened the binding of neutralizing candidates to cells expressing these mutants. This approach allowed for defining which amino acids were critical to the binding of each neutralizing antibody. As shown in FIGS. 7A-7C, most neutralizing mAbs bound the RBD, although the overall binding pattern of the VHH RBD-binding mAbs (6-1, 6-3, and 6-63) differed from that of the IgG RBD-binding mAbs (2-5, 2-2, 2-6). Whereas residues in the ACE2-binding site of the RBD were critical for the VHH RBD-binders, more occluded residues mediated the binding of the 2-5 and 2-2 IgG candidates. 2-6, an IgG, bound a unique site that extended beyond the RBD (FIG. 7A and FIG. 7C). Although most of the neutralizing mAbs mapped bound to S1 RBD, there was one notable exception: 1-12. The inability of this candidate to inhibit the binding of S1 RBD to ACE2 in the Vero E6 flow cytometry assay indicated the position of a binding epitope outside the RBD. To clarify this, the shotgun mutagenesis approach was extended beyond the RBD. Critical residues for the binding of 1-12 were found in the NTD of the S1 subunit (FIGS. 7A-7C).

Materials and Methods

Epitope mapping was performed essentially as described previously, using a SARS-CoV-2 (strain Wuhan-Hu-1) S protein RBD shotgun mutagenesis mutation library, made using a full-length expression construct for S protein, where residues of S1 were individually mutated to alanine, and alanine residues to serine. Mutations were confirmed by DNA sequencing, and clones arrayed in a 384-well plate, one mutant per well. Binding of mAbs to each mutant clone in the alanine scanning library was determined, in duplicate, by high-throughput flow cytometry. Each S protein mutant was transfected into HEK-293T cells and allowed to express for 22 hrs. Cells were fixed in 4% (v/v) paraformaldehyde (Electron Microscopy Sciences), and permeabilized with 0.1% (w/v) saponin (Sigma-Aldrich) in PBS plus calcium and magnesium (PBS++) before incubation with mAbs diluted in PBS++, 10% normal goat serum (Sigma), and 0.1% saponin. MAb screening concentrations were determined using an independent immunofluorescence titration curve against cells expressing wild-type S protein to ensure that signals were within the linear range of detection. Antibodies were detected using 3.75 μg/mL of AlexaFluor488-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) in 10% normal goat serum with 0.1% saponin. Cells were washed three times with PBS++/0.1% saponin followed by two washes in PBS and mean cellular fluorescence was detected using a high-throughput Intellicyte iQue flow cytometer (Sartorius). Antibody reactivity against each mutant S protein clone was calculated relative to wild-type S protein reactivity by subtracting the signal from mock-transfected controls and normalizing to the signal from wild-type S-transfected controls. Mutations within clones were identified as critical to the mAb epitope if they did not support reactivity of the test mAb but supported reactivity of other SARS-CoV-2 antibodies. This counter-screen strategy facilitates the exclusion of S mutants that are locally misfolded or have an expression defect. Validated critical residues represent amino acids whose side chains make the highest energetic contributions to the mAb-epitope interaction.

A subset of antibodies was also submitted for epitope mapping by high-throughput SPR and negative-stain electron microscopy by the CoVIC consortium. IgGs were cleaved by either IdeS (Promega) or papain (Sigma) and purified by ion exchange chromatography with MonoQ column (GE). Purified Spike trimer (in normal S buffer) was mixed with Fab fragments (1:2 or 2:1 molar ratio) at RT for 3 hours or overnight. Complexes were then purified with Superdex 6. Samples were stained by 0.75% uranyl formate with standard protocol. Datasets were collected by the Halo Titian electron microscope (Thermo Fisher Scientific).

Example 6: SARS-CoV-2 Variants

Different variants of SARS-CoV-2 are listed in Table 5. Different SARS-CoV-2 variants may alter the epitopes that a given antibody binds to and affect immunity.

TABLE 5
SARS-CoV-2 variants
	B.1.1.7	B.1.351	P.1
Alternate name	501Y.V1	501Y.V2	501Y.V3
Mutations	23	21	17
Spike mutations	8	9	10
Key RBD, spike	E69/70 deletion,	E484K, K417N,	E484, K417N/T,
mutations beyond	P681H 144Y deletion,	orflb deletion	orflb deletion
N501Y in all	A570D
Other mutations,	T7161, S982A,	L18F, D80A,	L18F, T2ON,
including N-terminal	D1118H	D215G, Δ242-244,	P26S and others
		R264I
Transmissibility A	>40% increased	Not established	Not established
Lethality A	concern raised, not	Not established	Not established
	resolved
Immune escape	Not established	Probable, extent	Not established
		unclear (in vitro)
Countries reported	62	26	7
(not = to local
transmission)

The tables in FIG. 8 highlight which mutations are located at the receptor binding domain (RBD). These mutations include G22813T, G23012A, A23063T, A23403g, K417N, E484K, N501Y, D641G for the 501Y.V2 variant (S. African), and A23063T and N501Y for the B.1.1.7, 501Y.V1 variant (UK).

Example 7: SPR Kinetics for 6-3 and 6-63

SPR kinetics were measured for SARS-COV-2 variant antibodies 6-3 and 6-63 against different SARS-COV-2 variant strains. The results are depicted in FIGS. 9A-9B and Table 6.

TABLE 6
Protein	6-3	6-63
SARS-CoV-2 S1	11 nM	65 nM
SARS-CoV-2 S1 (D614G)	19 nM	95 nM
SARS-CoV-2 S1 (P681H)	12 nM	16 nM
SARS-CoV-2 S1 (HV69-70del, N501Y, D614G)	9.3 nM	63 nM
SARS-CoV-2 S1 (HV69-70del, Y453F, D614G)	23 nM	96 nM
SARS-CoV-2 S1 (HV69-70del, Y144del, N501Y,	n/b*	80 nM
A570D, D614G, P681H)
SARS-CoV-2 S RBD (N501Y)	0.71 nM	3.8 nM
SARS-CoV-2 S RBD (Y453F)	5.7 nM	5.4 nM
SARS-CoV-2 S RBD (N439K)	9.5 nM	16 nM
SARS-CoV-2 S RBD (K417N)	5.9 nM	14 nM
SARS-CoV-2 S RBD (E484K)	4.6 nM	17 nM
SARS-CoV-2 S RBD (L452R)	n/b	n/b
SARS-CoV-2 S Trimer	2.4 nM	0.68 nM
SARS SI	n/b	n/b
SARS-CoV-2 Sl-Fc (South Africa 178-08)	6.1 nM	26.8 nM
SARS-CoV-2 Sl-Fc (UK 178-09)	857 pM	332 pM

Example 8: Analysis of COVIC Antibody Binding Epitopes Against Variant SARS-CoV-2 Variants

Antibodies that bind to the receptor-binding motif are highly sensitive to emerging mutations. However, antibodies that bind outside of the RBM (CoVIC-94, 6-3, RBD-6; CoVIC-23, 182-7, RBD-9; COVIC-21, 182-3, RBD-10) showed an increased resistance to emerging mutations. This includes antibodies that target the N-terminal domain (NT) of the 51 spike protein.

The IC50 of neutralizing antibodies against pseudoviruses with single mutations relative to the G614-parent virus was tested. Results are depicted in FIG. 10. Values about 2.5 and below −2.5 indicate an increase and decrease in potency, respectively. “KO” indicates a complete loss of neutralization for the virus-antibody pair. 4 of the 6 variant antibodies tested, COVIC-94 (6-3), COVIC-23, COVIC-21 and COVIC-20 were resistant to variant SARS-CoV-2 variant mutations.

Example 9: VSV-Pseudotype SARS-CoV2 Neutralization Analysis for Variant SARS-CoV-2 Strains

Serial semi-log dilutions of all test antibodies (TA) and control were prepared and mixed with the VSV-pseudotype virus in a 1:1 ratio for 1 h at RT followed by incubation over Vero cells (ATCC® CCL-81™) seeded at 60,000 cells per well at 37° C. The cells were lysed the following day and luciferase activity was measured to assess the potency of each TA to block viral entry into the Vero cells. All samples were run in triplicate. Data analysis was conducted using XLFit and Graphpad Prism.

The percent neutralization of 1-12, 6-3 and 6-63 were measured against single mutations and variant pseudovirus strains. Results are depicted in FIGS. 11A-11F. 6-63 showed the highest levels of neutralization against the α variant. 6-3 showed the highest levels of neutralization against the β variant. 1-12 showed the highest level of neutralization against the δ and E variant.

Example 10: Variant Live Testing

Two cell lines were used to test the ability of antibody variants to neutralize SARS-CoV-2 variants in live cells: Vero cells overexpressing human ACE2 and TMPRSS2 (VAT) and Vero cells without overexpression of ACE2 and TMPRSS2 (WHO cells). Vero cells were infected with variant strains of SARS-CoV-2 isolates. To assess the binding efficiency of this panel of antibodies, each antibody was incubated with 10⁵ VERO cells at 100 nM, a labeled secondary antibody was used to measure binding using flow cytometry. The binding of each antibody was compared to a baseline value, consisting of secondary antibody alone, to derive a Mean Fluorescence Intensity (MFI) over baseline (MFI/Baseline). Antibody variants 6-1, 6-3, 6-63, 1-12 and 2165 were tested against AZ1 (human SARS-CoV-2 isolate from Arizona 2020) and B.1.351 (South African variant)

The results are depicted in FIGS. 12A-12D and Table 7. The epitopes that 6-1 and 1-21 recognize in AZ1 is not present in B.1.351 as there is no detectable neutralization to B.1.351 but there is to AZ1. mAb 6-3 seems to have better efficacy of neutralization of B.1.351 in comparison to AZ1. Overall, the V.A.T cell assay provides a higher level of sensitivity while using much less virus.

TABLE 7
EC50 [ng/mL] V.A.T Cells	EC50 [ng/mL] WHO Cells
	AZ1	B.1.351		AZ1	B.1.351
202-1	211.9	ND	202-1	5313	ND
202-3	207	8.195	202-3	361.2	62.53
202-63	131.2	12760	202-63	1967	0
181-8	32450	ND	181-8	ND	ND
h2165	72.68	9154	h2165	260	46680

Example 11. Sequences

Tables 8-30 show exemplary sequences for CDRH1-H3 and CDRL1-L3 as well as variant heavy chains and variant light chains for the SARS-CoV-2 and ACE2 variants.

TABLE 8
ACE2 VHH Variable Heavy Chain CDRs
	SEQ		SEQ		SEQ
	ID		ID		ID
Variant	NO	CDRH1	NO	CDRH2	NO	CDRH3
4-1	1	RTFSDDTMG	51	GGISWSGGNTYYA	101	CATDPPLFW
4-2	2	RTFGDYIMG	52	AAINWSAGYTAYA	102	CARASPNTGWHFDRW
4-3	3	RTFSDDAMG	53	AAINWSGGTTRYA	103	CATDPPLFW
4-4	4	RTFGDYIMG	54	AAINWIAGYTADA	104	CAEPSPNTGWHFDHW
4-5	5	RTFGDDTMG	55	AAINWSGGNTYYA	105	CATDPPLFW
4-6	6	RTFGDDTMG	56	AAINWTGGYTPYA	106	CATDPPLFW
4-7	7	RTFGDYIMG	57	AAINWSGGYTAYA	107	CATASPNTGWHFDHW
4-8	8	RTFGDYIMG	58	GGINWSGGYTYYA	108	CATDPPLFW
4-9	9	RTFGDYIMG	59	AAINWSGGYTHYA	109	CATDPPLFW
4-10	10	RTFSDDTMG	60	AAIHWSGSSTRYA	110	CATDPPLFW
4-11	11	RTFGDYAMG	61	APINWSGGSTYYA	111	CATDPPLFW
4-12	12	RTFGDDTMG	62	AAINWSGGNTPYA	112	CATDPPLFW
4-13	13	RTFGDDTMG	63	AAINWSGDNTHYA	113	CATDPPLFW
4-14	14	RTFSDDTMG	64	AAINWSGGTTRYA	114	CATDPPLFW
4-15	15	RTFSDDTMG	65	AAINWSGDSTYYA	115	CATDPPLFW
4-16	16	RTFSDYTMG	66	AAINWSGGYTYYA	116	CATDPPLFW
4-17	17	RTFGDDTMG	67	AAINWSGGNTDYA	117	CATDPPLFW
4-18	18	RTFGDYIMG	68	AAINWSGGYTPYA	118	CATDPPLFW
4-19	19	RTFSDDTMG	69	AAINWSGGSTYYA	119	CATDPPLFW
4-20	20	RTFGDDIMG	70	AAIHWSAGYTRYA	120	CATDPPLFWGHVDLW
4-21	21	RTFSDDTMG	71	AGMTWSGSSTFYA	121	CATDPPLFW
4-22	22	RTFGDYIMG	72	AAINWSGDNTHYA	122	CATDPPLFW
4-23	23	RTFSDDAMG	73	AGISWNGGSIYYA	123	CATDPPLFW
4-24	24	RTFSDYTMG	74	AAINWSGGTTYYA	124	CATDPPLFW
4-25	25	GTFSRYAMG	75	SAVDSGGSTYYA	125	CAASPSLRSAWQW
4-26	26	RTFSDDTMG	76	AAVNWSGGSTYY	126	CATDPPLFW
				A
4-27	27	RTFGDYIMG	77	AAINWSAGYTAYA	127	CARATPNTGWHFDHW
4-28	28	RTFGDDTMG	78	AAINWNGGNTHY	128	CATDPPLFW
				A
4-29	29	RTFGDDTMG	79	AAINWSGGYTYYA	129	CATDPPLFW
4-30	30	RTFGDYTMG	80	AAINWTGGYTYYA	130	CATDPPLFW
4-31	31	RTFGDYIMG	81	AAINWSAGYTAYA	131	CATASPNTGWHFDHW
4-32	32	FTFDDYEMG	82	AAISWRGGTTYYA	132	CAADRRGLASTRAGDYD
						W
4-33	33	FTFSRHDMG	83	AGINWESGSTNYA	133	CAADRGVYGGRWYRTS
						QYTW
4-34	34	RTFGDYIMG	84	AAINWSADYTAYA	134	CATDPPLFCWHFDHW
4-35	35	QLANFASYAM	85	AAITRSGSSTVYA	135	CATTMNPNPRW
		G
4-36	36	RTFGDYIMG	86	AAINWSAGYTAYA	136	CATAPPLFCWHFDHW
4-37	37	RTFGDYGMG	87	ATINWSGALTHYA	137	CATLPFYDFWSGYYTGY
						YYMDVW
4-38	38	RTFSDDTMG	88	AAITWSGGRTRYA	138	CATDRPLFW
4-39	39	RTFSNAAMG	89	ARILWTGASRNYA	139	CATTENPNPRW
4-40	40	RTFSDDTMG	90	AGINWSGNGVYY	140	CATDPPLFW
				A
4-41	41	RTFGDYIMG	91	AAINWSGGTTPYA	141	CATDPPLFCCHVDLW
4-42	42	RTFGDDTMG	92	AAINWSGGYTPYA	142	CATDPPLFWGHVDLW
4-43	43	RTFSDDTMG	93	AAINWSGGSTDYA	143	CATDPPLFW
4-44	44	RTFGDYIMG	94	AAINWSAGYTAYA	144	CATARPNTGWHFDHW
4-45	45	RTFSDDAMG	95	AAINWSGGSTRYA	145	CATDPPLFW
4-46	46	RTFGDYIMG	96	AAINWSAGYTPYA	146	CATDPPLFWGHVDLW
4-47	47	FTFGDYVMG	97	AAINWNAGYTAYA	147	CAKASPNTGWHFDHW
4-48	48	RTFSDDAMG	98	GRINWSGGNTYYA	148	CATDPPLFW
4-49	49	RTFGDYIMG	99	AAINWSAGYTAYA	149	CARASPNTGWHFDHW
4-50	50	GTFSNSGMG	100	AVVNWSGRRTYYA	150	CAVPWMDYNRRDW

TABLE 9
SARS-CoV-2 S1 Variable Heavy Chain CDRs
	SEQ		SEQ		SEQ
	ID		ID		ID
Variant	NO	CDRH1	NO	CDRH2	NO	CDRH3
2-1	151	FTFSNYATD	166	SIISGSGGATYYA	181	CAKGGYCSSDTCWWEYWLDPW
2-2	152	FTFSRHAMN	167	SGISGSGDETYY	182	CARDLPASYYDSSGYYWHNGMD
				A		VW
2-3	153	FTFSDFAMA	168	SAISGSGDITYYA	183	CAREADCLPSPWYLDLW
2-4	154	FTFSDFAMA	169	SAITGTGDITYYA	184	CAREADGLHSPW
2-5	155	FTFSDFAMA	170	SAISGSGDITYYA	185	CAREADGLHSPWHFDLW
2-6	156	FTFSDFAMA	171	SAISGSGDITYYA	186	CAREADGLHSPWHFDLW
2-7	157	FTFSDFAMA	172	SAITGSGDITYYA	187	CAREADGLHSPWHFDLW
2-8	158	FTFSDFAMA	173	SAISGSGDITYYA	188	CAREADGLHSPWHFDLW
2-9	159	FTFPRYAMS	174	STISGSGSTTYYA	189	CARLIDAFDIW
2-10	160	FTFSAFAMG	175	SAITASGDITYYA	190	CARQSDGLPSPWHFDLG
2-11	161	FTFSNYPMN	176	STISGSGGNTFYA	191	CVRHDEYSFDYW
2-12	162	FTFSDYPMN	177	STISGSGGITFYA	192	CVRHDEYSFDYW
2-13	163	FTFSDYPMN	178	SAISGSGDNTYY	193	CVRHDEYSFDYW
				A
2-14	164	FTFSDYPMN	179	SAITGSGDITYYA	194	CVRHDEYSFDYW
2-15	165	FTFSDYPMN	180	STISGSGGITFYA	195	CVRHDEYSFDYW

TABLE 10
SARS-CoV-2 S1 Variable Light Chain CDRs
	SEQ		SEQ		SEQ
	ID		ID		ID
Variant	NO	CDRL1	NO	CDRL2	NO	CDRL3
2-1	196	RASQSIHRFL	211	AASNLH	226	CQQSYGLPPTF
		N		S
2-2	197	RASQTINTYL	212	SASTLQS	227	CQQSYSTFTF
		N
2-3	198	RASQNIHTYL	213	AASTFA	228	CQQSYSAPPYTF
		N		K
2-4	199	RASQSIDTYL	214	AASALA	229	CQQSYSAPPYTF
		N		S
2-5	200	RASQSIHTYL	215	AASALA	230	CQQSYSAPPYTF
		N		S
2-6	201	RASQSIDTYL	216	AASALA	231	CQQSYSAPPYTF
		N		S
2-7	202	RASQSIDTYL	217	AASALA	232	CQQSYSAPPYTF
		N		S
2-8	203	RASQSIDTYL	218	AASALA	233	CQQSYSAPPYTF
		N		S
2-9	204	RASQRIGTYL	219	AASNLE	234	CQQNYSTTWTF
		N		G
2-10	205	RASQSIHISLN	220	LASPLAS	235	CQQSYSAPPYTF
2-11	206	RASQSIGNYL	221	GVSSLQ	236	CQQSHSAPLTF
		N		S
2-12	207	RASQSIDNYL	222	GVSALQ	237	CQQSHSAPPYFF
		N		S
2-13	208	RASQSIDTYL	223	GASALE	238	CQQSHSAPPYFF
		N		S
2-14	209	RASQSIDTYL	224	GVSALQ	239	CQQSYSAPPYFF
		N		S
2-15	210	RASQSIDNYL	225	GVSALQ	240	CQQSHSAPLTF
		N		S

TABLE 11
ACE2 Variable Heavy Chain CDRs
	SEQ		SEQ		SEQ
	ID		ID		ID
Variant	NO	CDRH1	NO	CDRH2	NO	CDRH3
3-1	241	FMFGNYAMS	256	AAISGSGGSTYY	271	CAKDRGYSSSWYGGFDYW
				A
3-2	242	FTFRSHAMN	257	SAISGSGGSTNYA	272	CARGLKFLEWLPSAFDIW
3-3	243	FTFRNYAMA	258	SGISGSGGTTYY	273	CARGTRFLEWSLPLDVW
				G
3-4	244	FTFRNHAMA	259	SGISGSGGTTYY	274	CARGTRFLQWSLPLDVW
				G
3-5	245	FTITNYAMS	260	SGISGSGAGTYY	275	CARHAWWKGAGFFDHW
				A
3-6	246	FTIPNYAMS	261	SGISGAGASTYY	276	CARHTWWKGAGFFDHW
				A
3-7	247	FTIPNYAMS	262	SGISGSGASTYYA	277	CARHTWWKGAGFFDHW
3-8	248	FTITNYAMS	263	SGISGSGASTYYA	278	CARHTWWKGAGFFDHW
3-9	249	FTITNYAMS	264	SGISGSGAGTYY	279	CARHTWWKGAGFFDHW
				A
3-10	250	FTFRSHAMS	265	SSISGGGASTYYA	280	CARVKYLTTSSGWPRPYFDN
						W
3-11	251	FTIRNYAMS	266	SSISGGGASTYYA	281	CARVKYLTTSSGWPRPYFDN
						W
3-12	252	FTFRSHAMS	267	SSISGGGASTYYA	282	CARVKYLTTSSGWPRPYFDN
						W
3-13	253	FTFRSHAMS	268	SSISGGGASTYYA	283	CARVKYLTTSSGWPRPYFDN
						W
3-14	254	FTFRSYAMS	269	SSISGGGASTYYA	284	CARVKYLTTSSGWPRPYFDN
						W
3-15	255	FTFSAYSMS	270	SAISGSGGSRYYA	285	CGRSKWPQANGAFDIW

TABLE 12
ACE2 Variable Light Chain CDRs
	SEQ ID		SEQ ID		SEQ ID
Name	NO	CDRL1	NO	CDRL2	NO	CDRL3
3-1	286	RASQTIYSYLN	301	ATSTLQG	316	CQHRGTF
3-2	287	RTSQSINTYLN	302	GASNVQS	317	CQQSYRIPRTF
3-3	288	RASRSISRYLN	303	AASSLQA	318	CQQSYSSLLTF
3-4	289	RASRSIRRYLN	304	ASSSLQA	319	CQQSYSTLLTF
3-5	290	RASQSIGRYLN	305	AASSLKS	320	CQQSYSLPRTF
3-6	291	RASQSIGKYLN	306	ASSSLQS	321	CQQSYSPPFTF
3-7	292	RASQSIGRYLN	307	ASSSLQS	322	CQQSYSLPRTF
3-8	293	RASQSIGRYLN	308	AASSLKS	323	CQQSYSLPLTF
3-9	294	RASQSIGRYLN	309	AASSLKS	324	CQQSYSLPRTF
3-10	295	RASQSIRKYLN	310	ASSTLQR	325	CQQSLSTPFTF
3-11	296	RASQSIGKYLN	311	ASSTLQR	326	CQQSLSPPFTF
3-12	297	RASQSIGKYLN	312	ASSTLQR	327	CQQSLSTPFTF
3-13	298	RASQSIGKYLN	313	ASSTLQR	328	CQQSFSPPFTF
3-14	299	RASQSIGKYLN	314	ASSTLQR	329	CQQSFSTPFTF
3-15	300	RASQNIKTYLN	315	AASKLQS	330	CQQSYSTSPTF

TABLE 13
SARS-CoV-2 S1 Variable Heavy Chain CDRs
	SEQ		SEQ		SEQ
	ID		ID		ID
Name	NO	CDRH1	NO	CDRH2	NO	CDRH3
2-1	331	FTFSNYA	358	SIISGSGGA	385	CAKGGYCSSDTCWWEYWLD
		TD		TYYA		PW
2-10	332	FTFSAFA	359	SAITASGDI	386	CARQSDGLPSPWHFDLG
		MG		TYYA
2-5	333	FTFSDFA	360	SAISGSGDI	387	CAREADGLHSPWHFDLW
		MA		TYYA
2-2	334	FTFSRHA	361	SGISGSGDE	388	CARDLPASYYDSSGYYWHN
		MN		TYYA		GMDVW
2-4	335	FTFSDFA	362	SAISGSGDI	389	CAREADGLHSPWHFDLW
		MA		TYYA
2-6	336	FTFSNYP	363	STISGSGGN	390	CVRHDEYSFDYW
		MN		TFYA
2-11	337	FTFSDFA	364	SAITGSGDI	391	CAREADGLHSPWHFDLW
		MA		TYYA
2-12	338	FTFSDYP	365	STISGSGGI	392	CVRHDEYSFDYW
		MN		TFYA
2-13	339	FTFSDYP	366	SAISGSGDN	393	CVRHDEYSFDYW
		MN		TYYA
2-14	340	FTFSDFA	367	SAITGTGDI	394	CAREADGLHSPW
		MA		TYYA
2-7	341	FTFSDYP	368	SAITGSGDI	395	CVRHDEYSFDYW
		MN		TYYA
2-8	342	FTFSDFA	369	SAISGSGDI	396	CAREADGLHSPWHFDLW
		MA		TYYA
2-15	343	FTFSDFA	370	SAISGSGDI	397	CAREADGLHSPWHFDLW
		MA		TYYA
2-9	344	FTFPRYA	371	STISGSGST	398	CARLIDAFDIW
		MS		TYYA
2-16	345	FTFSSYA	372	SVISGSGGS	399	CAREGYRDYLWYFDLW
		MS		TYYA
2-17	346	FTFSNYA	373	SAISGSAGS	400	CARVRQGLRRTWYYFDYW
		MS		TYYA
2-18	347	FTFSSYA	374	SAISGSAGS	401	CARDTNDFWSGYSIFDPW
		MY		TYYA
2-19	348	FTFSSYT	375	SVISGSGGS	402	CAREGYRDYLWYFDLW
		MS		TYYA
2-2	349	FTFSSYD	376	SVISGSGGS	403	CAKGPLVGWYFDLW
		MS		TYYA
2-21	350	FTFPRYA	377	STISGSGST	404	CARLIDAFDIW
		MS		TYYA
2-22	351	FTFTTYA	378	SGISGSGDE	405	CTTGDDFWSGGNWFDPW
		LS		TYYA
2-23	352	FTFSRHA	379	SGITGSGDE	406	CARDLPASYYDSSGYYWHN
		MN		TYYA		GMDVW
2-24	353	FVFSSYA	380	SAISGSGGS	407	CARVGGGYWYGIDVW
		MS		SYYA
2-25	354	FTLSSYV	381	SGISGGGAS	408	CARGYSRNWYPSWFDPW
		MS		TYYA
2-26	355	FTFSTYA	382	SSIGGSGST	409	CAGGWYLDYW
		MS		TYYA
2-27	356	FTYSNYA	383	SAISGSSGS	410	CASLCIVDPFDIW
		MT		TYYA
2-28	357	FTFSNYP	384	STISGSGGN	411	CVRHDEYSFDYW
		MN		TFYA

TABLE 14
SARS-CoV-2 S1 Variable Light Chain CDRs
			SEQ
	SEQ ID		ID		SEQ ID
Name	NO	CDRL1	NO	CDRL2	NO	CDRL3
2-1	412	RASQSIHRFLN	439	AASNLHS	466	CQQSYGLPPTF
2-10	413	RASQSIHISLN	440	LASPLAS	467	CQQSYSAPPYTF
2-5	414	RASQSIHTYLN	441	AASALAS	468	CQQSYSAPPYTF
2-2	415	RASQTINTYLN	442	SASTLQS	469	CQQSYSTFTF
2-4	416	RASQSIDTYLN	443	AASALAS	470	CQQSYSAPPYTF
2-6	417	RASQSIGNYLN	444	GVSSLQS	471	CQQSHSAPLTF
2-11	418	RASQSIDTYLN	445	AASALAS	472	CQQSYSAPPYTF
2-12	419	RASQSIDNYLN	446	GVSALQS	473	CQQSHSAPPYFF
2-13	420	RASQSIDTYLN	447	GASALES	474	CQQSHSAPPYFF
2-14	421	RASQSIDTYLN	448	AASALAS	475	CQQSYSAPPYTF
2-7	422	RASQSIDTYLN	449	GVSALQS	476	CQQSYSAPPYFF
2-8	423	RASQSIDTYLN	450	AASALAS	477	CQQSYSAPPYTF
2-15	424	RASQSIDNYLN	451	GVSALQS	478	CQQSHSAPLTF
2-9	425	RASQRIGTYLN	452	AASNLEG	479	CQQNYSTTWTF
2-16	426	TGTSSDVGSYDLVS	453	EGNKRPS	480	CCSYAGSSVVF
2-17	427	TGTSSDVGSSNLVS	454	EGSKRPS	481	CCSYAGSLYVF
2-18	428	TGTSSDIGSYNLVS	455	EGTKRPS	482	CCSYAGSRTYVF
2-19	429	TGTSTDVGSYNLVS	456	EGTKRPS	483	CCSYAGSYTSVVF
2-2	430	TGTSSNVGSYNLVS	457	EGTKRPS	484	CCSYAGSSSFVVF
2-21	431	RASQSIHTYLN	458	AASALAS	485	CQQSYSAPPYTF
2-22	432	RASQSIHTYLN	459	AASALAS	486	CQQSYSAPPYTF
2-23	433	RASQTINTFLN	460	SASTLQS	487	CQQSYSTFTF
2-24	434	RASQTIRTYLN	461	DASTLQR	488	CQQSYRTPPWTF
2-25	435	RSSQSISSYLN	462	GASRLRS	489	CQQGYSAPWTF
2-26	436	RASQSISGSLN	463	AESRLHS	490	CQQSYSPPQTF
2-27	437	RASRSISTYLN	464	AASNLQG	491	CQQSHSIPRTF
2-28	438	RASQSIHTYLN	465	AASALAS	492	CQQSYSAPPYTF

TABLE 15
SARS-CoV-2 S1 Variant Sequences Variable Heavy Chain
	SEQ
	ID
Name	NO	Amino Acid Sequence
2-1	493	EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYATDWVRQAPGKGLEWVS
		IISGSGGATYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKG
		GYCSSDTCWWEYWLDPWGQGTLVTVSS
2-10	494	EVQLLESGGGLVQPGGSLRLSCAASGFTFSAFAMGWVRQAPGKGLEWV
		SAITASGDITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
		QSDGLPSPWHFDLGGQGTLVTVSS
2-5	495	EVQLLESGGGLVQPGGSLRLSCAASGFTFSDFAMAWVRQAPGKGLEWV
		SAISGSGDITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
		EADGLHSPWHFDLWGQGTLVTVSS
2-2	496	EVQLLESGGGLVQPGGSLRLSCAASGFTFSRHAMNWVRQAPGKGLEWV
		SGISGSGDETYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA
		RDLPASYYDSSGYYWHNGMDVWGQGTLVTVSS
2-4	497	EVQLLESGGGLVQPGGSLRLSCAASGFTFSDFAMAWVRQAPGKGLEWV
		SAISGSGDITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
		EADGLHSPWHFDLWGQGTLVTVSS
2-6	498	EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYPMNWVRQAPGKGLEWV
		STISGSGGNTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVR
		HDEYSFDYWGQGTLVTVSS
2-11	499	EVQLLESGGGLVQPGGSLRLSCAASGFTFSDFAMAWVRQAPGKGLEWV
		SAITGSGDITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
		EADGLHSPWHFDLWGQGTLVTVSS
2-12	500	EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYPMNWVRQAPGKGLEWV
		STISGSGGITFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVR
		HDEYSFDYWGQGTLVTVSS
2-13	501	EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYPMNWVRQAPGKGLEWV
		SAISGSGDNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCV
		RHDEYSFDYWGQGTLVTVSS
2-14	502	EVQLLESGGGLVQPGGSLRLSCAASGFTFSDFAMAWVRQAPGKGLEWV
		SAITGTGDITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
		EADGLHSPWGQGTLVTVSS
2-7	503	EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYPMNWVRQAPGKGLEWV
		SAITGSGDITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVR
		HDEYSFDYWGQGTLVTVSS
2-8	504	EVQLLESGGGLVQPGGSLRLSCAASGFTFSDFAMAWVRQAPGKGLEWV
		SAISGSGDITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
		EADGLHSPWHFDLWGQGTLVTVSS
2-15	505	EVQLLESGGGLVQPGGSLRLSCAASGFTFSDFAMAWVRQAPGKGLEWV
		SAISGSGDITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
		EADGLHSPWHFDLWGQGTLVTVSS
2-9	506	EVQLLESGGGLVQPGGSLRLSCAASGFTFPRYAMSWVRQAPGKGLEWVS
		TISGSGSTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARL
		IDAFDIWGQGTLVTVSS
2-16	507	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVS
		VISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
		EGYRDYLWYFDLWGQGTLVTVSS
2-17	508	EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWV
		SAISGSAGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
		VRQGLRRTWYYFDYWGQGTLVTVSS
2-18	509	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMYWVRQAPGKGLEWV
		SAISGSAGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
		DTNDFWSGYSIFDPWGQGTLVTVSS
2-19	510	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVS
		VISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
		EGYRDYLWYFDLWGQGTLVTVSS
2-2	511	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVS
		VISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
		GPLVGWYFDLWGQGTLVTVSS
2-21	512	EVQLLESGGGLVQPGGSLRLSCAASGFTFPRYAMSWVRQAPGKGLEWVS
		TISGSGSTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARL
		IDAFDIWGQGTLVTVSS
2-22	513	EVQLLESGGGLVQPGGSLRLSCAASGFTFTTYALSWVRQAPGKGLEWVS
		GISGSGDETYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTT
		GDDFWSGGNWFDPWGQGTLVTVSS
2-23	514	EVQLLESGGGLVQPGGSLRLSCAASGFTFSRHAMNWVRQAPGKGLEWV
		SGITGSGDETYYADSVKGRFTISRDNSKNTLYLQMNSLKAEDTAVYYCA
		RDLPASYYDSSGYYWHNGMDVWGQGTLVTVSS
2-24	515	EVQLLESGGGLVQPGGSLRLSCAASGFVFSSYAMSWVRQAPGKGLEWVS
		AISGSGGSSYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
		VGGGYWYGIDVWGQGTLVTVSS
2-25	516	EVQLLESGGGLVQPGGSLRLSCAASGFTLSSYVMSWVRQAPGKGLEWVS
		GISGGGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
		GYSRNWYPSWFDPWGQGTLVTVSS
2-26	517	EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVS
		SIGGSGSTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAG
		GWYLDYWGQGTLVTVSS
2-27	518	EVQLLGSGGGLVQPGGSLRLSCAASGFTYSNYAMTWVRQAPGKGLEWV
		SAISGSSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAS
		LCIVDPFDIWGQGTLVTVSS
2-28	519	EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYPMNWVRQAPGKGLEWV
		STISGSGGNTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVR
		HDEYSFDYWGQGTLVTVSS

TABLE 16
SARS-CoV-2 S1 Variant Sequences Variable Light Chain
	SEQ
	ID
Name	NO	Amino Acid Sequence
2-1	520	DIQMTQSPSSLSASVGDRVTITCRASQSIHRFLNWYQQKPGKAPKLLIYAA
		SNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYGLPP-
		TFGQGTKVEIK
2-10	521	DIQMTQSPSSLSASVGDRVTITCRASQSIHISLNWYQQKPGKAPKLLIYLAS
		PLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPPYTFGQGTK
		VEIK
2-5	522	DIQMTQSPSSLSASVGDRVTITCRASQSIHTYLNWYQQKPGKAPKLLIYAA
		SALASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPPYTFGQGT
		KVEIK
2-2	523	DIQMTQSPSSLSASVGDRVTITCRASQTINTYLNWYQQKPGKAPKLLIYSA
		STLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTFTFGQGTKV
		EIK
2-4	524	DIQMTQSPSSLSASVGDRVTITCRASQSIDTYLNWYQQKPGKAPKLLIYAA
		SALASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPPYTFGQGT
		KVEIK
2-6	525	DIQMTQSPSSLSASVGDRVTITCRASQSIDTYLNWYQQKPGKAPKLLIYAA
		SALASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPPYTFGQGT
		KVEIK
2-11	526	DIQMTQSPSSLSASVGDRVTITCRASQSIGNYLNWYQQKPGKAPKLLIYGV
		SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHSAPLTFGQGTK
		VEIK
2-12	527	DIQMTQSPSSLSASVGDRVTITCRASQSIDNYLNWYQQKPGKAPKLLIYGV
		SALQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHSAPPYFFGQGT
		KVEIK
2-13	528	DIQMTQSPSSLSASVGDRVTITCRASQSIDTYLNWYQQKPGKAPKLLIYGA
		SALESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHSAPPYFFGQGT
		KVEIK
2-14	529	DIQMTQSPSSLSASVGDRVTITCRASQSIDTYLNWYQQKPGKAPKLLIYGV
		SALQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPPYFFGQGT
		KVEIK
2-7	530	DIQMTQSPSSLSASVGDRVTITCRASQSIDTYLNWYQQKPGKAPKLLIYAA
		SALASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPPYTFGQGT
		KVEIK
2-8	531	DIQMTQSPSSLSASVGDRVTITCRASQSIDTYLNWYQQKPGKAPKLLIYAA
		SALASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPPYTFGQGT
		KVEIK
2-15	532	DIQMTQSPSSLSASVGDRVTITCRASQSIDNYLNWYQQKPGKAPKLLIYGV
		SALQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHSAPLTFGQGTK
		VEIK
2-9	533	DIQMTQSPSSLSASVGDRVTITCRASQRIGTYLNWYQQKPGKAPKLLIYAA
		SNLEGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNYSTTWTFGQGT
		KVEIK
2-16	534	DIQMTQSPSSLSASVGDRVTITCTGTSSDVGSYDLVSWYQQKPGKAPKLLI
		YEGNKRPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCCSYAGSSWFG
		QGTKVEIK
2-17	535	DIQMTQSPSSLSASVGDRVTITCTGTSSDVGSSNLVSWYQQKPGKAPKLLI
		YEGSKRPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCCSYAGSLYVFGQ
		GTKVEIK
2-18	536	DIQMTQSPSSLSASVGDRVTITCTGTSSDIGSYNLVSWYQQKPGKAPKLLI
		YEGTKRPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCCSYAGSRTYVFG
		QGTKVEIK
2-19	537	DIQMTQSPSSLSASVGDRVTITCTGTSTDVGSYNLVSWYQQKPGKAPKLLI
		YEGTKRPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCCSYAGSYTSVVF
		GQGTKVEIK
2-2	538	DIQMTQSPSSLSASVGDRVTITCTGTSSNVGSYNLVSWYQQKPGKAPKLLI
		YEGTKRPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCCSYAGSSSFVVF
		GQGTKVEIK
2-21	539	DIQMTQSPSSLSASVGDRVTITCRASQSIHTYLNWYQQKPGKAPKLLIYAA
		SALASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPPYTFGQGT
		KVEIK
2-22	540	DIQMTQSPSSLSASVGDRVTITCRASQSIHTYLNWYQQKPGKAPKLLIYAA
		SALASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPPYTFGQGT
		KVEIK
2-23	541	DIQMTQSPSSLSASVGDRVTITCRASQTINTFLNWYQQKPGKAPKLLIYSA
		STLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTFTFGGGTKV
		EIK
2-24	542	DIQMTQSPSSLSASVGDRVTITCRASQTIRTYLNWYRQKPGKAPKLLIYDA
		STLQRGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYRTPPWTFGGGT
		KVEIK
2-25	543	DIQMTQSPSSLSASVGDRVTITCRSSQSISSYLNWYQQKPGEAPKLLIYGAS
		RLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYSAPWTFGGGTK
		VEIK
2-26	544	DIQMTQSPSSLSASVGDRVTITCRASQSISGSLNWYQQKPGKAPKLLIYAES
		RLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSPPQTFGGGTKV
		EIK
2-27	545	DIQMTQSPSSLSASVGDRVTITCRASRSISTYLNWYQQKPGKAPKLLIYAA
		SNLQGGVPSRLSGSGSGTDFTLTISSLQPEDFATYYCQQSHSIPRTFGGGTK
		VEIK
2-28	546	DIQMTQSPSSLSASVGDRVTITCRASQSIHTYLNWYQQKPGKAPKLLIYAA
		SALASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPPYTFGQGT
		KVEIK

TABLE 17
ACE2 Variable Heavy Chain CDRs
	SEQ		SEQ
	ID		ID		SEQ
Name	NO	CDRH1	NO	CDRH2	ID NO	CDRH3
3-10	547	FTFRSHAMS	576	SSISGGGAST	605	CARVKYLTTSSGWPR
				YYA		PYFDNW
3-4	548	FTFSAYSMS	577	SAISGSGGSR	606	CGRSKWPQANGAFDI
				YYA		W
3-7	549	FMFGNYAM	578	AAISGSGGST	607	CAKDRGYSSSWYGG
		S		YYA		FDYW
3-1	550	FTFRNHAM	579	SGISGSGGTT	608	CARGTRFLQWSLPLD
		A		YYG		VW
3-5	551	FTIPNYAMS	580	SGISGAGAST	609	CARHTWWKGAGFFD
				YYA		HW
3-6	552	FTFRNYAM	581	SGISGSGGTT	610	CARGTRFLEWSLPLD
		A		YYG		VW
3-15	553	FTIRNYAMS	582	SSISGGGAST	611	CARVKYLTTSSGWPR
				YYA		PYFDNW
3-3	554	FTIPNYAMS	583	SGISGSGAST	612	CARHTWWKGAGFFD
				YYA		HW
3-11	555	FTITNYAMS	584	SGISGSGAGT	613	CARHAWWKGAGFFD
				YYA		HW
3-8	556	FTFRSHAMS	585	SSISGGGAST	614	CARVKYLTTSSGWPR
				YYA		PYFDNW
3-2	557	FTITNYAMS	586	SGISGSGAST	615	CARHTWWKGAGFFD
				YYA		HW
3-12	558	FTFRSHAM	587	SAISGSGGST	616	CARGLKFLEWLPSAF
		N		NYA		DIW
3-14	559	FTFRSHAMS	588	SSISGGGAST	617	CARVKYLTTSSGWPR
				YYA		PYFDNW
3-9	560	FTFRSYAMS	589	SSISGGGAST	618	CARVKYLTTSSGWPR
				YYA		PYFDNW
3-13	561	FTITNYAMS	590	SGISGSGAGT	619	CARHTWWKGAGFFD
				YYA		HW
3-16	562	FTFTNFAMS	591	SAISGRGGG	620	CARDAHGYYYDSSG
				TYYA		YDDW
3-17	563	FTFRSYPMS	592	STISGSGGIT	621	CAKGVYGSTVTTCH
				YYA		W
3-18	564	FTLTSYAMS	593	SAISGSGVDT	622	CARPTNWGFDYW
				YYA
3-19	565	FTFINYAMS	594	STISTSGGNT	623	CARADSNWASSAYW
				YYA
3-2	566	FPFSTYAMS	595	SGISVSGGFT	624	CARDPYSYGYYYYY
				YYA		GMDVW
3-21	567	FTFSTYAM	596	SGISGGGVST	625	CARARNWGPSDYW
		G		YYA
3-22	568	FIFSDYAMT	597	SAISGSAFYA	626	CARDATYSSSWYNW
						FDPW
3-23	569	FTFSDYAM	598	SDISGSGGST	627	CARGTVTSFDFW
		T		YYA
3-24	570	FTFSIYAMG	599	SFISGSGGST	628	CAKDYHSASWFSAA
				YYA		ADYW
3-25	571	FTFASYAM	600	SAISESGGST	629	CAREGQEYSSGSSYF
		T		YYA		DYW
3-26	572	FTFSEYAMS	601	SAITGSGGST	630	CARGSQTPYCGGDCP
				YYG		ETFDYW
3-27	573	FTFDDYAM	602	SGISGGGTST	631	CARDLYSSGWYGFD
		S		YYA		YW
3-28	574	FTFNNYAM	603	SAISGSVGST	632	CARDNYDFWSGYYT
		N		YYA		NWFDPW
3-29	575	FTFTNHAM	604	SAISGSGSNI	633	CARDSLSVTMGRGV
		s		YYA		VTYYYYGMDFW

TABLE 18
ACE2 Variant Sequences Variable Light Chain
	SEQ		SEQ		SEQ
	ID		ID		ID
Name	NO	CDRL1	NO	CDRL2	NO	CDRL3
3-10	634	RASQSIRKYLN	663	ASSTLQR	692	CQQSLSTPFTF
3-4	635	RASQNIKTYLN	664	AASKLQS	693	CQQSYSTSPTF
3-7	636	RASQTIYSYLN	665	ATSTLQG	694	CQHRGTF
3-1	637	RASRSIRRYLN	666	ASSSLQA	695	CQQSYSTLLTF
3-5	638	RASQSIGKYLN	667	ASSSLQS	696	CQQSYSPPFTF
3-6	639	RASRSISRYLN	668	AASSLQA	697	CQQSYSSLLTF
3-15	640	RASQSIGKYLN	669	ASSTLQR	698	CQQSLSPPFTF
3-3	641	RASQSIGRYLN	670	ASSSLQS	699	CQQSYSLPRTF
3-11	642	RASQSIGRYLN	671	AASSLKS	700	CQQSYSLPRTF
3-8	643	RASQSIGKYLN	672	ASSTLQR	701	CQQSLSTPFTF
3-2	644	RASQSIGRYLN	673	AASSLKS	702	CQQSYSLPLTF
3-12	645	RTSQSINTYLN	674	GASNVQS	703	CQQSYRIPRTF
3-14	646	RASQSIGKYLN	675	ASSTLQR	704	CQQSFSPPFTF
3-9	647	RASQSIGKYLN	676	ASSTLQR	705	CQQSFSTPFTF
3-13	648	RASQSIGRYLN	677	AASSLKS	706	CQQSYSLPRTF
3-16	649	RASQIIGSYLN	678	TTSNLQS	707	CQQSYITPWTF
3-17	650	RASQSISRYIN	679	EASSLES	708	CQQSHITPLTF
3-18	651	RASQSIYTYLN	680	SASNLHS	709	CQQSDTTPWTF
3-19	652	RASQSIATYLN	681	GASSLEG	710	CQQTFSSPFTF
3-2	653	RASQNINTYLN	682	SASSLQS	711	CQQSSLTPWTF
3-21	654	RASQGIATYLN	683	YASNLQS	712	CQQSYSTRFTF
3-22	655	RASERISNYLN	684	TASNLES	713	CQQSYTPPRTF
3-23	656	RASQSISSSLN	685	AASRLQD	714	CQQSYSTPRSF
3-24	657	RASQSISSHLN	686	RASTLQS	715	CQQTYNTPQTF
3-25	658	RASQSISSYLI	687	AASRLHS	716	CQQGYNTPRTF
3-26	659	RASPSISTYLN	688	TASRLQT	717	CQQTYSTPSSF
3-27	660	RASQNIAKYLN	689	GASGLQS	718	CQQSHSPPITF
3-28	661	RASQSIGTYLN	690	AASNLHS	719	CQESYSAPYTF
3-29	662	RASQSISPYLN	691	KASSLQS	720	CQQSSSTPYTF

TABLE 19
ACE2 Variant Sequences Variable Heavy Chain
	SEQ ID
Name	NO	Amino Acid Sequence
3-10	721	EVQLLESGGGLVQPGGSLRLSCAASGFTFRSHAMSWVRQAPGKGL
		EWVSSISGGGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT
		AVYYCARVKYLTTSSGWPRPYFDNWGQGTLVTVSS
3-4	722	EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYSMSWVRQAPGKGL
		EWVSAISGSGGSRYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT
		AVYYCGRSKWPQANGAFDIWGQGTLVTVSS
3-7	723	EVQLLESGGGLVQPGGSLRLSCAASGFMFGNYAMSWVRQAPGKG
		LEWVAAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAE
		DTAVYYCAKDRGYSSSWYGGFDYWGQGTLVTVSS
3-1	724	EVQLLESGGGLVQPGGSLRLSCAASGFTFRNHAMAWVRQAPGKG
		LEWVSGISGSGGTTYYGDSVKGRFTISRDNSKNTLYLQMNSLRAED
		TAVYYCARGTRFLQWSLPLDVWGQGTLVTVSS
3-5	725	EVQLLESGGGLVQPGGSLRLSCAASGFTIPNYAMSWVRQAPGKGL
		EWVSGISGAGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED
		TAVYYCARHTWWKGAGFFDHWGQGTLVTVSS
3-6	726	EVQLLESGGGLVQPGGSLRLSCAASGFTFRNYAMAWVRQAPGKG
		LEWVSGISGSGGTTYYGDSVKGRFTISRDNSKNTLYLQMNSLRAED
		TAVYYCARGTRFLEWSLPLDVWGQGTLVTVSS
3-15	727	EVQLLESGGGLVQPGGSLRLSCAASGFTIRNYAMSWVRQAPGKGL
		EWVSSISGGGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT
		AVYYCARVKYLTTSSGWPRPYFDNWGQGTLVTVSS
3-3	728	EVQLLESGGGLVQPGGSLRLSCAASGFTIPNYAMSWVRQAPGKGL
		EWVSGISGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT
		AVYYCARHTWWKGAGFFDHWGQGTLVTVSS
3-11	729	EVQLLESGGGLVQPGGSLRLSCAASGFTITNYAMSWVRQAPGKGL
		EWVSGISGSGAGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED
		TAVYYCARHAWWKGAGFFDHWGQGTLVTVSS
3-8	730	EVQLLESGGGLVQPGGSLRLSCAASGFTFRSHAMSWVRQAPGKGL
		EWVSSISGGGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT
		AVYYCARVKYLTTSSGWPRPYFDNWGQGTLVTVSS
3-2	731	EVQLLESGGGLVQPGGSLRLSCAASGFTITNYAMSWVRQAPGKGL
		EWVSGISGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT
		AVYYC ARHTWWKGAGFFDHWGQGTLVTVSS
3-12	732	EVQLLESGGGLVQPGGSLRLSCAASGFTFRSHAMNWVRQAPGKGL
		EWVSAISGSGGSTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT
		AVYYCARGLKFLEWLPSAFDIWGQGTLVTVSS
3-14	733	EVQLLESGGGLVQPGGSLRLSCAASGFTFRSHAMSWVRQAPGKGL
		EWVSSISGGGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT
		AVYYCARVKYLTTSSGWPRPYFDNWGQGTLVTVSS
3-9	734	EVQLLESGGGLVQPGGSLRLSCAASGFTFRSYAMSWVRQAPGKGL
		EWVSSISGGGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT
		AVYYCARVKYLTTSSGWPRPYFDNWGQGTLVTVSS
3-13	735	EVQLLESGGGLVQPGGSLRLSCAASGFTITNYAMSWVRQAPGKGL
		EWVSGISGSGAGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED
		TAVYYCARHTWWKGAGFFDHWGQGTLVTVSS
3-16	736	EVQLLESGGGLVQPGGSLRLSCAASGFTFTNFAMSWVRQAPGKGL
		EWVSAISGRGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED
		TAVYYCARDAHGYYYDSSGYDDWGQGTLVTVSS
3-17	737	EVQLLESGGGLVQPGGSLRLSCAASGFTFRSYPMSWVRQAPGKGL
		EWVSTISGSGGITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT
		AVYYCAKGVYGSTVTTCHWGQGTLVTVSS
3-18	738	EVQLLESGGGLVQPGGSLRLSCAASGFTLTSYAMSWVRQAPGKGL
		EWVSAISGSGVDTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED
		TAVYYCARPTNWGFDYWGQGTLVTVSS
3-19	739	EVQLLESGGGLVQPGGSLRLSCAASGFTFINYAMSWVRQAPGKGL
		EWVSTISTSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT
		AVYYCARADSNWASSAYWGQGTLVTVSS
3-2	740	EVQLLESGGGLVQPGGSLRLSCAASGFPFSTYAMSWVRQAPGKGL
		EWVSGISVSGGFTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT
		AVYYCARDPYSYGYYYYYGMDVWGQGTLVTVSS
3-21	741	EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMGWVRQAPGKGL
		EWVSGISGGGVSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED
		TAVYYCARARNWGPSDYWGQGTLVTVSS
3-22	742	EVQLLESGGGLVQPGGSLRLSCAASGFIFSDYAMTWVRQAPGKGL
		EWVSAISGSAFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAV
		YYCARDATYSSSWYNWFDPWGQGTLVTVSS
3-23	743	EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYAMTWVRQAPGKGL
		EWVSDISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT
		AVYYCARGTVTSFDFWGQGTLVTVSS
3-24	744	EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYAMGWVRQAPGKGL
		EWVSFISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT
		AVYYCAKDYHSASWFSAAADYWGQGTLVTVSS
3-25	745	EVQLLESGGGLVQPGGSLRLSCAASGFTFASYAMTWVRQAPGKGL
		EWVSAISESGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT
		AVYYCAREGQEYSSGSSYFDYWGQGTLVTVSS
3-26	746	EVQLLESGGGLVQPGGSLRLSCAASGFTFSEYAMSWVRQAPGKGL
		EWVSAITGSGGSTYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDT
		AVYYCARGSQTPYCGGDCPETFDYWGQGTLVTVSS
3-27	747	EVQLLESGGGLVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGL
		EWVSGISGGGTSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT
		AVYYCARDLYSSGWYGFDYWGQGTLVTVSS
3-28	748	EVQLLESGGGLVQPGGSLRLSCAASGFTFNNYAMNWVRQAPGKG
		LEWVSAISGSVGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED
		TAVYYCARDNYDFWSGYYTNWFDPWGQGTLVTVSS
3-29	749	EVQLLESGGGLVQPGGSLRLSCAASGFTFTNHAMSWVRQAPGKGL
		EWVSAISGSGSNIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT
		AVYYCARDSLSVTMGRGVVTYYYYGMDFWGQGTLVTVSS

TABLE 20
ACE2 Variant Sequences Variable Light Chain
	SEQ
Name	ID NO	Amino Acid Sequence
3-10	750	DIQMTQSPSSLSASVGDRVTITCRASQSIRKYLNWYQQKPGKAPKLLIY
		ASSTLQRGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLSTPFTFG
		GGTKVEIK
3-4	751	DIQMTQSPSSLSASVGDRVTITCRASRSIRRYLNWYQQKPGKAPKLLIY
		ASSSLQAGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTLLTFG
		QGTKVEIK
3-7	752	DIQMTQSPSSLSASVGDRVTITCRASQSIGRYLNWYQQKPGKAPKLLIY
		ASSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSLPRTFG
		QGTKVEIK
3-1	753	DIQMTQSPSSLSASVGDRVTITCRASQTIYSYLNWYQQKPGKAPKLLIY
		ATSTLQGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHRGTFGQGT
		KVEIK
3-5	754	DIQMTQSPSSLSASVGDRVTITCRASQSIGRYLNWYQQKPGKAPKLLIY
		AASSLKSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSLPRTFG
		QGTKVEIK
3-6	755	DIQMTQSPSSLSASVGDRVTITCRASQSIGKYLNWYQQKPGKAPKLLIY
		ASSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSPPFTFG
		QGTKVEIK
3-15	756	DIQMTQSPSSLSASVGDRVTITCRASQNIKTYLNWYQQKPGKAPKLLIY
		AASKLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTSPTFG
		QGTKVEIK
3-3	757	DIQMTQSPSSLSASVGDRVTITCRASRSISRYLNWYQQKPGKAPKLLIY
		AASSLQAGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSLLTFG
		QGTKVEIK
3-11	758	DIQMTQSPSSLSASVGDRVTITCRASQSIGKYLNWYQQKPGKAPKLLIY
		ASSTLQRGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLSPPFTFG
		QGTKVEIK
3-8	759	DIQMTQSPSSLSASVGDRVTITCRASQSIGRYLNWYQQKPGKAPKLLIY
		AASSLKSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSLPLTFG
		QGTKVEIK
3-2	760	DIQMTQSPSSLSASVGDRVTITCRTSQSINTYLNWYQQKPGKAPKLLIY
		GASNVQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYRIPRTFG
		QGTKVEIK
3-12	761	DIQMTQSPSSLSASVGDRVTITCRASQSIGKYLNWYQQKPGKAPKLLIY
		ASSTLQRGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLSTPFTFG
		QGTKVEIK
3-14	762	DIQMTQSPSSLSASVGDRVTITCRASQSIGKYLNWYQQKPGKAPKLLIY
		ASSTLQRGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSFSTPFTFG
		QGTKVEIK
3-9	763	DIQMTQSPSSLSASVGDRVTITCRASQSIGRYLNWYQQKPGKAPKLLIY
		AASSLKSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSLPRTFG
		QGTKVEIK
3-13	764	DIQMTQSPSSLSASVGDRVTITCRASQSIGKYLNWYQQKPGKAPKLLIY
		ASSTLQRGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSFSPPFTFG
		QGTKVEIK
3-16	765	DIQMTQSPSSLSASVGDRVTITCRASQIIGSYLNWYQQKPGKAPKLLIY
		TTSNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYITPWTFG
		QGTKVEIK
3-17	766	DIQMTQSPSSLSASVGDRVTITCRASQSISRYINWYQQKPGKAPKLLIY
		EASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHITPLTFGQ
		GTKVEIK
3-18	767	DIQMTQSPSSLSASVGDRVTITCRASQSIYTYLNWYQQKPGKAPKLLIY
		SASNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSDTTPWTF
		GQGTKVEIK
3-19	768	DIQMTQSPSSLSASVGDRVTITCRASQSIATYLNWYQQKPGKAPKLLIY
		GASSLEGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTFSSPFTFG
		QGTKVEIK
3-2	769	DIQMTQSPSSLSASVGDRVTITCRASQNINTYLNWYQQKPGKAPKLLIY
		SASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSLTPWTFG
		QGTKVEIK
3-21	770	DIQMTQSPSSLSASVGDRVTITCRASQGIATYLNWYQQKPGKAPKLLIY
		YASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTRFTFG
		QGTKVEIK
3-22	771	DIQMTQSPSSLSASVGDRVTITCRASERISNYLNWYQQKPGKAPKLLIY
		TASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTPPRTFG
		QGTKVEIK
3-23	772	DIQMTQSPSSLSASVGDRVTITCRASQSISSSLNWYQQKPGKAPKLLIY
		AASRLQDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPRSFG
		QGTKVEIK
3-24	773	DIQMTQSPSSLSASVGDRVTITCRASQSISSHLNWYQQKPGKAPKLLIY
		RASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYNTPQTF
		GQGTKVEIK
3-25	774	DIQMTQSPSSLSASVGDRVTITCRASQSISSYLIWYQQKPGKAPKLLIYA
		ASRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYNTPRTFG
		QGTKVEIK
3-26	775	DIQMTQSPSSLSASVGDRVTITCRASPSISTYLNWYQQKPGKAPKLLIY
		TASRLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYSTPSSFG
		QGTKVEIK
3-27	776	DIQMTQSPSSLSASVGDRVTITCRASQNIAKYLNWYQQKPGKAPKLLI
		YGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHSPPITF
		GQGTKVEIK
3-28	777	DIQMTQSPSSLSASVGDRVTITCRASQSIGTYLNWYQQKPGKAPKLLIY
		AASNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQESYSAPYTFG
		QGTKVEIK
3-29	778	DIQMTQSPSSLSASVGDRVTITCRASQSISPYLNWYQQKPGKAPKLLIY
		KASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSSTPYTFG
		QGTKVEIK

TABLE 21
ACE2 Variable Heavy Chain CDRs
	SEQ		SEQ		SEQ
	ID		ID		ID
Name	NO	CDRH1	NO	CDRH2	NO	CDRH3
4-51	779	PGTAIMG	920	ARISTSGGSTKYA	1062	CARTTVTTPPLIW
4-52	780	RSFSNSVMG	921	ARITWNGGSTYYA	1063	CATTENPNPRW
4-53	781	RTFGDDTMG	922	AAVSWSGSGVYY	1064	CATDPPLFW
				A
4-54	782	RTFSDARMG	923	GAVSWSGGTTVY	1065	CATTEDPYPRW
				A
4-49	783	RTFGDYIMG	924	AAINWSAGYTAY	1066	CARASPNTGWHFDHW
				A
4-55	784	SGLSINAMG	925	AAISWSGGSTYTA	1067	CAAYQAGWGDW
				YA
4-39	785	RTFSNAAMG	926	ARILWTGASRNYA	1068	CATTENPNPRW
4-56	786	FSLDYYGMG	927	AAISWNGDFTAYA	1069	CAKRANPTGAYFDYW
4-33	787	FTFSRHDMG	928	AGINWESGSTNYA	1070	CAADRGVYGGRWYRTSQ
						YTW
4-57	788	LTFRNYAMG	929	AAIGSGGYTDYA	1071	CAVKPGWVARDPSQYNW
4-25	789	GTFSRYAMG	930	SAVDSGGSTYYA	1072	CAASPSLRSAWQW
4-58	790	FTLDYYDM	931	AAVTWSGGSTYY	1073	CAADRRGLASTRAADYD
		G		A		W
4-59	791	RTFGDYIMG	932	AAINWSAGYTPYA	1074	CATAPPLFCWHFDLW
4-6	792	RTFGDDIMG	933	AAIHWSAGYTRY	1075	CATDPPLFWGHVDLW
				A
4-61	793	RTFGDYIMG	934	AAINWSADYTPYA	1076	CATAPPNTGWHFDHW
4-3	794	RTFGDYIMG	935	AAINWSAGYTAY	1077	CATATPNTGWHFDHW
				A
4-62	795	RTFSDDTMG	936	AAINWSGGSTDYA	1078	CATDPPLFW
4-43	796	RTFGDDTMG	937	AGINWSGGNTYY	1079	CATDPPLFW
				A
4-5	797	RTFGDYIMG	938	AAINWTGGYTSYA	1080	CATDPPLFW
4-42	798	RTFGDDTMG	939	AAINWSGGNTYY	1081	CATDPPLFW
				A
4-63	799	RTFSDYTMG	940	AAINWSGGYTYY	1082	CATDPPLFW
				A
4-6	800	RTFGDYGM	941	ATINWSGALTHYA	1083	CATLPFYDFWSGYYTGYY
		G				YMDVW
4-40	801	RTFSDDTMG	942	AGVTWSGSSTFYA	1084	CATDPPLFW
4-21	802	RTFSDDIMG	943	AAISWSGGNTHYA	1085	CATDPPLFW
4-64	803	RTFGDYIMG	944	AAINWSAGYTAY	1086	CATASPNTGWHFDHW
				A
4-47	804	FTFDDDYVM	945	AAVSGSGDDTYY	1087	CAADRRGLASTRAADYD
		G		A		W
4-65	805	RTFGDYIMG	946	AAINWSAGYTAY	1088	CATEPPLSCWHFDLW
				A
4-18	806	RTFGDYIMG	947	AAINWSGGYTPYA	1089	CATAPPNTGWHFDHW
4-66	807	RTFGDDTMG	948	AAINWSAGYTPYA	1090	CATDPPLFCCHFDLW
4-36	808	RTFSDDTMG	949	AAISWSGGTTRYA	1091	CATDPPLFW
4-67	809	RTFSDDTMG	950	AAINWSGDSTYYA	1092	CATDPPLFW
4-16	810	RTFSDDTMG	951	AAINWSGGTTRYA	1093	CATDPPLFW
4-11	811	RTFSDDAMG	952	AAIHWSGSSTRYA	1094	CATDPPLFW
4-68	812	RTFSDDTMG	953	GTINWSGGSTYYA	1095	CATDPPLFW
4-34	813	RTFGDYIMG	954	AAINWSGGYTPYA	1096	CATDPPLFW
4-28	814	RTFGDDTMG	955	AAINWNGGNTHY	1097	CATDPPLFW
				A
4-69	815	RTFSDDAMG	956	AAINWSGGTTRYA	1098	CATDPPLFW
4-7	816	RTFGDYIMG	957	AAINWSAGYTPYA	1099	CATDPPLFWGHVDLW
4-71	817	RTFSDDTMG	958	ASINWSGGSTYYA	1100	CATDPPLFW
4-23	818	RTFSDDAMG	959	AGISWNGGSIYYA	1101	CATDPPLFW
4-9	819	FTFDDYEMG	960	AAISWRGGTTYYA	1102	CAADRRGLASTRAGDYD
						W
4-72	820	RTFGDDTMG	961	AAINWSGGYTPYA	1103	CATDPPLFWGHVDLW
4-73	821	RTFSDDAMG	962	AAINWSGGSTRYA	1104	CATDPPLFW
4-29	822	VTLDDYAM	963	AVINWSGGSTDYA	1105	CARGGGWVPSSTSESLNW
		G				YFDRW
4-41	823	RTFGDYIMG	964	AAINWSGGTTPYA	1106	CATDPPLFCCHVDLW
4-74	824	LTFSDDTMG	965	AAVSWSGGNTYY	1107	CATDPPLFW
				A
4-75	825	RTFGDDTMG	966	AAINWTGGYTPYA	1108	CATDPPLFW
4-31	826	RTFGDYIMG	967	ATINWTAGYTYY	1109	CATDPPLFCWHFDHW
				A
4-32	827	RTFGDDTMG	968	AAINWSGGNTDY	1110	CATDPPLFW
				A
4-15	828	RTFGDYTMG	969	AAINWSGGNTYY	1111	CATDPPLFW
				A
4-14	829	RTFSDDTMG	970	AGINWSGNGVYY	1112	CATDPPLFW
				A
4-76	830	RTFGDYAM	971	APINWSGGSTYYA	1113	CATDPPLFW
		G
4-50	831	GTFSNSGMG	972	AVVNWSGRRTYY	1114	CAVPWMDYNRRDW
				A
4-17	832	QLANFASYA	973	AAITRSGSSTVYA	1115	CATTMNPNPRW
		MG
4-37	833	RTFSDDIMG	974	AAINWTGGSTYYA	1116	CATDPPLFW
4-44	834	RTFGDYIMG	975	AAINWSAGYTAY	1117	CATARPNTGWHFDHW
				A
4-77	835	RTFSDDTMG	976	GSINWSGGSTYYA	1118	CATDPPLFW
4-78	836	RTFSDDTMG	977	AGMTWSGSSTFY	1119	CATDPPLFW
				A
4-79	837	RTFGDYIMG	978	AAINWSGDYTDY	1120	CATDPPLFW
				A
4-8	838	RTFGDYIMG	979	GGINWSGGYTYY	1121	CATDPPLFW
				A
4-81	839	RTFSDDTMG	980	AAVNWSGGSTYY	1122	CATDPPLFW
				A
4-82	840	RTFGDYAM	981	AAINWSGGYTRY	1123	CATDPPLFW
		G		A
4-83	841	RTFGDDTMG	982	AAINWSGGYTPYA	1124	CATDPPLFW
4-35	842	RTFGDYIMG	983	AAINWSAGYTAY	1125	CARASPNTGWHFDRW
				A
4-45	843	RTFGDYIMG	984	AAINWSGGYTHY	1126	CATDPPLFW
				A
4-84	844	RTFSDDTMG	985	AAITWSGGRTRYA	1127	CATDRPLFW
4-85	845	RTFGDYIMG	986	AAINWSGGYTAY	1128	CATASPNTGWHFDHW
				A
4-86	846	RTFSDDTMG	987	AAIHWSGSSTRYA	1129	CATDPPLFW
4-87	847	RTFSDYTMG	988	AAINWSGGTTYYA	1130	CATDPPLFW
4-88	848	RTFGDDTMG	989	AAINWSGDNTHY	1131	CATDPPLFW
				A
4-89	849	FAFGDNWIG	990	ASISSGGTTAYA	1132	CAHRGGWLRPWGYW
4-9	850	RTFSDDAMG	991	GRINWSGGNTYY	1133	CATDPPLFW
				A
4-91	851	RTFSDDTMG	992	GGISWSGGNTYYA	1134	CATDPPLFW
4-92	852	RTFSDDTMG	993	AAINWSGGSTYYA	1135	CATDPPLFW
4-46	853	RTFGDDTMG	994	AAINWSGGYTYY	1136	CATDPPLFW
				A
4-20	854	RTFGDYIMG	995	AAINWSADYTAY	1137	CATDPPLFCWHFDHW
				A
4-93	855	RTFSDDAMG	996	AAINWSGSSTYYA	1138	CATDPPLFW
4-4	856	RTFGDYIMG	997	AAINWIAGYTADA	1139	CAEPSPNTGWHFDHW
4-2	857	RTFGDDTMG	998	AAINWSGGNTPYA	1140	CATDPPLFW
4-94	858	RTFSDDTMG	999	AAINWSGDNTHY	1141	CATDPPLFW
				A
4-95	859	RTFGDYIMG	1000	AAINWSAGYTAY	1142	CATAPPLFCWHFDHW
				A
4-12	860	FTFGDYVMG	1001	AAINWNAGYTAY	1143	CAKASPNTGWHFDHW
				A
4-30	861	RTFGDYTMG	1002	AAINWTGGYTYY	1144	CATDPPLFW
				A
4-27	862	RTFGDYIMG	1003	AAINWSAGYTAY	1145	CARATPNTGWHFDHW
				A
4-22	863	RTFGDYIMG	1004	AAINWSGDNTHY	1146	CATDPPLFW
				A
4-96	864	RTFGDYIMG	1005	AAINWSAGYTPYA	1147	CATDPPLFCCHFDHW
4-97	865	RTFGDYIMG	1006	AAINWSAGYTAY	1148	CATAPPNTGWHFDHW
				A
4-98	866	FTWGDYTM	1007	AAINWSGGNTYY	1149	CAADRRGLASTRAADYD
		G		A		W
4-99	867	IPSTLRAMG	1008	AAVSSLGPFTRYA	1150	CAAKPGWVARDPSQYNW
4-100	868	FSFDDDYVM	1009	AAINWSGGSTYYA	1151	CAADRRGLASTRAADYD
		G				W
4-101	869	RTFSNAAMG	1010	ARILWTGASRSYA	1152	CATTENPNPRW
4-102	870	GTFGVYHM	1011	AAINMSGDDSAYA	1153	CAILVGPGQVEFDHW
		G
4-103	871	FTFSSYYMG	1012	ARISGSTFYA	1154	CAALPFVCPSGSYSDYGD
						EYDW
4-104	872	RTFSGDFMG	1013	GRINWSGGNTYY	1155	CPTDPPLFW
				A
4-105	873	STLRDYAMG	1014	AAITWSGGSTAYA	1156	CASLLAGDRYFDYW
4-106	874	FTFDDYTMG	1015	AAITDNGGSKYYA	1157	CAADRRGLASTRAADYD
						W
4-107	875	GTFSSYGMG	1016	AAINWSGASTYYA	1158	CARDWRDRTWGNSLDY
						W
4-108	876	FSFDDDYVM	1017	AAISWSEDNTYYA	1159	CAADRRGLASTRAADYD
		G				W
4-109	877	FSFDDDYVM	1018	AAVSGSGDDTYY	1160	CAADRRGLASTRAADYD
		G		A		W
4-110	878	NIAAINVMG	1019	AAISASGRRTDYA	1161	CARRVYYYDSSGPPGVTF
						DIW
4-111	879	IITSRYVMG	1020	AAISTGGSTIYA	1162	CARQDSSSPYFDYW
4-112	880	FSFDDDYVM	1021	AAISNSGLSTYYA	1163	CAADRRGLASTRAADYD
		G				W
4-113	881	SISSINVMG	1022	ATMRWSTGSTYY	1164	CAQRVRGFFGPLRTTPSW
				A		YEW
4-114	882	LTFILYRMG	1023	AAINNFGTTKYA	1165	CARTHYDFWSGYTSRTPN
						YFDYW
4-115	883	GTFSVYHMG	1024	AAISWSGGSTAYA	1166	CAAVNTWTSPSFDSW
4-116	884	RAFSTYGMG	1025	AGINWSGDTPYYA	1167	CAREVGPPPGYFDLW
4-117	885	RTFSDIAMG	1026	ASINWGGGNTYY	1168	CAAKGIWDYLGRRDFGD
				A		W
4-118	886	RTFSSARMG	1027	AAISWSGDNTHYA	1169	CATTENPNPRW
4-119	887	FAFSSYAMG	1028	ATINGDDYTYYA	1170	CVATPGGYGLW
4-120	888	ITFRRHDMG	1029	AAIRWSSSSTVYA	1171	CAADRGVYGGRWYRTSQ
						YTW
4-121	889	TAASFNPMG	1030	AAITSGGSTNYA	1172	CAAIAYEEGVYRWDW
4-122	890	NINIINYMG	1031	AAIHWNGDSTAY	1173	CASGPPYSNYFAYW
				A
4-123	891	FTFDDYAMG	1032	AAISGSGGSTAYA	1174	CAKIMGSGRPYFDHW
4-124	892	NIFTRNVMG	1033	AAITSSGSTNYA	1175	CARPSSDLQGGVDYW
4-125	893	RTFSSIAMG	1034	ASINWGGGNTIYA	1176	CAAKGIWDYLGRRDFGD
						W
4-126	894	IPSTLRAMG	1035	AAVSSLGPFTRYA	1177	CAAKPGWVARDPSEYNW
4-127	895	FTLDDSAMG	1036	AAITNGGSTYYA	1178	CAREARGSPYFDFW
4-128	896	SISSFNAMG	1037	AAIDWDGSTAYA	1179	CARGGGYYGSGSFEYW
4-129	897	NIFSDNIIG	1038	AYYTSGGSIDYA	1180	CARGTAVGRPPPGGMDV
						W
4-130	898	SISSIGAMG	1039	AAISSSGSSTVYA	1181	CARVPPGQAYFDSW
4-131	899	FTFDDYGMG	1040	ATITWSGDSTYYA	1182	CAKGGSWYYDSSGYYGR
						W
4-132	900	RTFSNYTMG	1041	SAISWSTGSTYYA	1183	CAADRYGPPWYDW
4-133	901	STNYMG	1042	AAISMSGDDTIYA	1184	CARIGLRGRYFDLW
4-134	902	GTFSSVGMG	1043	AVINWSGARTYY	1185	CAVPWMDYNRRDW
				A
4-135	903	RIFTNTAMG	1044	AAINWSGGSTAYA	1186	CARTSGSYSFDYW
4-136	904	EEFSDHWM	1045	GAIHWSGGRTYY	1187	CAADRRGLASTRAADYD
		G		A		W
4-137	905	RTFSSIAMG	1046	AAINWSGARTAY	1188	CAAKGIWDYLGRRDFGD
				A		W
4-138	906	STSSLRTMG	1047	AAISSRDGSTIYA	1189	CARDDSSSPYFDYW
4-139	907	GGTFGSYAM	1048	AAISIASGASGGTT	1190	CATTMNPNPRW
		G		NYA
4-140	908	RTFSNAAMG	1049	ARITWNGGSTFYA	1191	CATTENPNPRW
4-141	909	IILSDNAMG	1050	AAISWLGESTYYA	1192	CAADRRGLASTRAADYD
						W
4-142	910	RTFGDYIMG	1051	AAINWNGGYTAY	1193	CATTSPNTGWHYYRW
				A
4-143	911	FNFNWYPM	1052	AAISWTGVSTYTA	1194	CARWGPGPAGGSPGLVGF
		G		YA		DYW
4-144	912	SIRSVSVMG	1053	AAISWSGVGTAYA	1195	CAAYQRGWGDW
4-145	913	MTFRLYAM	1054	GAINWLSESTYYA	1196	CAAKPGWVARDPSEYNW
		G
4-146	914	RTFSDDAMG	1055	AAINWSGGSTYYA	1197	CATDPPLFW
4-147	915	GTFSVYAMG	1056	AAISMSGDDAAYA	1198	CAKISKDDGGKPRGAFFD
						SW
4-148	916	FALGYYAM	1057	AAISSRDGSTAYA	1199	CARLATGPQAYFHHW
		G
4-149	917	FNLDDYAM	1058	AAISWDGGATAY	1200	CARVGRGTTAFDSW
		G		A
4-150	918	NTFSGGFMG	1059	ASIRSGARTYYA	1201	CAQRVRGFFGPLRTTPSW
						YEW
4-151	919	SIRSINIMG	1060	AAISWSGGSTVYA	1202	CASLLAGDRYFDYW

TABLE 22
ACE2 Variant Sequences Variable Heavy Chain
	SEQ
Name	ID	Amino Acid Sequence
4-51	1203	EVQLVESGGGLVQPGGSLRLSCAASGPGTAIMGWFRQAPGKEREFVARISTSG
		GSTKYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARTTVTTPPLI
		WGQGTLVTVSS
4-52	1204	EVQLVESGGGLVQPGGSLRLSCAASGRSFSNSVMGWFRQAPGKEREFVARIT
		WNGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATTENPN
		PRWGQGTLVTVSS
4-53	1205	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDDTMGWFRQAPGKEREFVAAV
		SWSGSGVYYADSVKGRFTITADNSKNTAYLQMNSLKPENTAVYYCATDPPLF
		WGQGTLVTVSS
4-54	1206	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDARMGWFRQAPGKEREFVGAVS
		WSGGTTVYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATTEDPYP
		RWGQGTLVTVSS
4-49	1207	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKERESVAAIN
		WSAGYTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARASPNT
		GWHFDHWGQGTLVTVSS
4-55	1208	EVQLVESGGGLVQPGGSLRLSCAASGSGLSINAMGWFRQAPGKERESVAAIS
		WSGGSTYTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYQA
		GWGDWGQGTLVTVSS
4-39	1209	EVQLVESGGGLVQPGGSLRLSCAASGRTFSNAAMGWFRQAPGKEREFVARIL
		WTGASRNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATTENPNP
		RWGQGTLVTVSS
4-56	1210	EVQLVESGGGLVQPGGSLRLSCAASGFSLDYYGMGWFRQAPGKERESVAAIS
		WNGDFTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKRANPT
		GAYFDYWGQGTLVTVSS
4-33	1211	EVQLVESGGGLVQPGGSLRLSCAASGFTFSRHDMGWFRQAPGKEREFVAGIN
		WESGSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADRGVY
		GGRWYRTSQYTWGQGTLVTVSS
4-57	1212	EVQLVESGGGLVQPGGSLRLSCAASGLTFRNYAMGWFRQAPGKEREFVAAIG
		SGGYTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVKPGWVA
		RDPSQYNWGQGTLVTVSS
4-25	1213	EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYAMGWFRQAPGKEREWVSAV
		DSGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASPSLRS
		AWQWGQGTLVTVSS
4-58	1214	EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYDMGWFRQAPGKEREFVAAV
		TWSGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADRRG
		LASTRAADYDWGQGTLVTVSS
4-59	1215	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREFVAAIN
		WSAGYTPYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATAPPLFC
		WHFDLWGQGTLVTVSS
4-6	1216	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDDIMGWFRQAPGKEREFVAAIH
		WSAGYTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGHVDLWGQGTLVTVSS
4-61	1217	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREIVAAIN
		WSADYTPYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATAPPNTG
		WHFDHWGQGTLVTVSS
4-3	1218	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREIVAAIN
		WSAGYTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATATPNT
		GWHFDHWGQGTLVTVSS
4-62	1219	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKEREFVAAIN
		WSGGSTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-43	1220	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDDTMGWFRQAPGKEREFVAGIN
		WSGGNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-5	1221	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREFVAAIN
		WTGGYTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-42	1222	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDDTMGWFRQAPGKERECVAAIN
		WSGGNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-63	1223	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDYTMGWFRQAPGKEREFVAAIN
		WSGGYTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-6	1224	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYGMGWFRQAPGKEREFVATIN
		WSGALTHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATLPFYDF
		WSGYYTGYYYMDVWGQGTLVTVSS
4-40	1225	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKEREFLAGVT
		WSGSSTFYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLFW
		GQGTLVTVSS
4-21	1226	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDIMGWFRQAPGKEREFVAAIS
		WSGGNTHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-64	1227	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKERESVAAIN
		WSAGYTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATASPNT
		GWHFDHWGQGTLVTVSS
4-47	1228	EVQLVESGGGLVQPGGSLRLSCAASGFTFDDDYVMGWFRQAPGKEREFVAA
		VSGSGDDTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADRR
		GLASTRAADYDWGQGTLVTVSS
4-65	1229	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREFVAAIN
		WSAGYTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATEPPLSC
		WHFDLWGQGTLVTVSS
4-18	1230	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREIVAAIN
		WSGGYTPYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATAPPNTG
		WHFDHWGQGTLVTVSS
4-66	1231	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDDTMGWFRQAPGKEREIVAAIN
		WSAGYTPYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLFC
		CHFDLWGQGTLVTVSS
4-36	1232	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKEREFVAAIS
		WSGGTTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-67	1233	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKEREFVAAIN
		WSGDSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-16	1234	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKEREFVAAIN
		WSGGTTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-11	1235	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDAMGWFRQAPGKEREFVAAIH
		WSGSSTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLFW
		GQGTLVTVSS
4-68	1236	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKERELVGTIN
		WSGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-34	1237	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREFVAAIN
		WSGGYTPYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-28	1238	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDDTMGWFRQAPGKERELVAAIN
		WNGGNTHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-69	1239	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDAMGWFRQAPGKEREFVAAIN
		WSGGTTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-7	1240	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKERESVAAIN
		WSAGYTPYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGHVDLWGQGTLVTVSS
4-71	1241	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKEREWVASIN
		WSGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-23	1242	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDAMGWFRQAPGKEREFVAGIS
		WNGGSIYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLFW
		GQGTLVTVSS
4-9	1243	EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYEMGWFRQAPGKEREFVAAIS
		WRGGTTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADRRGL
		ASTRAGDYDWGQGTLVTVSS
4-72	1244	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDDTMGWFRQAPGKEREFVAAIN
		WSGGYTPYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGHVDLWGQGTLVTVSS
4-73	1245	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDAMGWFRQAPGKEREFVAAIN
		WSGGSTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLFW
		GQGTLVTVSS
4-29	1246	EVQLVESGGGLVQPGGSLRLSCAASGVTLDDYAMGWFRQAPGKEREFVAVI
		NWSGGSTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGGGW
		VPSSTSESLNWYFDRWGQGTLVTVSS
4-41	1247	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKERESVAAIN
		WSGGTTPYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLFC
		CHVDLWGQGTLVTVSS
4-74	1248	EVQLVESGGGLVQPGGSLRLSCAASGLTFSDDTMGWFRQAPGKEREFVAAVS
		WSGGNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-75	1249	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDDTMGWFRQAPGKEREFVAAIN
		WTGGYTPYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-31	1250	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREFVATIN
		WTAGYTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLFC
		WHFDHWGQGTLVTVSS
4-32	1251	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDDTMGWFRQAPGKEREFVAAIN
		WSGGNTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-15	1252	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYTMGWFRQAPGKEREFVAAIN
		WSGGNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-14	1253	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKEREFVAGIN
		WSGNGVYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-76	1254	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYAMGWFRQAPGKERELVAPIN
		WSGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-50	1255	EVQLVESGGGLVQPGGSLRLSCAASGGTFSNSGMGWFRQAPGKERELVAVV
		NWSGRRTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVPWM
		DYNRRDWGQGTLVTVSS
4-17	1256	EVQLVESGGGLVQPGGSLRLSCAASGQLANFASYAMGWFRQAPGKEREFVA
		AITRSGSSTVYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATTMN
		PNPRWGQGTLVTVSS
4-37	1257	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDIMGWFRQAPGKEREFVAAIN
		WTGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-44	1258	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREIVAAIN
		WSAGYTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATARPNT
		GWHFDHWGQGTLVTVSS
4-77	1259	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKEREWVGSIN
		WSGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-78	1260	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKEREFVAGM
		TWSGSSTFYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-79	1261	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKERECVAAIN
		WSGDYTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-8	1262	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREFVGGIN
		WSGGYTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-81	1263	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKEREFVAAV
		NWSGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-82	1264	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYAMGWFRQAPGKEREFVAAIN
		WSGGYTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-83	1265	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDDTMGWFRQAPGKEREFVAAIN
		WSGGYTPYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-35	1266	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKERESVAAIN
		WSAGYTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARASPNT
		GWHFDRWGQGTLVTVSS
4-45	1267	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREFVAAIN
		WSGGYTHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-84	1268	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKEREFVAAIT
		WSGGRTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDRPLF
		WGQGTLVTVSS
4-85	1269	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKERESVAAIN
		WSGGYTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATASPNT
		GWHFDHWGQGTLVTVSS
4-86	1270	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKEREFVAAIH
		WSGSSTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLFW
		GQGTLVTVSS
4-87	1271	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDYTMGWFRQAPGKEREWVAAI
		NWSGGTTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-88	1272	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDDTMGWFRQAPGKEREFVAAIN
		WSGDNTHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-89	1273	EVQLVESGGGLVQPGGSLRLSCAASGFAFGDNWIGWFRQAPGKEREWVASIS
		SGGTTAYADNVKGRFTIIADNSKNTAYLQMNSLKPEDTAVYYCAHRGGWLR
		PWGYWGQGTLVTVSS
4-9	1274	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDAMGWFRQAPGKEREFVGRIN
		WSGGNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-91	1275	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKEREFVGGIS
		WSGGNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-92	1276	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKEREFVAAIN
		WSGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-46	1277	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDDTMGWFRQAPGKEREFVAAIN
		WSGGYTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-20	1278	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREFVAAIN
		WSADYTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLFC
		WHFDHWGQGTLVTVSS
4-93	1279	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDAMGWFRQAPGKEREFVAAIN
		WSGSSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLFW
		GQGTLVTVSS
4-4	1280	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREMVAAIN
		WIAGYTADADSVRRLFTITADNNKNTAHLMMNLLKPENTAVYYCAEPSPNT
		GWHFDHWGQGTLVTVSS
4-2	1281	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDDTMGWFRQAPGKEREFVAAIN
		WSGGNTPYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-94	1282	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKEREFVAAIN
		WSGDNTHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-95	1283	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREIVAAIN
		WSAGYTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATAPPLFC
		WHFDHWGQGTLVTVSS
4-12	1284	EVQLVESGGGLVQPGGSLRLSCAASGFTFGDYVMGWFRQAPGKEREIVAAIN
		WNAGYTAYADSVRGLFTITADNSKNTAYLQMNSLKPEDTAVYYCAKASPNT
		GWHFDHWGQGTLVTVSS
4-30	1285	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYTMGWFRQAPGKEREFVAAIN
		WTGGYTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-27	1286	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREIVAAIN
		WSAGYTAYADSVKGLFTITADNSKNTAYLQMNILKPEDTAVYYCARATPNT
		GWHFDHWGQGTLVTVSS
4-22	1287	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREFVAAIN
		WSGDNTHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTLVTVSS
4-96	1288	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREIVAAIN
		WSAGYTPYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLFC
		CHFDHWGQGTLVTVSS
4-97	1289	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKERESVAAIN
		WSAGYTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATAPPNT
		GWHFDHWGQGTLVTVSS
4-98	1290	EVQLVESGGGLVQPGGSLRLSCAASGFTWGDYTMGWFRQAPGKEREFVAAI
		NWSGGNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADRRG
		LASTRAADYDWGQGTLVTVSS
4-99	1291	EVQLVESGGGLVQPGGSLRLSCAASGIPSTLRAMGWFRQAPGKEREFVAAVS
		SLGPFTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAKPGWV
		ARDPSQYNWGQGTLVTVSS
4-100	1292	EVQLVESGGGLVQPGGSLRLSCAASGFSFDDDYVMGWFRQAPGKEREFVAAI
		NWSGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADRRG
		LASTRAADYDWGQGTLVTVSS
4-101	1293	EVQLVESGGGLVQPGGSLRLSCAASGRTFSNAAMGWFRQAPGKEREFVARIL
		WTGASRSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATTENPNP
		RWGQGTLVTVSS
4-102	1294	EVQLVESGGGLVQPGGSLRLSCAASGGTFGVYHMGWFRQAPGKEREGVAAI
		NMSGDDSAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAILVGPG
		QVEFDHWGQGTLVTVSS
4-103	1295	EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMGWFRQAPGKEREFVARI--
		SGSTFYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAALPFVCPSGS
		YSDYGDEYDWGQGTLVTVSS
4-104	1296	EVQLVESGGGLVQPGGSLRLSCAASGRTFSGDFMGWFRQAPGKEREFVGRIN
		WSGGNTYYADSVRGLFTITADNNKNTAYLMMNLLKPEDTAVYYCPTDPPLF
		WGLGTLVTWSS
4-105	1297	EVQLVESGGGLVQPGGSLRLSCAASGSTLRDYAMGWFRQAPGKERESVAAIT
		WSGGSTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASLLAGD
		RYFDYWGQGTLVTVSS
4-106	1298	EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYTMGWFRQAPGKEREFVAAIT
		DNGGSKYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADRRGL
		ASTRAADYDWGQGTLVTVSS
4-107	1299	EVQLVESGGGLVQPGGSLRLSCAASGGTFSSYGMGWFRQAPGKEREFVAAIN
		WSGASTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARDWRDR
		TWGNSLDYWGQGTLVTVSS
4-108	1300	EVQLVESGGGLVQPGGSLRLSCAASGFSFDDDYVMGWFRQAPGKEREFVAAI
		SWSEDNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADRRG
		LASTRAADYDWGQGTLVTVSS
4-109	1301	EVQLVESGGGLVQPGGSLRLSCAASGFSFDDDYVMGWFRQAPGKEREFVAA
		VSGSGDDTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADRR
		GLASTRAADYDWGQGTLVTVSS
4-110	1302	EVQLVESGGGLVQPGGSLRLSCAASGNIAAINVMGWFRQAPGKEREFVAAIS
		ASGRRTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARRVYYY
		DSSGPPGVTFDIWGQGTLVTVSS
4-111	1303	EVQLVESGGGLVQPGGSLRLSCAASGIITSRYVMGWFRQAPGKEREGVAAIST
		GGSTIYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARQDSSSPYFD
		YWGQGTLVTVSS
4-112	1304	EVQLVESGGGLVQPGGSLRLSCAASGFSFDDDYVMGWFRQAPGKEREFVAAI
		SNSGLSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADRRGL
		ASTRAADYDWGQGTLVTVSS
4-113	1305	EVQLVESGGGLVQPGGSLRLSCAASGSISSINVMGWFRQAPGKEREFVATMR
		WSTGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAQRVRGFF
		GPLRTTPSWYEWGQGTLVTVSS
4-114	1306	EVQLVESGGGLVQPGGSLRLSCAASGLTFILYRMGWFRQAPGKEREFVAAIN
		NFGTTKYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARTHYDFW
		SGYTSRTPNYFDYWGQGTLVTVSS
4-115	1307	EVQLVESGGGLVQPGGSLRLSCAASGGTFSVYHMGWFRQAPGKEREPVAAIS
		WSGGSTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVNTWT
		SPSFDSWGQGTLVTVSS
4-116	1308	EVQLVESGGGLVQPGGSLRLSCAASGRAFSTYGMGWFRQAPGKEREFVAGIN
		WSGDTPYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAREVGPPP
		GYFDLWGQGTLVTVSS
4-117	1309	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDIAMGWFRQAPGKEREFVASIN
		WGGGNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAKGIWD
		YLGRRDFGDWGQGTLVTVSS
4-118	1310	EVQLVESGGGLVQPGGSLRLSCAASGRTFSSARMGWFRQAPGKEREFVAAIS
		WSGDNTHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATTENPN
		PRWGQGTLVTVSS
4-119	1311	EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYAMGWFRQAPGKEREWVATIN
		GDDYTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCVATPGGYG
		LWGQGTLVTVSS
4-120	1312	EVQLVESGGGLVQPGGSLRLSCAASGITFRRHDMGWFRQAPGKEREFVAAIR
		WSSSSTVYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADRGVY
		GGRWYRTSQYTWGQGTLVTVSS
4-121	1313	EVQLVESGGGLVQPGGSLRLSCAASGTAASFNPMGWFRQAPGKEREFVAAIT
		SGGSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIAYEEGV
		YRWDWGQGTLVTVSS
4-122	1314	EVQLVESGGGLVQPGGSLRLSCAASGNINIINYMGWFRQAPGKEREGVAAIH
		WNGDSTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASGPPYS
		NYFAYWGQGTLVTVSS
4-123	1315	EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQAPGKERESVAAIS
		GSGGSTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKIMGSGR
		PYFDHWGQGTLVTVSS
4-124	1316	EVQLVESGGGLVQPGGSLRLSCAASGNIFTRNVMGWFRQAPGKEREFVAAIT
		SSGSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARPSSDLQG
		GVDYWGQGTLVTVSS
4-125	1317	EVQLVESGGGLVQPGGSLRLSCAASGRTFSSIAMGWFRQAPGKEREFVASIN
		WGGGNTIYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAKGIWD
		YLGRRDFGDWGQGTLVTVSS
4-126	1318	EVQLVESGGGLVQPGGSLRLSCAASGIPSTLRAMGWFRQAPGKEREFVAAVS
		SLGPFTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAKPGWV
		ARDPSEYNWGQGTLVTVSS
4-127	1319	EVQLVESGGGLVQPGGSLRLSCAASGFTLDDSAMGWFRQAPGKEREWVAAI
		TNGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARFARGSP
		YFDFWGQGTLVTVSS
4-128	1320	EVQLVESGGGLVQPGGSLRLSCAASGSISSFNAMGWFRQAPGKERESVAAID
		WDGSTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGGGYYG
		SGSFEYWGQGTLVTVSS
4-129	1321	EVQLVESGGGLVQPGGSLRLSCAASGNIFSDNIIGWFRQAPGKEREMVAYYTS
		GGSIDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGTAVGRPP
		PGGMDVWGQGTLVTVSS
4-130	1322	EVQLVESGGGLVQPGGSLRLSCAASGSISSIGAMGWFRQAPGKEREGVAAISS
		SGSSTVYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARVPPGQAY
		FDSWGQGTLVTVSS
4-131	1323	EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMGWFRQAPGKERELVATIT
		WSGDSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKGGSWY
		YDSSGYYGRWGQGTLVTVSS
4-132	1324	EVQLVESGGGLVQPGGSLRLSCAASGRTFSNYTMGWFRQAPGKEREWVSAIS
		WSTGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADRYGP
		PWYDWGQGTLVTVSS
4-133	1325	EVQLVESGGGLVQPGGSLRLSCAASGSTNYMGWFRQAPGKEREGVAAISMS
		GDDTIYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARIGLRGRYF
		DLWGQGTLVTVSS
4-134	1326	EVQLVESGGGLVQPGGSLRLSCAASGGTFSSVGMGWFRQAPGKERELVAVIN
		WSGARTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVPWMD
		YNRRDWGQGTLVTVSS
4-135	1327	EVQLVESGGGLVQPGGSLRLSCAASGRIFTNTAMGWFRQAPGKEREGVAAIN
		WSGGSTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARTSGSYS
		FDYWGQGTLVTVSS
4-136	1328	EVQLVESGGGLVQPGGSLRLSCAASGEEFSDHWMGWFRQAPGKEREFVGAIH
		WSGGRTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADRRGL
		ASTRAADYDWGQGTLVTVSS
4-137	1329	EVQLVESGGGLVQPGGSLRLSCAASGRTFSSIAMGWFRQAPGKEREFVAAIN
		WSGARTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAKGIWD
		YLGRRDFGDWGQGTLVTVSS
4-138	1330	EVQLVESGGGLVQPGGSLRLSCAASGSTSSLRTMGWFRQAPGKEREGVAAISS
		RDGSTIYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARDDSSSPYF
		DYWGQGTLVTVSS
4-139	1331	EVQLVESGGGLVQPGGSLRLSCAASGGGTFGSYAMGWFRQAPGKEREFVAAI
		SIASGASGGTTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATT
		MNPNPRWGQGTLVTVSS
4-140	1332	EVQLVESGGGLVQPGGSLRLSCAASGRTFSNAAMGWFRQAPGKEREFVARIT
		WNGGSTFYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATTENPNP
		RWGQGTLVTVSS
4-141	1333	EVQLVESGGGLVQPGGSLRLSCAASGIILSDNAMGWFRQAPGKEREFVAAIS
		WLGESTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADRRGL
		ASTRAADYDWGQGTLVTVSS
4-142	1334	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKERESVAAIN
		WNGGYTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATTSPNT
		GWHYYRWGQGTLVTVSS
4-143	1335	EVQLVESGGGLVQPGGSLRLSCAASGFNFNWYPMGWFRQAPGKERESVAAIS
		WTGVSTYTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARWGP
		GPAGGSPGLVGFDYWGQGTLVTVSS
4-144	1336	EVQLVESGGGLVQPGGSLRLSCAASGSIRSVSVMGWFRQAPGKEREAVAAIS
		WSGVGTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYQRG
		WGDWGQGTLVTVSS
4-145	1337	EVQLVESGGGLVQPGGSLRLSCAASGMTFRLYAMGWFRQAPGKEREFVGAI
		NWLSESTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAKPGW
		VARDPSEYNWGQGTLVTVSS
4-146	1338	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDAMGWFRQAPGKEREFVAAIN
		WSGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPPLF
		WGQGTMVTVSS
4-147	1339	EVQLVESGGGLVQPGGSLRLSCAASGGTFSVYAMGWFRQAPGKEREGVAAIS
		MSGDDAAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKISKDD
		GGKPRGAFFDSWGQGTLVTVSS
4-148	1340	EVQLVESGGGLVQPGGSLRLSCAASGFALGYYAMGWFRQAPGKERESVAAIS
		SRDGSTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARLATGPQ
		AYFHHWGQGTLVTVSS
4-149	1341	EVQLVESGGGLVQPGGSLRLSCAASGFNLDDYAMGWFRQAPGKERESVAAIS
		WDGGATAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARVGRGT
		TAFDSWGQGTLVTVSS
4-150	1342	EVQLVESGGGLVQPGGSLRLSCAASGNTFSGGFMGWFRQAPGKEREFVASIR
		SGARTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAQRVRGFFG
		PLRTTPSWYEWGQGTLVTVSS
4-151	1343	EVQLVESGGGLVQPGGSLRLSCAASGSIRSINIMGWFRQAPGKEREAVAAISW
		SGGSTVYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASLLAGDRY
		FDYWGQGTLVTVSS

TABLE 23
SARS-CoV-2 S1 Variable Heavy Chain CDRs
	SEQ		SEQ		SEQ
	ID		ID		ID
Name	NO	CDRH1	NO	CDRH2	NO	CDRH3
5-1	1344	GTFSSIGMG	1524	AAISWDGGATAY	1704	CAKEDVGKPFDW
				A
5-2	1345	LRFDDYAM	1525	AIKFSGGTTDYA	1705	CASWDGLIGLDAYEYDW
		G
5-3	1346	SIFSIDVMG	1526	AGISWSGDSTLYA	1706	CAAFDGYTGSDW
5-4	1347	FTLADYAM	1527	AVITCSGGSTDYA	1707	CAADDCYIGCGW
		G
5-5	1348	RTFSSIAMG	1528	AEITEGGISPSGDNI	1708	CAAELHSSDYTSPGAESD
				YYA		YGW
5-6	1349	PTFSSYAMM	1529	AAINNFGTTKYA	1709	CAASASDYGLGLELFHDE
		G				YNW
5-7	1350	STGYMG	1530	AAIHSGGSTNYA	1710	CATVATALIW
5-8	1351	RPFSEYTMG	1531	SSIHWGGRGTNYA	1711	CAAELHSSDYTSPGAYAW
5-9	1352	LTLSTYGMG	1532	AHIPRSTYSPYYA	1712	CAAIGDGAVW
5-10	1353	FTFNNHNMG	1533	AAISSYSHTAYA	1713	CALQPFGASNYRW
5-11	1354	GIYRVMG	1534	ASISSGGGINYA	1714	CAAESWGRQW
5-12	1355	YTDSNLWM	1535	AINRSTGSTSYA	1715	CATSGSGSPNW
		G
5-13	1356	FTFDYYTMG	1536	AAIRSSGGLFYA	1716	CAAYLDGYSGSW
5-14	1357	GIFSINVMG	1537	SAIRWNGGNTAY	1717	CAGFDGYTGSDW
				A
5-15	1358	FTFDGAAMG	1538	ATIRWTNSTDYA	1718	CARGRYGIVERW
5-16	1359	RTHSIYPMG	1539	AAIHSGGATVYA	1719	CAARRWIPPGPIW
5-17	1360	PTFSIYAMG	1540	AGIRWSDVYTQY	1720	CALDIDYRDW
				A
5-18	1361	LTFDDNIHV	1541	AAIHWSGGSTIYA	1721	CAADVYPQDYGLGYVEG
		MG				KMYYGMDW
5-19	1362	LTLDYYAM	1542	ASINWSGGSTYYA	1722	CAAYGSGEFDW
		G
5-20	1363	RTIVPYTMG	1543	AAISPSAFTEYA	1723	CAARRWGYDW
5-21	1364	gtfttyhmg	1544	AHISTGGATNYA	1724	CATFPAIVTDSDYDLGND
						W
5-22	1365	FTFNVFAMG	1545	AAINWSDSRTDYA	1725	CASGSDNRARELSRYEYV
						W
5-23	1366	SIFSIDVMG	1546	AAISWSGESTLYA	1726	CAAFDGYSGSDW
5-24	1367	FTFSSYSMG	1547	AAISSYSHTAYA	1727	CALQPFGASSYRW
5-25	1368	NTFSINVMG	1548	AAIHWSGDSTLYA	1728	CAAFDGYSGNHW
5-26	1369	RTISSYIMG	1549	ARIYTGGDTIYA	1729	C AARTS YNGRYDYIDDYS
						W
5-27	1370	RANSINWM	1550	ATITPGGNTNYA	1730	CAAAAGSTWYGTLYEYD
		G				W
5-28	1371	GTFSVFAMG	1551	AEITAGGSTYYA	1731	CAVDGPFGW
5-29	1372	FTFDDYPMG	1552	ASVLRGGYTWYA	1732	CAKDWATGLAW
5-30	1373	FALGYYAM	1553	AGIRWTDAYTEYA	1733	CAADVSPSYGSRWYW
		G
5-31	1374	RTLDIHVMG	1554	AVINWTGESTLYA	1734	CAAFDGYTGNYW
5-32	1375	FTPDNYAMG	1555	AALGWSGVTTYH	1735	CASDESDAANW
				YYA
5-33	1376	FTFDDYAMG	1556	ATIMWSGNTTYY	1736	CATNDDDV
				A
5-34	1377	RTFSRYIMG	1557	AAISWSGGDNTYY	1737	CAAYRIVVGGTSPGDWR
				A		W
5-35	1378	PTFSIYAMG	1558	AGISWNGGSTNYA	1738	CALRRRFGGQEW
5-36	1379	RTFSLNAMG	1559	AAISCGGGSTYA	1739	CAADNDMGYCSW
5-37	1380	STFSINAMG	1560	GGISRSGATTNYA	1740	CAADGVPEYSDYASGPV
						W
5-38	1381	RTFSMHAM	1561	ASISSQGRTNYA	1741	CAAEVRNGSDYLPIDW
		G
5-39	1382	VTLDLYAM	1562	AGIRWTDAYTEYA	1742	CAVDIDYRDW
		G
5-40	1383	LPFTINVMG	1563	AAIHWSGLTTFYA	1743	CAELDGYFFAHW
5-41	1384	RAFSNYAM	1564	AWINNRGTTDYA	1744	CASTDDYGVDW
		G		DSGSTYYA
5-42	1385	FTPDDYAMG	1565	ASIGYSGRSNSYN	1745	CAIAHGSSTYNW
				YYA
5-43	1386	FTLNYYGM	1566	AAITSGGAPHYA	1746	CASAYDRGIGYDW
		G
5-44	1387	LPFSTKSMG	1567	AAIHWSGLTSYA	1747	CAADRAADFFAQRDEYD
						W
5-45	1388	RTFSINAMG	1568	AAISWSGESTQYA	1748	CAAFDGGSGTQW
5-46	1389	EEFSDHWM	1569	AAIHWSGDSTHRN	1749	CATVGITLNW
		G		YA
5-47	1390	FTFGSYDMG	1570	TAINWSGARTAYA	1750	CAARSVYSYEYNW
5-48	1391	LPLDLYAMG	1571	AGIRWSDAYTEYA	1751	CALDIDYRHW
5-49	1392	RTSTVNGMG	1572	ASISQSGAATAYA	1752	CAADRTYSYSSTGYYW
5-50	1393	FSLDYYGMG	1573	AAITSGGTPHYA	1753	CASAYNPGIGYDW
5-51	1394	RPNSINWMG	1574	ATITPGGNTNYA	1754	CAAAAGTTWYGTLYEYD
						W
5-52	1395	EKFSDHWM	1575	ATITFSGARTAYA	1755	CAALIKPSSTDRIFEEW
		G
5-53	1396	LTVVPYAM	1576	AAIRRSAVTNYA	1756	CAARRWGYHYW
		G
5-54	1397	TTFNFNVMG	1577	AVISWTGESTLYA	1757	CAAFDGYTGRDW
5-55	1398	IDVNRNAMG	1578	AAITWSGGWRYY	1758	CATTFGDAGIPDQYDFGW
				A
5-56	1399	RTFSSNMG	1579	ARIFGGDRTLYA	1759	CADINGDW
5-57	1400	GTFSMGWIR	1580	GCIGWITYYA	1760	CAPFGW
5-58	1401	CTLDYYAM	1581	AGIRWTDAYTEYA	1761	CAADVSPSYGGRWYW
		G
5-59	1402	LTFSLYRMC	1582	SCISNIDGSTYYA	1762	CAADLLGDSDYEPSSGFG
						W
5-60	1403	RSFSSHRMG	1583	AAIMWSGSHRNY	1763	CAAIAYEEGVYRWDW
				A
5-61	1404	RIIVPNTMG	1584	TGISPSAFTEYA	1764	CAAHGWGCHW
5-62	1405	SIFIISMG	1585	TGINWSGGSTTYA	1765	CAASAIGSGALRRFEYDW
5-63	1406	FSLDYYDMG	1586	AALGWSGGSTDY	1766	CAAGNGGRYGIVERW
				A
5-64	1407	TSISNRVMG	1587	ARIYTGGDTLYA	1767	CAARKIYRSLSYYGDYDW
5-65	1408	NIDRLYAMG	1588	AAIDSDGSTDYA	1768	CAALIDYGLGFPIEW
5-66	1409	NTFTINVMG	1589	AAINWNGGTTLY	1769	CAAFDGYSGIDW
				A
5-67	1410	FNVNDYAM	1590	AGITSSVGVTNYA	1770	CAADIFFVNW
		G
5-68	1411	FTFDHYTMG	1591	AAISGSENVTSYA	1771	CAAEPYIPVRTMRHMTFL
						TW
6-1	1412	RTFGNYNM	1592	ATINSLGGTSYA	1772	CARVDYYMDVW
		G
6-2	1413	FTMSSSWM	1593	TVISGVGTSYA	1773	CARGPDSSGYGFDYW
		G
6-3	1414	FTFSPSWMG	1594	ATINEYGGRNYA	1774	CARVDRDFDYW
6-4	1415	FTRDYYTMG	1595	AAISRSGSLTSYA	1775	CANLAYYDSSGYYDYW
6-5	1416	RTFTMG	1596	ASTNSAGSTNYA	1776	CTTVDQYFDYW
6-6	1417	TTLDYYAM	1597	AAISWSGGSTAYA	1777	CARED YYDSSGYSW
		G
6-7	1418	FTFSSYWMG	1598	ATINWSGVTAYA	1778	CARADDYFDYW
6-8	1419	FTLSGIWMG	1599	AIITTGGRTTYA	1779	CAGYSTFGSSSAYYYYSM
						DVG
6-9	1420	FTFDYYAMG	1600	SAIDSEGRTSYA	1780	CARWGPFDIW
6-10	1421	SIASIHAMG	1601	AAISRSGGFGSYA	1781	CARDDKYYDSSGYPAYFQ
						HW
6-11	1422	LAFNAYAM	1602	ATIGWSGANTYYA	1782	CASDPPGW
		G
6-12	1423	STYTTYSMG	1603	AAISGSENVTSYA	1783	CARVDDYMDVW
6-13	1424	LTFNDYAM	1604	AHIPRSTYSPYYA	1784	CAFLVGPQGVDHGAFDV
		G				W
6-14	1425	ITFRFKAMG	1605	AAVSWDGRNTYY	1785	CASDYYYMDVW
				A
6-15	1426	STVLINAMG	1606	AAVRWSDDYTYY	1786	CAKEGRAGSLDYW
				A
6-16	1427	FTFDDAAMG	1607	AHISWSGGSTYYA	1787	CATFGATVTATNDAFDIW
6-17	1428	NTGSTGYM	1608	AGVINDGSTVYA	1788	CARLATSHQDGTGYLFDY
		G				W
6-18	1429	LTFRNYAMG	1609	AGMMWSGGTTTY	1789	CAREGYYYDSSGYLNYFD
				A		YW
6-19	1430	SILSIAVMG	1610	AAISPSAVTTYYA	1790	CAIGYYDSSGYFDYW
6-20	1431	STLPYHAMG	1611	AAITWNGASTSYA	1791	CARDRYYDTSASYFESET
						W
6-21	1432	TLFKINAMG	1612	AAITSSGSNIDYTY	1792	CARSNTGWYSFDYW
				YA
6-22	1433	RTFSEVVMG	1613	ATIHSSGSTSYA	1793	CVRVTSDYSMDSW
6-23	1434	SIFSMNTMG	1614	ALINRSGGGINYA	1794	CVRLSSGYYDFDYW
6-24	1435	FTLDYYAM	1615	AAINWSGDNTHY	1795	CARAPFYCTTTKCQDNYY
		G		A		YMDVW
6-25	1436	ltfgtytmg	1616	AAISRFGSTYYA	1796	CARGGDYDFWSVDYMDV
						W
6-26	1437	DTFSTSWMG	1617	ATINTGGGTNYA	1797	CARVTTSFDYW
6-27	1438	ITFRFKAMG	1618	ASISRSGTTYYA	1798	CATDYSAFDMW
6-28	1439	DTYGSYWM	1619	ATITSDDRTNYA	1799	CARVTSSLSGMDVW
		G
6-29	1440	YTLKNYYA	1620	AAIIWTGESTLDA	1800	CAREGYYDSSGYYW
		MG
6-30	1441	FAFGDSWM	1621	ATINWSGVTAYA	1801	CARADGYFDYW
		G
6-31	1442	DTFSANRMG	1622	ASITWSSANTYYA	1802	CATFNWNDEGFDFW
6-32	1443	FTLDYYDM	1623	ALISWSGGSTYYA	1803	CATDFYGWGTRERDAFDI
		G				W
6-33	1444	TFQRINHMG	1624	ATINTGGQPNYA	1804	CASLIAAQDYYFDYW
6-34	1445	SAFRSNAMG	1625	AHISWSSKSTYYA	1805	CATYCSSTSCFDYW
6-35	1446	FTLAYYAM	1626	AAISMSGDDTIYA	1806	C ARELGYSSTVWPW
		G
6-36	1447	FDFSVSWMG	1627	TAITWSGDSTNYA	1807	CASLLHTGPSGGNYFDYW
6-37	1448	HTFSTSWMG	1628	ATINSLGGTNYA	1808	CARVSSGDYGMDVW
6-38	1449	NTFSGGFMG	1629	AVISSLSSKSYA	1809	CAKVDSGYDYW
6-39	1450	FTFSPSWMG	1630	AAISWSGGSTAYA	1810	CHGLGEGDPYGDYEGYF
						DLW
6-40	1451	FTFSDYWM	1631	ARVWWNGGSAY	1811	CAREVLRQQVVLDYW
		G		YA
6-41	1452	FTFSTSWMG	1632	ASINEYGGRNYA	1812	CAGLHYYYDSSGYNPTEY
						YGMDVW
6-42	1453	DTYGSYWM	1633	AVITSGGSTNYA	1813	CTHVQNSYYYAMDVW
		G
6-43	1454	RTFSSYAMM	1634	ASVNWDASQINY	1814	CTTLGAVYFDSSGYHDYF
		G		A		DYW
6-44	1455	GTFGVYHM	1635	GRITWTDGSTYYA	1815	CFGLLEVYDMTFDYW
		G
6-45	1456	NMFSINAMG	1636	TLISWSSGRTSYA	1816	CASLGYCSGGSCFDYW
6-46	1457	LTFSAMG	1637	ALIRRDGSTIYA	1817	CAALGILFGYDAFDIW
6-47	1458	RTFSMHAM	1638	ASITYGGNINYA	1818	CAKEGYYDSTGYRTYFQQ
		G				W
6-48	1459	FTVSNYAMG	1639	ASVNWSGGTTSY	1819	CATTGTVTLGYW
				A
6-49	1460	STVLINAMG	1640	AAISWSPGRTDYA	1820	CARDCSGGSCYSGDYW
6-50	1461	FSFDRWAM	1641	ASLATGGNTNYA	1821	CARVTNYDAFDIW
		G
6-51	1462	YTYSSYVMG	1642	AAISRFGSTYYA	1822	CARDSGEHFWDSGYIDH
						W
6-52	1463	DTYGSYWM	1643	AAITSGGSTVYA	1823	CARVDSRFDYW
		G
6-53	1464	ISINTNVMG	1644	AAISTGSVTIYA	1824	CARVDDFGYFDLW
6-54	1465	FEFENHWM	1645	AHITAGGLSNYA	1825	CGRHWGIYDSSGFSSFDY
		G				W
6-55	1466	FTMSSSWM	1646	ARITSGGSTGYA	1826	CASVDGYFDYW
		G
6-56	1467	NIFRSNMG	1647	AGITWNGDTTYY	1827	CARALGVTYQFDYW
				A
6-57	1468	LTFDDHSMG	1648	AAVPLSGNTYYA	1828	CASFSGGPADFDYW
6-58	1469	RAVSTYAM	1649	AAISGSENVTSYA	1829	CLSVTGDTEDYGVFDTW
		G
6-59	1470	ISGSVFSRTP	1650	SSIYSDGSNTYYA	1830	CAHWSWELGDWFDPW
		MG
6-60	1471	DTYGSYWM	1651	ATISQSGAATAYA	1831	CAGLLRYSGTYYDAFDV
		G				W
6-61	1472	DTYGSYWM	1652	AAINWSGGSTNYA	1832	CAGLGWNYMDYW
		G
6-62	1473	STFSGNWM	1653	AVISWTGGSTYYA	1833	CATHNSLSGFDYW
		G
6-63	1474	QTFNMG	1654	AAIGSGGSTSYA	1834	CWRLGNDYFDYW
6-64	1475	IPSIHAMG	1655	AAINWSHGVTYY	1835	CGGGYGYHFDYW
				A
6-65	1476	LPFSTLHMG	1656	ASLSIFGATGYA	1836	CWMYYYDSSGYYGNYY
						YGMDVW
6-66	1477	LTFSLFAMG	1657	AAISSGGSTDYA	1837	CARGNTKYYYDSSGYSSA
						FDYW
6-67	1478	SFSNYAMG	1658	AAISSSGALTSYA	1838	CWIVGPGPLDGMDVW
6-68	1479	FTLSDRAMG	1659	AHITAGGLSNYA	1839	CVHLASQTGAGYFDLW
6-69	1480	GTFSSVGMG	1660	AGISRSGGTYYA	1840	CARYDFWSGYPYW
6-70	1481	FNLDDYAD	1661	AAIGWGGGSTRY	1841	CAREILWFGEFGEPNVW
		MG		A
6-71	1482	ITFSNDAMG	1662	AIITSSDTNDTTNY	1842	CARLHYYDSSGYFDYW
				A
6-72	1483	STLSINAMG	1663	AAIDWSGGSTAYA	1843	CARDSSATRTGPDYW
6-73	1484	HTFSGYAMG	1664	AVITREGSTYYA	1844	CARLGGEGFDYW
6-74	1485	FAFGDSWM	1665	AAITSGGSTDYA	1845	CARGLLWFGELFGYW
		G
6-75	1486	GTFSTYWM	1666	AAISRSGGNTYYA	1846	CVRHSGTDGDSSFDYW
		G
6-76	1487	LAFDFDGMG	1667	AAINSGGSTYYA	1847	CARFFRAHDYW
6-77	1488	FTFDRSWMG	1668	AAVTEGGTTSYA	1848	CARADYDFDYW
6-78	1489	RTYDAMG	1669	ASVTSGGYTHYA	1849	CAKFGRKIVGATELDYW
6-79	1490	SISSIDYMG	1670	SWISSSDGSTYYA	1850	CARSPSFSQIYYYYYMDV
						W
6-80	1491	GTFSFYNMG	1671	AFISGNGGTSYA	1851	CAVVAMRMVTTEGPDVL
						DVW
6-81	1492	FIGNYHAMG	1672	AAVTWSGGTTNY	1852	CAREGYYYDSSGYPYYFD
				A		YW
6-82	1493	SSLDAYGMG	1673	AAISWGGGSIYYA	1853	CARLSQGMVALDYW
6-83	1494	SIASIHAMG	1674	AAITWSGAITSYA	1854	CAKDGGYGELHYGMEV
						W
6-84	1495	FTPDDYAMG	1675	AAINSGGSYTYYA	1855	CARDRGPW
6-85	1496	GTFSVFAMG	1676	SAINWSGGSLLYA	1856	CALFGDFDYW
6-86	1497	PISGINRMG	1677	AVITSNGRPSYA	1857	CVRLSSGYFDFDYW
6-87	1498	TSIMVGAMG	1678	AIIRGDGRTSYA	1858	CARFAGWDAFDIW
6-88	1499	RTFSTHWM	1679	AVINWSGGSIYYA	1859	CARLSSDGYNYFDFW
		G
6-89	1500	TIFASAMG	1680	AVVNWNGSSTVY	1860	CTTVDQYFNYW
				A
6-90	1501	FPFSIWPMG	1681	AAVRWSSTYYA	1861	CATGECDGGSCSLAYW
6-91	1502	RTFGNYAM	1682	ASISSSGVSKHYA	1862	CVRFGSSWARDLDQW
		G
6-92	1503	FLFDSYASM	1683	ATIWRRGNTYYA	1863	CTETGTAAW
		G		NYA
6-93	1504	LPFSTKSMG	1684	AAISMSGLTSYA	1864	CLKVLGGDYEADNWFDY
						W
6-94	1505	NIFRIETMG	1685	AGIIRSGGETLYA	1865	CARSLYYDRSGSYYFDY
						W
6-95	1506	IPSSIRAMG	1686	AVIRWTGGSTYYA	1866	CARDIGYYDSSGYYNDGG
						FDYW
6-96	1507	FTLSGNWM	1687	AIITSGGRTNYA	1867	CAGHATFGGSSSSYYYGM
		G				DVW
6-97	1508	FTFSSLAMG	1688	AAITWSGDITNYA	1868	CLRLSSSGFDHW
6-98	1509	TFGHYAMG	1689	AAINWSSRSTVYA	1869	CAKSDGLMGELRSASAFD
						IW
6-99	1510	IPFRSRTMG	1690	AGISRSGASTAYA	1870	CTHANDYGDYW
6-100	1511	GTFSTSWMG	1691	AHITAGGLSNYA	1871	CARLLVREDWYFDLW
6-101	1512	GTFSLFAMG	1692	AAISWTGDSTYYK	1872	CAYNNSSGEYW
				YYA
6-102	1513	SSFSAYAMG	1693	SAIDSEGTTTYA	1873	CAGDYNFWSGFDHW
6-103	1514	RTSSPIAMG	1694	AVRWSDDYTYYA	1874	CAKKLGGYYAFDIW
6-104	1515	LTFNQYTMG	1695	ASITDGGSTYYA	1875	CARDSRYMDVW
6-105	1516	PTFSSMG	1696	AAISWDGGATAY	1876	CAIEIVVGGIYW
				A
6-106	1517	IPSTLRAMG	1697	AATSWSGGSKYY	1877	CATDLYYMDVW
				A
6-107	1518	GVGFSVTNM	1698	AVISSSSSTNYA	1878	CTTFNWNDEGFDYW
		G
6-108	1519	GTFGSYGMG	1699	AAIRWSGGITYYA	1879	CARERYWNPLPYYYYGM
						DVW
6-109	1520	GTFSTYAMG	1700	ASIDWSGLTSYA	1880	CARGPFYMYCSGTKCYST
						NWFDPW
6-110	1521	PIYAVNRMG	1701	AGIWRSGGHRDY	1881	CARGEIDILTGYWYDYW
				A
6-111	1522	FTFSNYWM	1702	GGISRSGVSTSYA	1882	CTTLLYYYDSSGYSFDAF
		G				DIW
6-112	1523	GTFSAYHMG	1703	TIIDNGGPTSYA	1883	CTALLYYFDNSGYNFDPF
						DIW

TABLE 24
SARS-CoV-2 S1 Variant Variably Heavy Chain
	SEQ
	ID
Name	NO	Amino Acid Sequence
5-1	1884	EVQLVESGGGLVQPGGSLRLSCAASGGTFSSIGMGWFRQAPGKEREFVAA
		ISWDGGATAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKE
		DVGKPFDWGQGTLVTVSS
5-2	1885	EVQLVESGGGLVQPGGSLRLSCAASGLRFDDYAMGWFRQAPGKERELVA
		IKFSGGTTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASWD
		GLIGLDAYEYDWGQGTLVTVSS
5-3	1886	EVQLVESGGGLVQPGGSLRLSCAASGSIFSIDVMGWFRQAPGKEREFVAGI
		SWSGDSTLYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAFD
		GYTGSDWGQGTLVTVSS
5-4	1887	EVQLVESGGGLVQPGGSLRLSCAASGFTLADYAMGWFRQAPGKEREFVA
		VITCSGGSTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAD
		DCYIGCGWGQGTLVTVSS
5-5	1888	EVQLVESGGGLVQPGGSLRLSCAASGRTFSSIAMGWFRQAPGKERELVAE
		ITEGGISPSGDNIYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYC
		AAELHSSDYTSPGAESDYGWGQGTLVTVSS
5-6	1889	EVQLVESGGGLVQPGGSLRLSCAASGPTFSSYAMMGWFRQAPGKEREWV
		AAINNFGTTKYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAS
		ASDYGLGLELFHDEYNWGQGTLVTVSS
5-7	1890	EVQLVESGGGLVQPGGSLRLSCAASGSTGYMGWFRQAPGKEREFVAAIH
		SGGSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATVATA
		LIWGQGTLVTVSS
5-8	1891	EVQLVESGGGLVQPGGSLRLSCAASGRPFSEYTMGWFRQAPGKEREFVSS
		IHWGGRGTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAE
		LHSSDYTSPGAYAWGQGTLVTVSS
5-9	1892	EVQLVESGGGLVQPGGSLRLSCAASGLTLSTYGMGWFRQAPGKEREFVA
		HIPRSTYSPYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAI
		GDGAVWGQGTLVTVSS
5-10	1893	EVQLVESGGGLVQPGGSLRLSCAASGFTFNNHNMGWFRQAPGKEREFVA
		AISSYSHTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCALQP
		FGASNYRWGQGTLVTVSS
5-11	1894	EVQLVESGGGLVQPGGSLRLSCAASGGIYRVMGWFRQAPGKERELVASIS
		SGGGINYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAESWG
		RQWGQGTLVTVSS
5-12	1895	EVQLVESGGGLVQPGGSLRLSCAASGYTDSNLWMGWFRQAPGKEREFVA
		INRSTGSTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATSG
		SGSPNWGQGTLVTVSS
5-13	1896	EVQLVESGGGLVQPGGSLRLSCAASGFTFDYYTMGWFRQAPGKEREFVA
		AIRSSGGLFYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYL
		DGYSGSWGQGTLVTVSS
5-14	1897	EVQLVESGGGLVQPGGSLRLSCAASGGIFSINVMGWFRQAPGKEREWVSA
		IRWNGGNTAYADSVKGRFTITADNSKNTAYLQMNSLKPEDTAVYYCAGF
		DGYTGSDWGQGTLVTVSS
5-15	1898	EVQLVESGGGLVQPGGSLRLSCAASGFTFDGAAMGWFRQAPGKEREFVA
		TIRWTNSTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARG
		RYGIVERWGQGTLVTVSS
5-16	1899	EVQLVESGGGLVQPGGSLRLSCAASGRTHSIYPMGWFRQAPGKERELVAA
		IHSGGATVYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAARR
		WIPPGPIWGQGTLVTVSS
5-17	1900	EVQLVESGGGLVQPGGSLRLSCAASGPTFSIYAMGWFRQAPGKEREFVAG
		IRWSDVYTQYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCALDI
		DYRDWGQGTLVTVSS
5-18	1901	EVQLVESGGGLVQPGGSLRLSCAASGLTFDDNIHVMGWFPQAPGKEREFV
		AAIHWSGGSTIYADSVKGRFTINADNSKNTAYLQMNSLKPEDTAVYYCA
		ADVYPQDYGLGYVEGKMYYGMDWGQGTLVTVSS
5-19	1902	EVQLVESGGGLVQPGGSLRLSCAASGLTLDYYAMGWFRQAPGKEREWV
		ASINWSGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCA
		AYGSGEFDWGQGTLVTVSS
5-20	1903	EVQLVESGGGLVQPGGSLRLSCAASGRTIVPYTMGWFRQAPGKERELVA
		AISPSAFTEYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAARR
		WGYDWGQGTLVTVSS
5-21	1904	EVQLVESGGGLVQPGGSLRLSCAASGGTFTTYHMGWFRQAPGKEREFVA
		HISTGGATNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATFP
		AIVTDSDYDLGNDWGQGTLVTVSS
5-22	1905	EVQLVESGGGLVQPGGSLRLSCAASGFTFNVFAMGWFRQAPGKEREFVA
		AINWSDSRTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAS
		GSDNRARELSRYEYVWGQGTLVTVSS
5-23	1906	EVQLVESGGGLVQPGGSLRLSCAASGSIFSIDVMGWFRQAPGKEREFVAAI
		SWSGESTLYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAFD
		GYSGSDWGQGTLVTVSS
5-24	1907	EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMGWFRQAPGKEREFVAA
		ISSYSHTAYADSVKGRFTIIADNSKNTAYLQMNSLKPEDTAVYYCALQPFG
		ASSYRWGQGTLVTVSS
5-25	1908	EVQLVESGGGLVQPGGSLRLSCAASGNTFSINVMGWFRQAPGKEREFVA
		AIHWSGDSTLYADSGKGRFTIIADNNKNTAYLQMISLKPEDTAVYYCAAF
		DGYSGNHWGQGTLVTVSS
5-26	1909	EVQLVESGGGLVQPGGSLRLSCAASGRTISSYIMGWFRQAPGKERELVARI
		YTGGDTIYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAARTSY
		NGRYDYIDDYSWGQGTLVTVSS
5-27	1910	EVQLVESGGGLVQPGGSLRLSCAASGRANSINWMGWFRQAPGKEREFVA
		TITPGGNTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAAA
		GSTWYGTLYEYDWGQGTLVTVSS
5-28	1911	EVQLVESGGGLVQPGGSLRLSCAASGGTFSVFAMGWFRQVPGKERELVA
		EITAGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVDG
		PFGWGQGTLVTVSS
5-29	1912	EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYPMGWFRQAPGKEREGVA
		SVLRGGYTWYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKD
		WATGLAWGQGTLVTVSS
5-30	1913	EVQLVESGGGLVQPGGSLRLSCAASGFALGYYAMGWFRQAPGKEREFVA
		GIRWTDAYTEYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAA
		DVSPSYGSRWYWGQGTLVTVSS
5-31	1914	EVQLVESGGGLVQPGGSLRLSCAASGRTLDIHVMGWFRQAPGKEREFVA
		VINWTGESTLYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAF
		DGYTGNYWGQGTLVTVSS
5-32	1915	EVQLVESGGGLVQPGGSLRLSCAASGFTPDNYAMGWFRQAPGKEREFVA
		ALGWSGVTTYHYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYC
		ASDESDAANWGQGTLVTVSS
5-33	1916	EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQAPGKERELVA
		TIMWSGNTTYYADSVRRRFIIRDNNNKNTAHLQMNSLKPEDTAVYYCAT
		NDDDVWGQGTLVTVSS
5-34	1917	EVQLVESGGGLVQPGGSLRLSCAASGRTFSRYIMGWFRQAPGKEREFVAA
		ISWSGGDNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAA
		YRIVVGGTSPGDWRWGQGTLVTVSS
5-35	1918	EVQLVESGGGLVQPGGSLRLSCAASGPTFSIYAMGWFRQAPGKERELVAG
		ISWNGGSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCALR
		RRFGGQEWGQGTLVTVSS
5-36	1919	EVQLVESGGGLVQPGGSLRLSCAASGRTFSLNAMGWFRQAPGKERELVA
		AISCGGGSTYADNGKGRFTIITDNSKNTAYLQMMNLKPEDTAAYYCAAD
		NDMGYCSWGQGTLVTVSS
5-37	1920	EVQLVESGGGLVQPGGSLRLSCAASGSTFSINAMGWFRQAPGKEREFVGG
		ISRSGATTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADG
		VPEYSDYASGPVWGQGTLVTVSS
5-38	1921	EVQLVESGGGLVQPGGSLRLSCAASGRTFSMHAMGWFRQAPGKERELVA
		SISSQGRTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAEV
		RNGSDYLPIDWGQGTLVTVSS
5-39	1922	EVQLVESGGGLVQPGGSLRLSCAASGVTLDLYAMGWFRQAPGKEREFVA
		GIRWTDAYTEYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAV
		DIDYRDWGQGTLVTVSS
5-40	1923	EVQLVESGGGLVQPGGSLRLSCAASGLPFTINVMGWFRQAPGKEREFVAA
		IHWSGLTTFYADSVKGLFTITEDNSKNTAHLMMNLLKPEDTAVYCCAELD
		GYFFAHWGQGTLVTVSS
5-41	1924	EVQLVESGGGLVQPGGSLRLSCAASGRAFSNYAMGWFRQAPGKEREFVA
		WINNRGTTDYADSGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDT
		AVYYCASTDDYGVDWGQGTLVTVSS
5-42	1925	EVQLVESGGGLVQPGGSLRLSCAASGFTPDDYAMGWFRQAPGKEREFVA
		SIGYSGRSNSYNYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYC
		AIAHGSSTYNWGQGTLVTVSS
5-43	1926	EVQLVESGGGLVQPGGSLRLSCAASGFTLNYYGMGWFPQAPGKEREFVA
		AITSGGAPHYADSVKGRFTINADNSKNTAYLQMNSLKPEDTAVYYCASA
		YDRGIGYDWGQGTLVTVSS
5-44	1927	EVQLVESGGGLVQPGGSLRLSCAASGLPFSTKSMGWFRQAPGKEREFVAA
		IHWSGLTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADR
		AADFFAQRDEYDWGQGTLVTVSS
5-45	1928	EVQLVESGGGLVQPGGSLRLSCAASGRTFSINAMGWFPQAPGKERELVAA
		ISWSGESTQYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAFD
		GGSGTQWGQGTLVTVSS
5-46	1929	EVQLVESGGGLVQPGGSLRLSCAASGEEFSDHWMGWFRQAPGKEREFVA
		AIHWSGDSTHRNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYC
		ATVGITLNWGQGTLVTVSS
5-47	1930	EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYDMGWFRQAPGKEREFVT
		AINWSGARTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAA
		RSVYSYEYNWGQGTLVTVSS
5-48	1931	EVQLVESGGGLVQPGGSLRLSCAASGLPLDLYAMGWFPPAPGKELEFVAG
		IRWSDAYTEYADSVKGRFTINADNSKNPANLQMNSLKPEDTAVYYCALDI
		DYRHWGQGTLVTVSS
5-49	1932	EVQLVESGGGLVQPGGSLRLSCAASGRTSTVNGMGWFRQAPGKEREFVA
		SISQSGAATAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAD
		RTYSYSSTGYYWGQGTLVTVSS
5-50	1933	EVQLVESGGGLVQPGGSLRLSCAASGFSLDYYGMGWFRQAPGKEREFVA
		AITSGGTPHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASAY
		NPGIGYDWGQGTLVTVSS
5-51	1934	EVQLVESGGGLVQPGGSLRLSCAASGRPNSINWMGWFRQAPGKERQFVA
		TITPGGNTNYADSVKGRFTISADNSKNTAYLLMNSLKPEDTAVYYCAAAA
		GTTWYGTLYEYDWGQGTLVTVSS
5-52	1935	EVQLVESGGGLVQPGGSLRLSCAASGEKFSDHWMGWFRQAPGKEREFVA
		TITFSGARTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAALI
		KPSSTDRIFEEWGQGTLVTVSS
5-53	1936	EVQLVESGGGLVQPGGSLRLSCAASGLTVVPYAMGWFRQAPGKEREFVA
		AIRRSAVTNYADSVKGRFTIIADNSKNTAYLLMNSLKPEDTAVYYCAARR
		WGYHYWGQGTLVTVSS
5-54	1937	EVQLVESGGGLVQPGGSLRLSCAASGTTFNFNVMGWFRQAPGKERELVA
		VISWTGESTLYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAF
		DGYTGRDWGQGTLVTVSS
5-55	1938	EVQLVESGGGLVQPGGSLRLSCAASGIDVNRNAMGWFRQAPGKEREFVA
		AITWSGGWRYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAT
		TFGDAGIPDQYDFGWGQGTLVTVSS
5-56	1939	EVQLVESGGGLVQPGGSLRLSCAASGRTFSSNMGWFRQAPGKEREFVARI
		FGGDRTLYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCADING
		DWGQGTLVTVSS
5-57	1940	EVQLVESGGGLVQPGGSLRLSCAASGGTFSMGWIRWVPQAQGKELEFMG
		CIGWITYYADYAKSRFSLFTDNADNTKNPPNMHMNPQKPEDTAVYYCAP
		FGWGQGTLVTVSS
5-58	1941	EVQLVESGGGLVQPGGSLRLSCAASGCTLDYYAMGWFRQAPGKEREFVA
		GIRWTDAYTEYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAA
		DVSPSYGGRWYWGQGTLVTVSS
5-59	1942	EVQLVESGGGLVQPGGSLRLSCAASGLTFSLYRMCWFRQAPGKEREEVSC
		ISNIDGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADL
		LGDSDYEPSSGFGWGQGTLVTVSS
5-60	1943	EVQLVESGGGLVQPGGSLRLSCAASGRSFSSHRMGWFRQAPGKEREFVA
		AIMWSGSHRNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAA
		IAYEEGVYRWDWGQGTLVTVSS
5-61	1944	EVQLVESGGGLVQPGGSLRLSCAASGRIIVPNTMGWFRQAPGKERERVTG
		ISPSAFTEYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAHGW
		GCHWGQGTLVTVSS
5-62	1945	EVQLVESGGGLVQPGGSLRLSCAASGSIFIISMGWFRQAPGKEHEFVTGIN
		WSGGSTTYADSVKGRFTINADNSKNTAYLQMNSLKPEDTAVYYCAASAI
		GSGALRRFEYDWGQGTLVTVSS
5-63	1946	EVQLVESGGGLVQPGGSLRLSCAASGFSLDYYDMGWFRQAPGKEREFVA
		ALGWSGGSTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAA
		GNGGRYGIVERWGQGTLVTVSS
5-64	1947	EVQLVESGGGLVQPGGSLRLSCAASGTSISNRVMGWFRQAPGKERELVAR
		IYTGGDTLYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAARKI
		YRSLSYYGDYDWGQGTLVTVSS
5-65	1948	EVQLVESGGGLVQPGGSLRLSCAASGNIDRLYAMGWFRQAPGKEREGVA
		AIDSDGSTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAALI
		DYGLGFPIEWGQGTLVTVSS
5-66	1949	EVQLVESGGGLVQPGGSLRLSCAASGNTFTINVMGWFRQAPGKEREFVA
		AINWNGGTTLYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAA
		FDGYSGIDWGQGTLVTVSS
5-67	1950	EVQLVESGGGLVQPGGSLRLSCAASGFNVNDYAMGWFRQAPGKEREFVA
		GITSSVGVTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAD
		IFFVNWGRGTLVTVSS
5-68	1951	EVQLVESGGGLVQPGGSLRLSCAASGFTFDHYTMGWFRQAPGKEREFVA
		AISGSENVTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAE
		PYIPVRTMRHMTFLTWGQGTLVTVSS
6-1	1952	EVQLVESGGGLVQPGGSLRLSCAASGRTFGNYNMGWFRQAPGKEREFVA
		TINSLGGTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARVD
		YYMDVWGQGTLVTVSS
6-2	1953	EVQLVESGGGLVQPGGSLRLSCAASGFTMSSSWMGWFRQAPGKEREFVT
		VISGVGTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGPD
		SSGYGFDYWGQGTLVTVSS
6-3	1954	EVQLVESGGGLVQPGGSLRLSCAASGFTFSPSWMGWFRQAPGKEREFVA
		TINEYGGRNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARV
		DRDFDYWGQGTLVTVSS
6-4	1955	EVQLVESGGGLVQPGGSLRLSCAASGFTRDYYTMGWFRQAPGKEREFVA
		AISRSGSLTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCANL
		AYYDSSGYYDYWGQGTLVTVSS
6-5	1956	EVQLVESGGGLVQPGGSLRLSCAASGRTFTMGWFRQAPGKEREFVASTNS
		AGSTNYADSVNGRFTISADNSKNTAYLQMNSLKPEDTAVYYCTTVDQYF
		DYWGQGTLVTVSS
6-6	1957	EVQLVESGGGLVQPGGSLRLSCAASGTTLDYYAMGWFRQAPGKERELVA
		AISWSGGSTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARE
		DYYDSSGYSWGQGTLVTVSS
6-7	1958	EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMGWFRQAPGKEREFVA
		TINWSGVTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARA
		DDYFDYWGQGTLVTVSS
6-8	1959	EVQLVESGGGLVQPGGSLRLSCAASGFTLSGIWMGWFLQAPGKEHEFVAI
		ITTGGRTTYADSXKGRFTSSSDNSKNTAYLQMNLLKPEDTAEYYCAGYST
		FGSSSAYYYYSMDVGWGQGTLVTVSS
6-9	1960	EVQLVESGGGLVQPGGSLRLSCAASGFTFDYYAMGWFRQAPGKEREFVS
		AIDSEGRTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARW
		GPFDIWGQGTLVTVSS
6-10	1961	EVQLVESGGGLVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAA
		ISRSGGFGSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARDD
		KYYDSSGYPAYFQHWGQGTLVTVSS
6-11	1962	EVQLVESGGGLVQPGGSLRLSCAASGLAFNAYAMGWFRQAPGKEREEVA
		TIGWSGANTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAS
		DPPGWGQGTLVTVSS
6-12	1963	EVQLVESGGGLVQPGGSLRLSCAASGSTYTTYSMGWFRQAPGKEREFVA
		AISGSENVTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARV
		DDYMDVWGQGTLVTVSS
6-13	1964	EVQLVESGGGLVQPGGSLRLSCAASGLTFNDYAMGWFRQAPGKEREFVA
		HIPRSTYSPYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAFL
		VGPQGVDHGAFDVWGQGTLVTVSS
6-14	1965	EVQLVESGGGLVQPGGSLRLSCAASGITFRFKAMGWFRQAPGKEREFVAA
		VSWDGRNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASD
		YYYMDVWGQGTLVTVSS
6-15	1966	EVQLVESGGGLVQPGGSLRLSCAASGSTVLINAMGWFRQAPGKEREFVA
		AVRWSDDYTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCA
		KEGRAGSLDYWGQGTLVTVSS
6-16	1967	EVQLVESGGGLVQPGGSLRLSCAASGFTFDDAAMGWFRQAPGKEREFVA
		HISWSGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATF
		GATVTATNDAFDIWGQGTLVTVSS
6-17	1968	EVQLVESGGGLVQPGGSLRLSCAASGNTGSTGYMGWFRQAPGKEREMV
		AGVINDGSTVYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAR
		LATSHQDGTGYLFDYWGQGTLVTVSS
6-18	1969	EVQLVESGGGLVQPGGSLRLSCAASGLTFRNYAMGWFRQAPGKEREFIAG
		MMWSGGTTTYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAR
		EGYYYDSSGYLNYFDYWGQGTLVTVSS
6-19	1970	EVQLVESGGGLVQPGGSLRLSCAASGSILSIAVMGWFRQAPGKEREFVAAI
		SPSAVTTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAIGYY
		DSSGYFDYWGQGTLVTVSS
6-20	1971	EVQLVESGGGLVQPGGSLRLSCAASGSTLPYHAMGWFRQAPGKEREFVA
		AITWNGASTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAR
		DRYYDTSASYFESETWGQGTLVTVSS
6-21	1972	EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWFRQAPGKEREFVA
		AITSSGSNIDYTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYC
		ARSNTGWYSFDYWGQGTLVTVSS
6-22	1973	EVQLVESGGGLVQPGGSLRLSCAASGRTFSEVVMGWFRQAPGKEREFVA
		TIHSSGSTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCVRVT
		SDYSMDSWGQGTLVTVSS
6-23	1974	EVQLVESGGGLVQPGGSLRLSCAASGSIFSMNTMGWFRQAPGKEREFVAL
		INRSGGGINYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCVRLS
		SGYYDFDYWGQGTLVTVSS
6-24	1975	EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAMGWFRQAPGKEREFVA
		AINWSGDNTHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAR
		APFYCTTTKCQDNYYYMDVWGQGTLVTVSS
6-25	1976	EVQLVESGGGLVQPGGSLRLSCAASGLTFGTYTMGWFRQAPGKEREFVA
		AISRFGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGG
		DYDFWSVDYMDVWGQGTLVTVSS
6-26	1977	EVQLVESGGGLVQPGGSLRLSCAASGDTFSTSWMGWFRQAPGKEREFVA
		TINTGGGTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARVT
		TSFDYWGQGTLVTVSS
6-27	1978	EVQLVESGGGLVQPGGSLRLSCAASGITFRFKAMGWFRQAPGKEREFVAS
		ISRSGTTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDYS
		AFDMWGQGTLVTVSS
6-28	1979	EVQLVESGGGLVQPGGSLRLSCAASGDTYGSYWMGWFRQAPGKEREFV
		ATITSDDRTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARV
		TSSLSGMDVWGQGTLVTVSS
6-29	1980	EVQLVESGGGLVQPGGSLRLSCAASGYTLKNYYAMGWFRQAPGKERXL
		VAAIIWTGESTLDADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCA
		REGYYDSSGYYWGQGTLVTVSS
6-30	1981	EVQLVESGGGLVQPGGSLRLSCAASGFAFGDSWMGWFRQAPGKEREFVA
		TINWSGVTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARA
		DGYFDYWGQGTLVTVSS
6-31	1982	EVQLVESGGGLVQPGGSLRLSCAASGDTFSANRMGWFRQAPGKEREFVA
		SITWSSANTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATF
		NWNDEGFDFWGQGTLVTVSS
6-32	1983	EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYDMGWFRQAPGKEREFVA
		LISWSGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATD
		FYGWGTRERDAFDIWGQGTLVTVSS
6-33	1984	EVQLVESGGGLVQPGGSLRLSCAASGTFQRINHMGWFRQAPGKEREFVAT
		INTGGQPNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASLIA
		AQDYYFDYWGQGTLVTVSS
6-34	1985	EVQLVESGGGLVQPGGSLRLSCAASGSAFRSNAMGWFRQAPGKEREFVA
		HISWSSKSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATY
		CSSTSCFDYWGQGTLVTVSS
6-35	1986	EVQLVESGGGLVQPGGSLRLSCAASGFTLAYYAMGWFRQAPGKEREFVA
		AISMSGDDTIYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARE
		LGYSSTVWPWGQGTLVTVSS
6-36	1987	EVQLVESGGGLVQPGGSLRLSCAASGFDFSVSWMGWFRQAPGKEREFVT
		AITWSGDSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASL
		LHTGPSGGNYFDYWGQGTLVTVSS
6-37	1988	EVQLVESGGGLVQPGGSLRLSCAASGHTFSTSWMGWFRQAPGKEREFVA
		TINSLGGTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARVS
		SGDYGMDVWGQGTLVTVSS
6-38	1989	EVQLVESGGGLVQPGGSLRLSCAASGNTFSGGFMGWFRQAPGKEREFVA
		VISSLSSKSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKVD
		SGYDYWGQGTLVTVSS
6-39	1990	EVQLVESGGGLVQPGGSLRLSCAASGFTFSPSWMGWFRQAPGKEREFVA
		AISWSGGSTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCHGL
		GEGDPYGDYEGYFDLWGQGTLVTVSS
6-40	1991	EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMGWFRQAPGKERELVA
		RVWWNGGSAYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCA
		REVLRQQVVLDYWGQGTLVTVSS
6-41	1992	EVQLVESGGGLVQPGGSLRLSCAASGFTFSTSWMGWFRQAPGKEREFVA
		SINEYGGRNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAGL
		HYYYDSSGYNPTEYYGMDVWGQGTLVTVSS
6-42	1993	EVQLVESGGGLVQPGGSLRLSCAASGDTYGSYWMGWFRQAPGKEREFV
		AVITSGGSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCTHV
		QNSYYYAMDVWGQGTLVTVSS
6-43	1994	EVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMMGWFRQAPGKEREFV
		ASVNWDASQINYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCT
		TLGAVYFDSSGYHDYFDYWGQGTLVTVSS
6-44	1995	EVQLVESGGGLVQPGGSLRLSCAASGGTFGVYHMGWFRQAPGKEREFIG
		RITWTDGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCFGL
		LEVYDMTFDYWGQGTLVTVSS
6-45	1996	EVQLVESGGGLVQPGGSLRLSCAASGNMFSINAMGWFRQAPGKEREFVT
		LISWSSGRTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASL
		GYCSGGSCFDYWGQGTLVTVSS
6-46	1997	EVQLVESGGGLVQPGGSLRLSCAASGLTFSAMGWFRQAPGKEREFVALIR
		RDGSTIYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAALGILF
		GYDAFDIWGQGTLVTVSS
6-47	1998	EVQLVESGGGLVQPGGSLRLSCAASGRTFSMHAMGWFRQAPGKERELVA
		SITYGGNINYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKEG
		YYDSTGYRTYFQQWGQGTLVTVSS
6-48	1999	EVQLVESGGGLVQPGGSLRLSCAASGFTVSNYAMGWFRQAPGKEREFVA
		SVNWSGGTTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAT
		TGTVTLGYWGQGTLVTVSS
6-49	2000	EVQLVESGGGLVQPGGSLRLSCAASGSTVLINAMGWFRQAPGKEREFVA
		AISWSPGRTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARD
		CSGGSCYSGDYWGQGTLVTVSS
6-50	2001	EVQLVESGGGLVQPGGSLRLSCAASGFSFDRWAMGWFRQAPGKEREWV
		ASLATGGNTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAR
		VTNYDAFDIWGQGTLVTVSS
6-51	2002	EVQLVESGGGLVQPGGSLRLSCAASGYTYSSYVMGWFRQAPGKEREFVA
		AISRFGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARDS
		GEHFWDSGYIDHWGQGTLVTVSS
6-52	2003	EVQLVESGGGLVQPGGSLRLSCAASGDTYGSYWMGWFRQAPGKEREVV
		AAITSGGSTVYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARV
		DSRFDYWGQGTLVTVSS
6-53	2004	EVQLVESGGGLVQPGGSLRLSCAASGISINTNVMGWFRQAPGKEREFVAA
		ISTGSVTIYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARVDD
		FGYFDLWGQGTLVTVSS
6-54	2005	EVQLVESGGGLVQPGGSLRLSCAASGFEFENHWMGWFRQAPGKEREYVA
		HITAGGLSNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCGRH
		WGIYDSSGFSSFDYWGQGTLVTVSS
6-55	2006	EVQLVESGGGLVQPGGSLRLSCAASGFTMSSSWMGWFRQAPGKEREFVA
		RITSGGSTGYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASVD
		GYFDYWGQGTLVTVSS
6-56	2007	EVQLVESGGGLVQPGGSLRLSCAASGNIFRSNMGWFRQAPGKEREFVAGI
		TWNGDTTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARAL
		GVTYQFDYWGQGTLVTVSS
6-57	2008	EVQLVESGGGLVQPGGSLRLSCAASGLTFDDHSMGWFRQAPGKEREFVA
		AVPLSGNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASFS
		GGPADFDYWGQGTLVTVSS
6-58	2009	EVQLVESGGGLVQPGGSLRLSCAASGRAVSTYAMGWFRQAPGKEREFVA
		AISGSENVTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCLSV
		TGDTEDYGVFDTWGQGTLVTVSS
6-59	2010	EVQLVESGGGLVQPGGSLRLSCAASGISGSVFSRTPMGWFRQAPGKEREW
		VSSIYSDGSNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCA
		HWSWELGDWFDPWGQGTLVTVSS
6-60	2011	EVQLVESGGGLVQPGGSLRLSCAASGDTYGSYWMGWFRQAPGKEREFV
		ATISQSGAATAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAG
		LLRYSGTYYDAFDVWGQGTLVTVSS
6-61	2012	EVQLVESGGGLVQPGGSLRLSCAASGDTYGSYWMGWFRQAPGKEREFV
		AAINWSGGSTNYADSVKGRFTITADNNKNTAYLQMNSLKPEDTAVYYCA
		GLGWNYMDYWGQGTLVTVSS
6-62	2013	EVQLVESGGGLVQPGGSLRLSCAASGSTFSGNWMGWFRQAPGKEREFVA
		VISWTGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAT
		HNSLSGFDYWGQGTLVTVSS
6-63	2014	EVQLVESGGGLVQPGGSLRLSCAASGQTFNMGWFRQAPGKEREFVAAIG
		SGGSTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCWRLGND
		YFDYWGQGTLVTVSS
6-64	2015	EVQLVESGGGLVQPGGSLRLSCAASGIPSIHAMGWFRQAPGKERELVAAI
		NWSHGVTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCGGG
		YGYHFDYWGQGTLVTVSS
6-65	2016	EVQLVESGGGLVQPGGSLRLSCAASGLPFSTLHMGWFRQAPGKEREFVAS
		LSIFGATGYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCWMYY
		YDSSGYYGNYYYGMDVWGQGTLVTVSS
6-66	2017	EVQLVESGGGLVQPGGSLRLSCAASGLTFSLFAMGWFRQAPGKERELVA
		AISSGGSTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGN
		TKYYYDSSGYSSAFDYWGQGTLVTVSS
6-67	2018	EVQLVESGGGLVQPGGSLRLSCAASGSFSNYAMGWFRQAPGKEREFVAAI
		SSSGALTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCWIVGP
		GPLDGMDVWGQGTLVTVSS
6-68	2019	EVQLVESGGGLVQPGGSLRLSCAASGFTLSDRAMGWFRQAPGKEREYVA
		HITAGGLSNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCVHLA
		SQTGAGYFDLWGQGTLVTVSS
6-69	2020	EVQLVESGGGLVQPGGSLRLSCAASGGTFSSVGMGWFRQAPGKEREFVA
		GISRSGGTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARYD
		FWSGYPYWGQGTLVTVSS
6-70	2021	EVQLVESGGGLVQPGGSLRLSCAASGFNLDDYADMGWFRQAPGKEREFV
		AAIGWGGGSTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCA
		REILWFGEFGEPNVWGQGTLVTVSS
6-71	2022	EVQLVESGGGLVQPGGSLRLSCAASGITFSNDAMGWFRQAPGKEREFVAII
		TSSDTNDTTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARL
		HYYDSSGYFDYWGQGTLVTVSS
6-72	2023	EVQLVESGGGLVQPGGSLRLSCAASGSTLSINAMGWFRQAPGKEREFVAA
		IDWSGGSTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARD
		SSATRTGPDYWGQGTLVTVSS
6-73	2024	EVQLVESGGGLVQPGGSLRLSCAASGHTFSGYAMGWFRQAPGKEREFVA
		VITREGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARLG
		GEGFDYWGQGTLVTVSS
6-74	2025	EVQLVESGGGLVQPGGSLRLSCAASGFAFGDSWMGWFRQAPGKERELVA
		AITSGGSTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGL
		LWFGELFGYWGQGTLVTVSS
6-75	2026	EVQLVESGGGLVQPGGSLRLSCAASGGTFSTYWMGWFRQAPGKEREFVA
		AISRSGGNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCVRH
		SGTDGDSSFDYWGQGTLVTVSS
6-76	2027	EVQLVESGGGLVQPGGSLRLSCAASGLAFDFDGMGWFRQAPGKEREGVA
		AINSGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARFF
		RAHDYWGQGTLVTVSS
6-77	2028	EVQLVESGGGLVQPGGSLRLSCAASGFTFDRSWMGWFRQAPGKEREFVA
		AVTEGGTTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARA
		DYDFDYWGQGTLVTVSS
6-78	2029	EVQLVESGGGLVQPGGSLRLSCAASGRTYDAMGWFRQAPGKEREFVASV
		TSGGYTHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKFGR
		KIVGATELDYWGQGTLVTVSS
6-79	2030	EVQLVESGGGLVQPGGSLRLSCAASGSISSIDYMGWFRQAPGKEREGVSW
		ISSSDGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARSPS
		FSQIYYYYYMDVWGQGTLVTVSS
6-80	2031	EVQLVESGGGLVQPGGSLRLSCAASGGTFSFYNMGWFRQAPGKEREFVA
		FISGNGGTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVVA
		MRMVTTEGPDVLDVWGQGTLVTVSS
6-81	2032	EVQLVESGGGLVQPGGSLRLSCAASGFIGNYHAMGWFRQAPGKEREFVA
		AVTWSGGTTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAR
		EGYYYDSSGYPYYFDYWGQGTLVTVSS
6-82	2033	EVQLVESGGGLVQPGGSLRLSCAASGSSLDAYGMGWFRQAPGKEREFVA
		AISWGGGSIYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARL
		SQGMVALDYWGQGTLVTVSS
6-83	2034	EVQLVESGGGLVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAA
		ITWSGAITSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKDG
		GYGELHYGMEVWGQGTLVTVSS
6-84	2035	EVQLVESGGGLVQPGGSLRLSCAASGFTPDDYAMGWFRQAPGKEREFVA
		AINSGGSYTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARD
		RGPWGQGTLVTVSS
6-85	2036	EVQLVESGGGLVQPGGSLRLSCAASGGTFSVFAMGWFRQAPGKEREFVS
		AINWSGGSLLYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCALF
		GDFDYWGQGTLVTVSS
6-86	2037	EVQLVESGGGLVQPGGSLRLSCAASGPISGINRMGWFRQAPGKEREFVAV
		ITSNGRPSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCVRLSS
		GYFDFDYWGQGTLVTVSS
6-87	2038	EVQLVESGGGLVQPGGSLRLSCAASGTSIMVGAMGWFRQAPGKEREFVAI
		IRGDGRTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARFAG
		WDAFDIWGQGTLVTVSS
6-88	2039	EVQLVESGGGLVQPGGSLRLSCAASGRTFSTHWMGWFRQAPGKEREFVA
		VINWSGGSIYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARL
		SSDGYNYFDFWGQGTLVTVSS
6-89	2040	EVQLVESGGGLVQPGGSLRLSCAASGTIFASAMGWFRQAPGKEHQFVAV
		VNWNGSSTVYADNVKGRFTIIADNSKNTAYLQMNSLKPEDTAVYYCTTV
		DQYFNYWGQGTLVTVSS
6-90	2041	EVQLVESGGGLVQPGGSLRLSCAASGFPFSIWPMGWFRQAPGKEREFVAA
		VRWSSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATGEC
		DGGSCSLAYWGQGTLVTVSS
6-91	2042	EVQLVESGGGLVQPGGSLRLSCAASGRTFGNYAMGWFRQAPGKEREFVA
		SISSSGVSKHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCVRF
		GSSWARDLDQWGQGTLVTVSS
6-92	2043	EVQLVESGGGLVQPGGSLRLSCAASGFLFDSYASMGWFRQAPGKEREFV
		ATIWRRGNTYYANYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYY
		CTETGTAAWGQGTLVTVSS
6-93	2044	EVQLVESGGGLVQPGGSLRLSCAASGLPFSTKSMGWFRQAPGKEREFVAA
		ISMSGLTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCLKVLG
		GDYEADNWFDYWGQGTLVTVSS
6-94	2045	EVQLVESGGGLVQPGGSLRLSCAASGNIFRIETMGWFRQAPGKEREFVAGI
		IRSGGETLYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARSLY
		YDRSGSYYFDYWGQGTLVTVSS
6-95	2046	EVQLVESGGGLVQPGGSLRLSCAASGIPSSIRAMGWFRQAPGKEREFVAVI
		RWTGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARDI
		GYYDSSGYYNDGGFDYWGQGTLVTVSS
6-96	2047	EVQLVESGGGLVQPGGSLRLSCAASGFTLSGNWMGWFRQAPGKEREFVA
		IITSGGRTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAGHA
		TFGGSSSSYYYGMDVWGQGTLVTVSS
6-97	2048	EVQLVESGGGLVQPGGSLRLSCAASGFTFSSLAMGWFRQAPGKEREFVAA
		ITWSGDITNYADSVKGRFTITADNSKNTAYLQMNSLKPEDTAVYYCLRLS
		SSGFDHWGQGTLVTVSS
6-98	2049	EVQLVESGGGLVQPGGSLRLSCAASGTFGHYAMGWFRQAPGKEREFVAA
		INWSSRSTVYADSVKGRFTITADNSKNTAYLQMNSLKPEDTAVYYCAKSD
		GLMGELRSASAFDIWGQGTLVTVSS
6-99	2050	EVQLVESGGGLVQPGGSLRLSCAASGIPFRSRTMGWFRQAPGKEREFVAG
		ISRSGASTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCTHAN
		DYGDYWGQGTLVTVSS
6-100	2051	EVQLVESGGGLVQPGGSLRLSCAASGGTFSTSWMGWFRQAPGKEREYVA
		HITAGGLSNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARLL
		VREDWYFDLWGQGTLVTVSS
6-101	2052	EVQLVESGGGLVQPGGSLRLSCAASGGTFSLFAMGWFRQAPGKEREFVA
		AISWTGDSTYYKYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYY
		CAYNNSSGEYWGQGTLVTVSS
6-102	2053	EVQLVESGGGLVQPGGSLRLSCAASGSSFSAYAMGWFRQAPGKEREFVS
		AIDSEGTTTYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAGDY
		NFWSGFDHWGQGTLVTVSS
6-103	2054	EVQLVESGGGLVQPGGSLRLSCAASGRTSSPIAMGWFRQAPGKEREPVAV
		RWSDDYTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKKL
		GGYYAFDIWGQGTLVTVSS
6-104	2055	EVQLVESGGGLVQPGGSLRLSCAASGLTFNQYTMGWFRQAPGKEREFVA
		SITDGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARDS
		RYMDVWGQGTLVTVSS
6-105	2056	EVQLVESGGGLVQPGGSLRLSCAASGPTFSSMGWFRQAPGKEREFVAAIS
		WDGGATAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAIEIV
		VGGIYWGQGTLVTVSS
6-106	2057	EVQLVESGGGLVQPGGSLRLSCAASGIPSTLRAMGWFRQAPGKEREFVAA
		TSWSGGSKYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATD
		LYYMDVWGQGTLVTVSS
6-107	2058	EVQLVESGGGLVQPGGSLRLSCAASGGVGFSVTNMGWFRQAPGKEREFV
		AVISSSSSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCTTFN
		WNDEGFDYWGQGTLVTVSS
6-108	2059	EVQLVESGGGLVQPGGSLRLSCAASGGTFGSYGMGWFRQAPGKEREFVA
		AIRWSGGITYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARE
		RYWNPLPYYYYGMDVWGQGTLVTVSS
6-109	2060	EVQLVESGGGLVQPGGSLRLSCAASGGTFSTYAMGWFRQVPGKEREFVA
		SIDWSGLTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGP
		FYMYCSGTKCYSTNWFDPWGQGTLVTVSS
6-110	2061	EVQLVESGGGLVQPGGSLRLSCAASGPIYAVNRMGWFRQAPGKEREFVA
		GIWRSGGHRDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAR
		GEIDILTGYWYDYWGQGTLVTVSS
6-111	2062	EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMGWFRQAPGKEREFVG
		GISRSGVSTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCTTLL
		YYYDSSGYSFDAFDIWGQGTLVTVSS
6-112	2063	EVQLVESGGGLVQPGGSLRLSCAASGGTFSAYHMGWFRQAPGKERELVTI
		IDNGGPTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCTALLY
		YFDNSGYNFDPFDIWGQGTLVTGSS

TABLE 25
Reformatted SARS-CoV-2 S1 Variant Sequences
	SEQ
	ID
Name	NO	Amino Acid Sequence
2-H1	2064	EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYATDWVRQAPGKGLEWVSII
		SGSGGATYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGG
		YCSSDTCWWEYWLDPWGQGTLVTVSS
2-H2	2065	EVQLLESGGGLVQPGGSLRLSCAASGFTFSAFAMGWVRQAPGKGLEWVS
		AITASGDITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQS
		DGLPSPWHFDLGGQGTLVTVSS
2-H3	2066	EVQLLESGGGLVQPGGSLRLSCAASGFTFSDFAMAWVRQAPGKGLEWVS
		AISGSGDITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREA
		DGLHSPWHFDLWGQGTLVTVSS
2-H4	2067	EVQLLESGGGLVQPGGSLRLSCAASGFTFSRHAMNWVRQAPGKGLEWVS
		GISGSGDETYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARD
		LPASYYDSSGYYWHNGMDVWGQGTLVTVSS
2-H5	2068	EVQLLESGGGLVQPGGSLRLSCAASGFTFSDFAMAWVRQAPGKGLEWVS
		AISGSGDITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREA
		DCLPSPWYLDLWGQGTLVTVSS
2-H6	2069	EVQLLESGGGLVQPGGSLRLSCAASGFTFSDFAMAWVRQAPGKGLEWVS
		AISGSGDITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREA
		DGLHSPWHFDLWGQGTLVTVSS
2-H7	2070	EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYPMNWVRQAPGKGLEWVST
		ISGSGGNTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRHD
		EYSFDYWGQGTLVTVSS
2-H8	2071	EVQLLESGGGLVQPGGSLRLSCAASGFTFSDFAMAWVRQAPGKGLEWVS
		AITGSGDITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREA
		DGLHSPWHFDLWGQGTLVTVSS
2-H9	2072	EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYPMNWVRQAPGKGLEWVST
		ISGSGGITFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRHDE
		YSFDYWGQGTLVTVSS
2-	2073	EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYPMNWVRQAPGKGLEWVS
H10		AISGSGDNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRH
		DEYSFDYWGQGTLVTVSS
2-	2074	EVQLLESGGGLVQPGGSLRLSCAASGFTFSDFAMAWVRQAPGKGLEWVS
H11		AITGTGDITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREA
		DGLHSPWGQGTLVTVSS
2-	2075	EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYPMNWVRQAPGKGLEWVS
H12		AITGSGDITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRHD
		EYSFDYWGQGTLVTVSS
2-	2076	EVQLLESGGGLVQPGGSLRLSCAASGFTFSDFAMAWVRQAPGKGLEWVS
H13		AISGSGDITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREA
		DGLHSPWHFDLWGQGTLVTVSS
2-	2077	EVQLLESGGGLVQPGGSLRLSCAASGFTFSDFAMAWVRQAPGKGLEWVS
H14		AISGSGDITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREA
		DGLHSPWHFDLWGQGTLVTVSS
2-	2078	EVQLLESGGGLVQPGGSLRLSCAASGFTFPRYAMSWVRQAPGKGLEWVST
H15		ISGSGSTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLID
		AFDIWGQGTLVTVSS
2-L1	2079	DIQMTQSPSSLSASVGDRVTITCRASQSIHRFLNWYQQKPGKAPKLLIYAAS
		NLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYGLPPTFGQGTKV
		EIK
2-L2	2080	DIQMTQSPSSLSASVGDRVTITCRASQSIHISLNWYQQKPGKAPKLLIYLASP
		LASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPPYTFGQGTKV
		EIK
2-L3	2081	DIQMTQSPSSLSASVGDRVTITCRASQSIHTYLNWYQQKPGKAPKLLIYAAS
		ALASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPPYTFGQGTK
		VEIK
2-L4	2082	DIQMTQSPSSLSASVGDRVTITCRASQTINTYLNWYQQKPGKAPKLLIYSAS
		TLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTFTFGQGTKVEI
		K
2-L5	2083	DIQMTQSPSSLSASVGDRVTITCRASQNIHTYLNWYQQKPGKAPKLLIYAA
		STFAKGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPPYTFGQGT
		KVEIK
2-L6	2084	DIQMTQSPSSLSASVGDRVTITCRASQSIDTYLNWYQQKPGKAPKLLIYAAS
		ALASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPPYTFGQGTK
		VEIK
2-L7	2085	DIQMTQSPSSLSASVGDRVTITCRASQSIGNYLNWYQQKPGKAPKLLIYGV
		SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHSAPLTFGQGTKV
		EIK
2-L8	2086	DIQMTQSPSSLSASVGDRVTITCRASQSIDTYLNWYQQKPGKAPKLLIYAAS
		ALASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPPYTFGQGTK
		VEIK
2-L9	2087	DIQMTQSPSSLSASVGDRVTITCRASQSIDNYLNWYQQKPGKAPKLLIYGV
		SALQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHSAPPYFFGQGT
		KVEIK
2-L10	2088	DIQMTQSPSSLSASVGDRVTITCRASQSIDTYLNWYQQKPGKAPKLLIYGAS
		ALESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHSAPPYFFGQGTK
		VEIK
2-L11	2089	DIQMTQSPSSLSASVGDRVTITCRASQSIDTYLNWYQQKPGKAPKLLIYAAS
		ALASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPPYTFGQGTK
		VEIK
2-L12	2090	DIQMTQSPSSLSASVGDRVTITCRASQSIDTYLNWYQQKPGKAPKLLIYGVS
		ALQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPPYFFGQGTK
		VEIK
2-L13	2091	DIQMTQSPSSLSASVGDRVTITCRASQSIDTYLNWYQQKPGKAPKLLIYAAS
		ALASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPPYTFGQGTK
		VEIK
2-L14	2092	DIQMTQSPSSLSASVGDRVTITCRASQSIDNYLNWYQQKPGKAPKLLIYGV
		SALQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHSAPLTFGQGTK
		VEIK
2-L15	2093	DIQMTQSPSSLSASVGDRVTITCRASQRIGTYLNWYQQKPGKAPKLLIYAA
		SNLEGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNYSTTWTFGQGTK
		VEIK
2-	2094	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSV
H16		ISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREG
		YRDYLWYFDLWGQGTLVTVSS
2-	2095	EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVS
H17		AISGSAGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARV
		RQGLRRTWYYFDYWGQGTLVTVSS
2-	2096	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMYWVRQAPGKGLEWVS
H18		AISGSAGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARD
		TNDFWSGYSIFDPWGQGTLVTVSS
2-	2097	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVSV
H19		ISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREG
		YRDYLWYFDLWGQGTLVTVSS
2-	2098	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSV
H20		ISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGPL
		VGWYFDLWGQGTLVTVSS
2-L16	2099	DIQMTQSPSSLSASVGDRVTITCTGTSSDVGSYDLVSWYQQKPGKAPKLLI
		YEGNKRPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCCSYAGSSVVFGQ
		GTKVEIK
2-L17	2100	DIQMTQSPSSLSASVGDRVTITCTGTSSDVGSSNLVSWYQQKPGKAPKLLIY
		EGSKRPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCCSYAGSLYVFGQG
		TKVEIK
2-L18	2101	DIQMTQSPSSLSASVGDRVTITCTGTSSDIGSYNLVSWYQQKPGKAPKLLIY
		EGTKRPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCCSYAGSRTYVFGQ
		GTKVEIK
2-L19	2102	DIQMTQSPSSLSASVGDRVTITCTGTSTDVGSYNLVSWYQQKPGKAPKLLI
		YEGTKRPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCCSYAGSYTSVVF
		GQGTKVEIK
2-L20	2103	DIQMTQSPSSLSASVGDRVTITCTGTSSNVGSYNLVSWYQQKPGKAPKLLI
		YEGTKRPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCCSYAGSSSFVVFG
		QGTKVEIK

TABLE 26
Reformatted ACE2 Variant Sequences
	SEQ
	ID
Name	NO	Amino Acid Sequence
3-H1	2104	EVQLLESGGGLVQPGGSLRLSCAASGFTFRSHAMSWVRQAPGKGLEWVSSI
		SGGGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVKY
		LTTSSGWPRPYFDNWGQGTLVTVSS
3-H2	2105	EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYSMSWVRQAPGKGLEWVSA
		ISGSGGSRYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGRSK
		WPQANGAFDIWGQGTLVTVSS
3-H3	2106	EVQLLESGGGLVQPGGSLRLSCAASGFMFGNYAMSWVRQAPGKGLEWVA
		AISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDR
		GYSSSWYGGFDYWGQGTLVTVSS
3-H4	2107	EVQLLESGGGLVQPGGSLRLSCAASGFTFRNHAMAWVRQAPGKGLEWVS
		GISGSGGTTYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGT
		RFLQWSLPLDVWGQGTLVTVSS
3-H5	2108	EVQLLESGGGLVQPGGSLRLSCAASGFTIPNYAMSWVRQAPGKGLEWVSGI
		SGAGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHTW
		WKGAGFFDHWGQGTLVTVSS
3-H6	2109	EVQLLESGGGLVQPGGSLRLSCAASGFTFRNYAMAWVRQAPGKGLEWVS
		GISGSGGTTYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGT
		RFLEWSLPLDVWGQGTLVTVSS
3-H7	2110	EVQLLESGGGLVQPGGSLRLSCAASGFTIRNYAMSWVRQAPGKGLEWVSSI
		SGGGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVKY
		LTTSSGWPRPYFDNWGQGTLVTVSS
3-H8	2111	EVQLLESGGGLVQPGGSLRLSCAASGFTIPNYAMSWVRQAPGKGLEWVSGI
		SGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHTW
		WKGAGFFDHWGQGTLVTVSS
3-H9	2112	EVQLLESGGGLVQPGGSLRLSCAASGFTITNYAMSWVRQAPGKGLEWVSG
		ISGSGAGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHA
		WWKGAGFFDHWGQGTLVTVSS
3-	2113	EVQLLESGGGLVQPGGSLRLSCAASGFTFRSHAMSWVRQAPGKGLEWVSSI
H10		SGGGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVKY
		LTTSSGWPRPYFDNWGQGTLVTVSS
3-	2114	EVQLLESGGGLVQPGGSLRLSCAASGFTITNYAMSWVRQAPGKGLEWVSG
H11		ISGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHT
		WWKGAGFFDHWGQGTLVTVSS
3-	2115	EVQLLESGGGLVQPGGSLRLSCAASGFTFRSHAMNWVRQAPGKGLEWVSA
H12		ISGSGGSTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGLK
		FLEWLPSAFDIWGQGTLVTVSS
3-	2116	EVQLLESGGGLVQPGGSLRLSCAASGFTFRSHAMSWVRQAPGKGLEWVSSI
H13		SGGGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVKY
		LTTSSGWPRPYFDNWGQGTLVTVSS
3-	2117	EVQLLESGGGLVQPGGSLRLSCAASGFTFRSYAMSWVRQAPGKGLEWVSSI
H14		SGGGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVKY
		LTTSSGWPRPYFDNWGQGTLVTVSS
3-	2118	EVQLLESGGGLVQPGGSLRLSCAASGFTITNYAMSWVRQAPGKGLEWVSG
H15		ISGSGAGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHT
		WWKGAGFFDHWGQGTLVTVSS
3-L1	2119	DIQMTQSPSSLSASVGDRVTITCRASQSIRKYLNWYQQKPGKAPKLLIYASS
		TLQRGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLSTPFTFGQGTKVE
		IK
3-L2	2120	DIQMTQSPSSLSASVGDRVTITCRASQNIKTYLNWYQQKPGKAPKLLIYAAS
		KLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTSPTFGQGTKVE
		IK
3-L3	2121	DIQMTQSPSSLSASVGDRVTITCRASQTIYSYLNWYQQKPGKAPKLLIYATS
		TLQGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHRGTFGQGTKVEIK
3-L4	2122	DIQMTQSPSSLSASVGDRVTITCRASRSIRRYLNWYQQKPGKAPKLLIYASS
		SLQAGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTLLTFGQGTKVE
		IK
3-L5	2123	DIQMTQSPSSLSASVGDRVTITCRASQSIGKYLNWYQQKPGKAPKLLIYASS
		SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSPPFTFGQGTKVEI
		K
3-L6	2124	DIQMTQSPSSLSASVGDRVTITCRASRSISRYLNWYQQKPGKAPKLLIYAAS
		SLQAGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSLLTFGQGTKVE
		IK
3-L7	2125	DIQMTQSPSSLSASVGDRVTITCRASQSIGKYLNWYQQKPGKAPKLLIYASS
		TLQRGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLSPPFTFGQGTKVE
		IK
3-L8	2126	DIQMTQSPSSLSASVGDRVTITCRASQSIGRYLNWYQQKPGKAPKLLIYASS
		SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSLPRTFGQGTKVE
		IK
3-L9	2127	DIQMTQSPSSLSASVGDRVTITCRASQSIGRYLNWYQQKPGKAPKLLIYAAS
		SLKSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSLPRTFGQGTKVE
		IK
3-L10	2128	DIQMTQSPSSLSASVGDRVTITCRASQSIGKYLNWYQQKPGKAPKLLIYASS
		TLQRGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLSTPFTFGQGTKVE
		IK
3-L11	2129	DIQMTQSPSSLSASVGDRVTITCRASQSIGRYLNWYQQKPGKAPKLLIYAAS
		SLKSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSLPLTFGQGTKVE
		IK
3-L12	2130	DIQMTQSPSSLSASVGDRVTITCRTSQSINTYLNWYQQKPGKAPKLLIYGAS
		NVQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYRIPRTFGQGTKVE
		IK
3-L13	2131	DIQMTQSPSSLSASVGDRVTITCRASQSIGKYLNWYQQKPGKAPKLLIYASS
		TLQRGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSFSPPFTFGQGTKVEI
		K
3-L14	2132	DIQMTQSPSSLSASVGDRVTITCRASQSIGKYLNWYQQKPGKAPKLLIYASS
		TLQRGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSFSTPFTFGQGTKVE
		IK
3-L15	2133	DIQMTQSPSSLSASVGDRVTITCRASQSIGRYLNWYQQKPGKAPKLLIYAAS
		SLKSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSLPRTFGQGTKVE
		IK
3-	2134	EVQLLESGGGLVQPGGSLRLSCAASGFTFTNFAMSWVRQAPGKGLEWVSA
H16		ISGRGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDA
		HGYYYDSSGYDDWGQGTLVTVSS
3-	2135	EVQLLESGGGLVQPGGSLRLSCAASGFTFRSYPMSWVRQAPGKGLEWVSTI
H17		SGSGGITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGVY
		GSTVTTCHWGQGTLVTVSS
3-	2136	EVQLLESGGGLVQPGGSLRLSCAASGFTLTSYAMSWVRQAPGKGLEWVSA
H18		ISGSGVDTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARPTN
		WGFDYWGQGTLVTVSS
3-	2137	EVQLLESGGGLVQPGGSLRLSCAASGFTFINYAMSWVRQAPGKGLEWVSTI
H19		STSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARADS
		NWASSAYWGQGTLVTVSS
3-	2138	EVQLLESGGGLVQPGGSLRLSCAASGFPFSTYAMSWVRQAPGKGLEWVSG
H20		ISVSGGFTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDPY
		SYGYYYYYGMDVWGQGTLVTVSS
3-	2139	EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMGWVRQAPGKGLEWVSG
H21		ISGGGVSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAR
		NWGPSDYWGQGTLVTVSS
3-	2140	EVQLLESGGGLVQPGGSLRLSCAASGFIFSDYAMTWVRQAPGKGLEWVSAI
H22		SGSAFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDATYSSS
		WYNWFDPWGQGTLVTVSS
3-	2141	EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYAMTWVRQAPGKGLEWVSD
H23		ISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGTV
		TSFDFWGQGTLVTVSS
3-	2142	EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYAMGWVRQAPGKGLEWVSFI
H24		SGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDYH
		SASWFSAAADYWGQGTLVTVSS
3-	2143	EVQLLESGGGLVQPGGSLRLSCAASGFTFASYAMTWVRQAPGKGLEWVSA
H25		ISESGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGQ
		EYSSGSSYFDYWGQGTLVTVSS
3-	2144	EVQLLESGGGLVQPGGSLRLSCAASGFTFSEYAMSWVRQAPGKGLEWVSA
H26		ITGSGGSTYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSQ
		TPYCGGDCPETFDYWGQGTLVTVSS
3-	2145	EVQLLESGGGLVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVS
H27		GISGGGTSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDL
		YSSGWYGFDYWGQGTLVTVSS
3-	2146	EVQLLESGGGLVQPGGSLRLSCAASGFTFNNYAMNWVRQAPGKGLEWVS
H28		AISGSVGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDN
		YDFWSGYYTNWFDPWGQGTLVTVSS
3-	2147	EVQLLESGGGLVQPGGSLRLSCAASGFTFTNHAMSWVRQAPGKGLEWVSA
H29		ISGSGSNIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSLS
		VTMGRGVVTYYYYGMDFWGQGTLVTVSS
3-L16	2148	DIQMTQSPSSLSASVGDRVTITCRASQIIGSYLNWYQQKPGKAPKLLIYTTSN
		LQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYITPWTFGQGTKVEI
		K
3-L17	2149	DIQMTQSPSSLSASVGDRVTITCRASQSISRYINWYQQKPGKAPKLLIYEASS
		LESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHITPLTFGQGTKVEIK
3-L18	2150	DIQMTQSPSSLSASVGDRVTITCRASQSIYTYLNWYQQKPGKAPKLLIYSAS
		NLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSDTTPWTFGQGTKV
		EIK
3-L19	2151	DIQMTQSPSSLSASVGDRVTITCRASQSIATYLNWYQQKPGKAPKLLIYGAS
		SLEGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTFSSPFTFGQGTKVEI
		K
3-L20	2152	DIQMTQSPSSLSASVGDRVTITCRASQNINTYLNWYQQKPGKAPKLLIYSAS
		SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSLTPWTFGQGTKVE
		IK
3-L21	2153	DIQMTQSPSSLSASVGDRVTITCRASQGIATYLNWYQQKPGKAPKLLIYYAS
		NLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTRFTFGQGTKVE
		IK
3-L22	2154	DIQMTQSPSSLSASVGDRVTITCRASERISNYLNWYQQKPGKAPKLLIYTAS
		NLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTPPRTFGQGTKVE
		IK
3-L23	2155	DIQMTQSPSSLSASVGDRVTITCRASQSISSSLNWYQQKPGKAPKLLIYAAS
		RLQDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPRSFGQGTKV
		EIK
3-L24	2156	DIQMTQSPSSLSASVGDRVTITCRASQSISSHLNWYQQKPGKAPKLLIYRAS
		TLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYNTPQTFGQGTKV
		EIK
3-L25	2157	DIQMTQSPSSLSASVGDRVTITCRASQSISSYLIWYQQKPGKAPKLLIYAASR
		LHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYNTPRTFGQGTKVEI
		K
3-L26	2158	DIQMTQSPSSLSASVGDRVTITCRASPSISTYLNWYQQKPGKAPKLLIYTASR
		LQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYSTPSSFGQGTKVEI
		K
3-L27	2159	DIQMTQSPSSLSASVGDRVTITCRASQNIAKYLNWYQQKPGKAPKLLIYGA
		SGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHSPPITFGQGTKV
		EIK
3-L28	2160	DIQMTQSPSSLSASVGDRVTITCRASQSIGTYLNWYQQKPGKAPKLLIYAAS
		NLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQESYSAPYTFGQGTKV
		EIK
3-L29	2161	DIQMTQSPSSLSASVGDRVTITCRASQSISPYLNWYQQKPGKAPKLLIYKAS
		SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSSTPYTFGQGTKVE
		IK

TABLE 27
Reformatted ACE2 Variant Sequences
	SEQ
	ID
Name	NO	Amino Acid Sequence
4-51	2162	EVQLVESGGGLVQPGGSLRLSCAASGPGTAIMGWFRQAPGKEREFVARIST
		SGGSTKYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARTTVTT
		PPLIWGQGTLVTVSS
4-52	2163	EVQLVESGGGLVQPGGSLRLSCAASGRSFSNSVMGWFRQAPGKEREFVAR
		ITWNGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATTE
		NPNPRWGQGTLVTVSS
4-53	2164	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDDTMGWFRQAPGKEREFVA
		AVSWSGSGVYYADSVKGRFTITADNSKNTAYLQMNSLKPENTAVYYCAT
		DPPLFWGQGTLVTVSS
4-54	2165	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDARMGWFRQAPGKEREFVGA
		VSWSGGTTVYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATTE
		DPYPRWGQGTLVTVSS
4-49	2166	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKERESVAA
		INWSAGYTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARAS
		PNTGWHFDHWGQGTLVTVSS
4-55	2167	EVQLVESGGGLVQPGGSLRLSCAASGSGLSINAMGWFRQAPGKERESVAAI
		SWSGGSTYTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAA
		YQAGWGDWGQGTLVTVSS
4-39	2168	EVQLVESGGGLVQPGGSLRLSCAASGRTFSNAAMGWFRQAPGKEREFVAR
		ILWTGASRNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATTE
		NPNPRWGQGTLVTVSS
4-56	2169	EVQLVESGGGLVQPGGSLRLSCAASGFSLDYYGMGWFRQAPGKERESVA
		AISWNGDFTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKR
		ANPTGAYFDYWGQGTLVTVSS
4-33	2170	EVQLVESGGGLVQPGGSLRLSCAASGFTFSRHDMGWFRQAPGKEREFVAG
		INWESGSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADR
		GVYGGRWYRTSQYTWGQGTLVTVSS
4-57	2171	EVQLVESGGGLVQPGGSLRLSCAASGLTFRNYAMGWFRQAPGKEREFVA
		AIGSGGYTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVKP
		GWVARDPSQYNWGQGTLVTVSS
4-25	2172	EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYAMGWFRQAPGKEREWVS
		AVDSGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASP
		SLRSAWQWGQGTLVTVSS
4-58	2173	EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYDMGWFRQAPGKEREFVA
		AVTWSGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAA
		DRRGLASTRAADYDWGQGTLVTVSS
4-59	2174	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREFVAA
		INWSAGYTPYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATAP
		PLFCWHFDLWGQGTLVTVSS
4-6	2175	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDDIMGWFRQAPGKEREFVAA
		IHWSAGYTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDP
		PLFWGHVDLWGQGTLVTVSS
4-61	2176	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREIVAAI
		NWSADYTPYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATAPP
		NTGWHFDHWGQGTLVTVSS
4-3	2177	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREIVAAI
		NWSAGYTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATAT
		PNTGWHFDHWGQGTLVTVSS
4-62	2178	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKEREFVAA
		INWSGGSTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDP
		PLFWGQGTLVTVSS
4-43	2179	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDDTMGWFRQAPGKEREFVA
		GINWSGGNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATD
		PPLFWGQGTLVTVSS
4-5	2180	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREFVAA
		INWTGGYTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDP
		PLFWGQGTLVTVSS
4-42	2181	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDDTMGWFRQAPGKERECVA
		AINWSGGNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATD
		PPLFWGQGTLVTVSS
4-63	2182	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDYTMGWFRQAPGKEREFVAA
		INWSGGYTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDP
		PLFWGQGTLVTVSS
4-6	2183	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYGMGWFRQAPGKEREFVA
		TINWSGALTHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATL
		PFYDFWSGYYTGYYYMDVWGQGTLVTVSS
4-40	2184	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKEREFLAG
		VTWSGSSTFYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDP
		PLFWGQGTLVTVSS
4-21	2185	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDIMGWFRQAPGKEREFVAAI
		SWSGGNTHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPP
		LFWGQGTLVTVSS
4-64	2186	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKERESVAA
		INWSAGYTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATAS
		PNTGWHFDHWGQGTLVTVSS
4-47	2187	EVQLVESGGGLVQPGGSLRLSCAASGFTFDDDYVMGWFRQAPGKEREFV
		AAVSGSGDDTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCA
		ADRRGLASTRAADYDWGQGTLVTVSS
4-65	2188	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREFVAA
		INWSAGYTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATEP
		PLSCWHFDLWGQGTLVTVSS
4-18	2189	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREIVAAI
		NWSGGYTPYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATAPP
		NTGWHFDHWGQGTLVTVSS
4-66	2190	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDDTMGWFRQAPGKEREIVAA
		INWSAGYTPYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDP
		PLFCCHFDLWGQGTLVTVSS
4-36	2191	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKEREFVAA
		ISWSGGTTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDP
		PLFWGQGTLVTVSS
4-67	2192	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKEREFVAA
		INWSGDSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDP
		PLFWGQGTLVTVSS
4-16	2193	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKEREFVAA
		INWSGGTTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDP
		PLFWGQGTLVTVSS
4-11	2194	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDAMGWFRQAPGKEREFVAA
		IHWSGSSTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPP
		LFWGQGTLVTVSS
4-68	2195	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKERELVGT
		INWSGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDP
		PLFWGQGTLVTVSS
4-34	2196	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREFVAA
		INWSGGYTPYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDP
		PLFWGQGTLVTVSS
4-28	2197	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDDTMGWFRQAPGKERELVA
		AINWNGGNTHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAT
		DPPLFWGQGTLVTVSS
4-69	2198	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDAMGWFRQAPGKEREFVAA
		INWSGGTTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDP
		PLFWGQGTLVTVSS
4-7	2199	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKERESVAA
		INWSAGYTPYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDP
		PLFWGHVDLWGQGTLVTVSS
4-71	2200	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKEREWVA
		SINWSGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATD
		PPLFWGQGTLVTVSS
4-23	2201	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDAMGWFRQAPGKEREFVAG
		ISWNGGSIYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPP
		LFWGQGTLVTVSS
4-9	2202	EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYEMGWFRQAPGKEREFVAA
		ISWRGGTTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADR
		RGLASTRAGDYDWGQGTLVTVSS
4-72	2203	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDDTMGWFRQAPGKEREFVA
		AINWSGGYTPYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATD
		PPLFWGHVDLWGQGTLVTVSS
4-73	2204	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDAMGWFRQAPGKEREFVAA
		INWSGGSTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDP
		PLFWGQGTLVTVSS
4-29	2205	EVQLVESGGGLVQPGGSLRLSCAASGVTLDDYAMGWFRQAPGKEREFVA
		VINWSGGSTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARG
		GGWVPSSTSESLNWYFDRWGQGTLVTVSS
4-41	2206	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKERESVAA
		INWSGGTTPYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDP
		PLFCCHVDLWGQGTLVTVSS
4-74	2207	EVQLVESGGGLVQPGGSLRLSCAASGLTFSDDTMGWFRQAPGKEREFVAA
		VSWSGGNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATD
		PPLFWGQGTLVTVSS
4-75	2208	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDDTMGWFRQAPGKEREFVA
		AINWTGGYTPYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATD
		PPLFWGQGTLVTVSS
4-31	2209	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREFVATI
		NWTAGYTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDP
		PLFCWHFDHWGQGTLVTVSS
4-32	2210	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDDTMGWFRQAPGKEREFVA
		AINWSGGNTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATD
		PPLFWGQGTLVTVSS
4-15	2211	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYTMGWFRQAPGKEREFVA
		AINWSGGNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATD
		PPLFWGQGTLVTVSS
4-14	2212	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKEREFVAG
		INWSGNGVYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDP
		PLFWGQGTLVTVSS
4-76	2213	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYAMGWFRQAPGKERELVA
		PINWSGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATD
		PPLFWGQGTLVTVSS
4-50	2214	EVQLVESGGGLVQPGGSLRLSCAASGGTFSNSGMGWFRQAPGKERELVAV
		VNWSGRRTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVP
		WMDYNRRDWGQGTLVTVSS
4-17	2215	EVQLVESGGGLVQPGGSLRLSCAASGQLANFASYAMGWFRQAPGKEREF
		VAAITRSGSSTVYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAT
		TMNPNPRWGQGTLVTVSS
4-37	2216	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDIMGWFRQAPGKEREFVAAI
		NWTGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPP
		LFWGQGTLVTVSS
4-44	2217	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREIVAAI
		NWSAGYTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATAR
		PNTGWHFDHWGQGTLVTVSS
4-77	2218	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKEREWVG
		SINWSGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATD
		PPLFWGQGTLVTVSS
4-78	2219	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKEREFVAG
		MTWSGSSTFYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDP
		PLFWGQGTLVTVSS
4-79	2220	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKERECVAA
		INWSGDYTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDP
		PLFWGQGTLVTVSS
4-8	2221	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREFVGG
		INWSGGYTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDP
		PLFWGQGTLVTVSS
4-81	2222	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKEREFVAA
		VNWSGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATD
		PPLFWGQGTLVTVSS
4-82	2223	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYAMGWFRQAPGKEREFVA
		AINWSGGYTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATD
		PPLFWGQGTLVTVSS
4-83	2224	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDDTMGWFRQAPGKEREFVA
		AINWSGGYTPYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATD
		PPLFWGQGTLVTVSS
4-35	2225	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKERESVAA
		INWSAGYTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARAS
		PNTGWHFDRWGQGTLVTVSS
4-45	2226	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREFVAA
		INWSGGYTHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDP
		PLFWGQGTLVTVSS
4-84	2227	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKEREFVAA
		ITWSGGRTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDR
		PLFWGQGTLVTVSS
4-85	2228	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKERESVAA
		INWSGGYTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATAS
		PNTGWHFDHWGQGTLVTVSS
4-86	2229	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKEREFVAA
		IHWSGSSTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPP
		LFWGQGTLVTVSS
4-87	2230	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDYTMGWFRQAPGKEREWVA
		AINWSGGTTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATD
		PPLFWGQGTLVTVSS
4-88	2231	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDDTMGWFRQAPGKEREFVA
		AINWSGDNTHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATD
		PPLFWGQGTLVTVSS
4-89	2232	EVQLVESGGGLVQPGGSLRLSCAASGFAFGDNWIGWFRQAPGKEREWVA
		SISSGGTTAYADNVKGRFTIIADNSKNTAYLQMNSLKPEDTAVYYCAHRG
		GWLRPWGYWGQGTLVTVSS
4-9	2233	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDAMGWFRQAPGKEREFVGR
		INWSGGNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDP
		PLFWGQGTLVTVSS
4-91	2234	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKEREFVGG
		ISWSGGNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDP
		PLFWGQGTLVTVSS
4-92	2235	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKEREFVAA
		INWSGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDP
		PLFWGQGTLVTVSS
4-46	2236	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDDTMGWFRQAPGKEREFVA
		AINWSGGYTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATD
		PPLFWGQGTLVTVSS
4-20	2237	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREFVAA
		INWSADYTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDP
		PLFCWHFDHWGQGTLVTVSS
4-93	2238	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDAMGWFRQAPGKEREFVAA
		INWSGSSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDP
		PLFWGQGTLVTVSS
4-4	2239	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREMVA
		AINWIAGYTADADSVRRLFTITADNNKNTAHLMMNLLKPENTAVYYCAEP
		SPNTGWHFDHWGQGTLVTVSS
4-2	2240	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDDTMGWFRQAPGKEREFVA
		AINWSGGNTPYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATD
		PPLFWGQGTLVTVSS
4-94	2241	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDTMGWFRQAPGKEREFVAA
		INWSGDNTHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDP
		PLFWGQGTLVTVSS
4-95	2242	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREIVAAI
		NWSAGYTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATAP
		PLFCWHFDHWGQGTLVTVSS
4-12	2243	EVQLVESGGGLVQPGGSLRLSCAASGFTFGDYVMGWFRQAPGKEREIVAA
		INWNAGYTAYADSVRGLFTITADNSKNTAYLQMNSLKPEDTAVYYCAKA
		SPNTGWHFDHWGQGTLVTVSS
4-30	2244	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYTMGWFRQAPGKEREFVA
		AINWTGGYTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAT
		DPPLFWGQGTLVTVSS
4-27	2245	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREIVAAI
		NWSAGYTAYADSVKGLFTITADNSKNTAYLQMNILKPEDTAVYYCARATP
		NTGWHFDHWGQGTLVTVSS
4-22	2246	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREFVAA
		INWSGDNTHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDP
		PLFWGQGTLVTVSS
4-96	2247	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKEREIVAAI
		NWSAGYTPYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDPP
		LFCCHFDHWGQGTLVTVSS
4-97	2248	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKERESVAA
		INWSAGYTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATAP
		PNTGWHFDHWGQGTLVTVSS
4-98	2249	EVQLVESGGGLVQPGGSLRLSCAASGFTWGDYTMGWFRQAPGKEREFVA
		AINWSGGNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAA
		DRRGLASTRAADYDWGQGTLVTVSS
4-99	2250	EVQLVESGGGLVQPGGSLRLSCAASGIPSTLRAMGWFRQAPGKEREFVAA
		VSSLGPFTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAKP
		GWVARDPSQYNWGQGTLVTVSS
4-100	2251	EVQLVESGGGLVQPGGSLRLSCAASGFSFDDDYVMGWFRQAPGKEREFVA
		AINWSGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAD
		RRGLASTRAADYDWGQGTLVTVSS
4-101	2252	EVQLVESGGGLVQPGGSLRLSCAASGRTFSNAAMGWFRQAPGKEREFVAR
		ILWTGASRSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATTE
		NPNPRWGQGTLVTVSS
4-102	2253	EVQLVESGGGLVQPGGSLRLSCAASGGTFGVYHMGWFRQAPGKEREGVA
		AINMSGDDSAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAIL
		VGPGQVEFDHWGQGTLVTVSS
4-103	2254	EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMGWFRQAPGKEREFVARI
		SGSTFYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAALPFVCPS
		GSYSDYGDEYDWGQGTLVTVSS
4-104	2255	EVQLVESGGGLVQPGGSLRLSCAASGRTFSGDFMGWFRQAPGKEREFVGR
		INWSGGNTYYADSVRGLFTITADNNKNTAYLMMNLLKPEDTAVYYCPTDP
		PLFWGLGTLVTWSS
4-105	2256	EVQLVESGGGLVQPGGSLRLSCAASGSTLRDYAMGWFRQAPGKERESVA
		AITWSGGSTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASL
		LAGDRYFDYWGQGTLVTVSS
4-106	2257	EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYTMGWFRQAPGKEREFVAA
		ITDNGGSKYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADR
		RGLASTRAADYDWGQGTLVTVSS
4-107	2258	EVQLVESGGGLVQPGGSLRLSCAASGGTFSSYGMGWFRQAPGKEREFVAA
		INWSGASTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARD
		WRDRTWGNSLDYWGQGTLVTVSS
4-108	2259	EVQLVESGGGLVQPGGSLRLSCAASGFSFDDDYVMGWFRQAPGKEREFVA
		AISWSEDNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAD
		RRGLASTRAADYDWGQGTLVTVSS
4-109	2260	EVQLVESGGGLVQPGGSLRLSCAASGFSFDDDYVMGWFRQAPGKEREFVA
		AVSGSGDDTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAA
		DRRGLASTRAADYDWGQGTLVTVSS
4-11	2261	EVQLVESGGGLVQPGGSLRLSCAASGNIAAINVMGWFRQAPGKEREFVAA
		ISASGRRTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARRV
		YYYDSSGPPGVTFDIWGQGTLVTVSS
4-111	2262	EVQLVESGGGLVQPGGSLRLSCAASGIITSRYVMGWFRQAPGKEREGVAAI
		STGGSTIYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARQDSSS
		PYFDYWGQGTLVTVSS
4-112	2263	EVQLVESGGGLVQPGGSLRLSCAASGFSFDDDYVMGWFRQAPGKEREFVA
		AISNSGLSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAD
		RRGLASTRAADYDWGQGTLVTVSS
4-113	2264	EVQLVESGGGLVQPGGSLRLSCAASGSISSINVMGWFRQAPGKEREFVATM
		RWSTGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAQRV
		RGFFGPLRTTPSWYEWGQGTLVTVSS
4-114	2265	EVQLVESGGGLVQPGGSLRLSCAASGLTFILYRMGWFRQAPGKEREFVAAI
		NNFGTTKYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARTHYD
		FWSGYTSRTPNYFDYWGQGTLVTVSS
4-115	2266	EVQLVESGGGLVQPGGSLRLSCAASGGTFSVYHMGWFRQAPGKEREPVA
		AISWSGGSTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAV
		NTWTSPSFDSWGQGTLVTVSS
4-116	2267	EVQLVESGGGLVQPGGSLRLSCAASGRAFSTYGMGWFRQAPGKEREFVAG
		INWSGDTPYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAREV
		GPPPGYFDLWGQGTLVTVSS
4-117	2268	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDIAMGWFRQAPGKEREFVASI
		NWGGGNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAKG
		IWDYLGRRDFGDWGQGTLVTVSS
4-118	2269	EVQLVESGGGLVQPGGSLRLSCAASGRTFSSARMGWFRQAPGKEREFVAA
		ISWSGDNTHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATTE
		NPNPRWGQGTLVTVSS
4-119	2270	EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYAMGWFRQAPGKEREWVA
		TINGDDYTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCVATP
		GGYGLWGQGTLVTVSS
4-12	2271	EVQLVESGGGLVQPGGSLRLSCAASGITFRRHDMGWFRQAPGKEREFVAA
		IRWSSSSTVYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADR
		GVYGGRWYRTSQYTWGQGTLVTVSS
4-121	2272	EVQLVESGGGLVQPGGSLRLSCAASGTAASFNPMGWFRQAPGKEREFVAA
		ITSGGSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIAYE
		EGVYRWDWGQGTLVTVSS
4-122	2273	EVQLVESGGGLVQPGGSLRLSCAASGNINIINYMGWFRQAPGKEREGVAAI
		HWNGDSTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASGPP
		YSNYFAYWGQGTLVTVSS
4-123	2274	EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQAPGKERESVA
		AISGSGGSTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKI
		MGSGRPYFDHWGQGTLVTVSS
4-124	2275	EVQLVESGGGLVQPGGSLRLSCAASGNIFTRNVMGWFRQAPGKEREFVAA
		ITSSGSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARPSSD
		LQGGVDYWGQGTLVTVSS
4-125	2276	EVQLVESGGGLVQPGGSLRLSCAASGRTFSSIAMGWFRQAPGKEREFVASI
		NWGGGNTIYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAKGI
		WDYLGRRDFGDWGQGTLVTVSS
4-126	2277	EVQLVESGGGLVQPGGSLRLSCAASGIPSTLRAMGWFRQAPGKEREFVAA
		VSSLGPFTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAKP
		GWVARDPSEYNWGQGTLVTVSS
4-127	2278	EVQLVESGGGLVQPGGSLRLSCAASGFTLDDSAMGWFRQAPGKEREWVA
		AITNGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARFA
		RGSPYFDFWGQGTLVTVSS
4-128	2279	EVQLVESGGGLVQPGGSLRLSCAASGSISSFNAMGWFRQAPGKERESVAAI
		DWDGSTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGGG
		YYGSGSFEYWGQGTLVTVSS
4-129	2280	EVQLVESGGGLVQPGGSLRLSCAASGNIFSDNIIGWFRQAPGKEREMVAYY
		TSGGSIDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGTAV
		GRPPPGGMDVWGQGTLVTVSS
4-13	2281	EVQLVESGGGLVQPGGSLRLSCAASGSISSIGAMGWFRQAPGKEREGVAAI
		SSSGSSTVYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARVPPG
		QAYFDSWGQGTLVTVSS
4-131	2282	EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMGWFRQAPGKERELVAT
		ITWSGDSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKGG
		SWYYDSSGYYGRWGQGTLVTVSS
4-132	2283	EVQLVESGGGLVQPGGSLRLSCAASGRTFSNYTMGWFRQAPGKEREWVS
		AISWSTGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAD
		RYGPPWYDWGQGTLVTVSS
4-134	2284	EVQLVESGGGLVQPGGSLRLSCAASGGTFSSVGMGWFRQAPGKERELVAV
		INWSGARTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVP
		WMDYNRRDWGQGTLVTVSS
4-135	2285	EVQLVESGGGLVQPGGSLRLSCAASGRIFTNTAMGWFRQAPGKEREGVAA
		INWSGGSTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARTS
		GSYSFDYWGQGTLVTVSS
4-136	2286	EVQLVESGGGLVQPGGSLRLSCAASGEEFSDHWMGWFRQAPGKEREFVG
		AIHWSGGRTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAA
		DRRGLASTRAADYDWGQGTLVTVSS
4-137	2287	EVQLVESGGGLVQPGGSLRLSCAASGRTFSSIAMGWFRQAPGKEREFVAAI
		NWSGARTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAKG
		IWDYLGRRDFGDWGQGTLVTVSS
4-138	2288	EVQLVESGGGLVQPGGSLRLSCAASGSTSSLRTMGWFRQAPGKEREGVAA
		ISSRDGSTIYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARDDS
		SSPYFDYWGQGTLVTVSS
4-139	2289	EVQLVESGGGLVQPGGSLRLSCAASGGGTFGSYAMGWFRQAPGKEREFV
		AAISIASGASGGTTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYY
		CATTMNPNPRWGQGTLVTVSS
4-14	2290	EVQLVESGGGLVQPGGSLRLSCAASGRTFSNAAMGWFRQAPGKEREFVAR
		ITWNGGSTFYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATTE
		NPNPRWGQGTLVTVSS
4-141	2291	EVQLVESGGGLVQPGGSLRLSCAASGIILSDNAMGWFRQAPGKEREFVAAI
		SWLGESTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADRR
		GLASTRAADYDWGQGTLVTVSS
4-142	2292	EVQLVESGGGLVQPGGSLRLSCAASGRTFGDYIMGWFRQAPGKERESVAA
		INWNGGYTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATTS
		PNTGWHYYRWGQGTLVTVSS
4-143	2293	EVQLVESGGGLVQPGGSLRLSCAASGFNFNWYPMGWFRQAPGKERESVA
		AISWTGVSTYTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCA
		RWGPGPAGGSPGLVGFDYWGQGTLVTVSS
4-144	2294	EVQLVESGGGLVQPGGSLRLSCAASGSIRSVSVMGWFRQAPGKEREAVAA
		ISWSGVGTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYQ
		RGWGDWGQGTLVTVSS
4-145	2295	EVQLVESGGGLVQPGGSLRLSCAASGMTFRLYAMGWFRQAPGKEREFVG
		AINWLSESTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAK
		PGWVARDPSEYNWGQGTLVTVSS
4-146	2296	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDDAMGWFRQAPGKEREFVAA
		INWSGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDP
		PLFWGQGTMVTVSS
4-147	2297	EVQLVESGGGLVQPGGSLRLSCAASGGTFSVYAMGWFRQAPGKEREGVA
		AISMSGDDAAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKI
		SKDDGGKPRGAFFDSWGQGTLVTVSS
4-148	2298	EVQLVESGGGLVQPGGSLRLSCAASGFALGYYAMGWFRQAPGKERESVA
		AISSRDGSTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARL
		ATGPQAYFHHWGQGTLVTVSS
4-149	2299	EVQLVESGGGLVQPGGSLRLSCAASGFNLDDYAMGWFRQAPGKERESVA
		AISWDGGATAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAR
		VGRGTTAFDSWGQGTLVTVSS
4-15	2300	EVQLVESGGGLVQPGGSLRLSCAASGNTFSGGFMGWFRQAPGKEREFVAS
		IRSGARTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAQRVR
		GFFGPLRTTPSWYEWGQGTLVTVSS
4-151	2301	EVQLVESGGGLVQPGGSLRLSCAASGSIRSINIMGWFRQAPGKEREAVAAIS
		WSGGSTVYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASLLAG
		DRYFDYWGQGTLVTVSS

TABLE 28
SARS-CoV-2 Variant Variable Heavy Chain Sequences
7-1	2302	EVQLVESGGGLVQPGGSLRLSCAASGFTLGDYVMGWFRQAPGKEREFVA
		AIHSGGSTYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAKEY
		GGTRRYDRAYNWGQGTLVTVSS
7-2	2303	EVQLVESGGGLVQPGGSLRLSCAASGGGTFGSYAMGWFRQAPGKERELV
		AAISSGGSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARG
		DWRYGWGQGTLVTVSS
7-3	2304	EVQLVESGGGLVQPGGSLRLSCAASGRTYSISAMGWFRQAPGKEREFVAAI
		SMSGDDSAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQL
		GYESGYSLTYDYDWGQGTLVTVSS
7-4	2305	EVQLVESGGGLVQPGGSLRLSCAASGGTFSTYPMGWFRQAPGKEREFVAA
		ITSDGSTLYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAATDY
		NKAYAREGRRYDWGQGTLVTVSS
7-5	2306	EVQLVESGGGLVQPGGSLRLSCAASGSIFRINAMGWFRQAPGKEREFVAAI
		HWSGSSTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQD
		RRRGDYYTFDYHWGQGTLVTVSS
7-6	2307	EVQLVESGGGLVQPGGSLRLSCAASGGTFNNYAMGWFRQAPGKERELVA
		AITSGGSTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGD
		WRYGWGQGTLVTVSS
7-7	2308	EVQLVESGGGLVQPGGSLRLSCAASGTIVNINVMGWFRQAPGKEREFVAAI
		HWSGGLKAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAMNR
		AGIYEWGQGTLVTVSS
7-8	2309	EVQLVESGGGLVQPGGSLRLSCAASGSTFSNYAMGWFRQAPGKERELVAA
		ITSGGSTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGDW
		RYGWGQGTLVTVSS
7-9	2310	EVQLVESGGGLVQPGGSLRLSCAASGFSFDDYVMGWFRQAPGKEREFVAA
		ISRSGNLKSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAKE
		YGGTRRYDRAYNWGQGTLVTVSS
7-10	2311	EVQLVESGGGLVQPGGSLRLSCAASGSAFRSTVMGWFRQAPGKEREFVAA
		VIGSSGITDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGD
		WRYGWGQGTLVTVSS
7-11	2312	EVQLVESGGGLVQPGGSLRLSCAASGRTFSDAGMGWFRQAPGKEREFVAA
		ISRSGNLKAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVQV
		NGTWAWGQGTLVTVSS
7-12	2313	EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAMGWFRQAPGKERELVA
		AISWNGGSTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARG
		DWRYGWGQGTLVTVSS
7-13	2314	EVQLVESGGGLVQPGGSLRLSCAASGGTFSTYVMGWFRQAPGKEREFVAA
		ISWSGESTLYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADL
		MYGVDRRYDWGQGTLVTVSS
7-14	2315	EVQLVESGGGLVQPGGSLRLSCAASGISSSKRNMGWFRQAPGKEREFVAGI
		SWTGGITYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAIAGR
		GRWGQGTLVTVSS
7-15	2316	EVQLVESGGGLVQPGGSLRLSCAASGRRFSAYGMGWFRQAPGKEREFVA
		VISRSGTLTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASSG
		PADARNGERWHWGQGTLVTVSS
7-16	2317	EVQLVESGGGLVQPGGSLRLSCAASGLTFSSFVMGWFRQAPGKEREFVAAI
		SSNGGSTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAKEY
		GGTRRYDRAYNWGQGTLVTVSS
7-17	2318	EVQLVESGGGLVQPGGSLRLSCAASGTVFSISAMGWFRQAPGKEREFVAAI
		SMSGDDTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQL
		GYESGYSLTYDYDWGQGTLVTVSS
7-18	2319	EVQLVESGGGLVQPGGSLRLSCAASGSIFSPNVMGWFRQAPGKEREFVAAI
		TNGGSTKYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQRW
		RGGSYEWGQGTLVTVSS
7-19	2320	EVQLVESGGGLVQPGGSLRLSCAASGIPASIRVMGWFRQAPGKEREFVAAI
		HWSGSSTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCALSRA
		IVPGDSEYDYRWGQGTLVTVSS
7-20	2321	EVQLVESGGGLVQPGGSLRLSCAASGRTFSMSAMGWFRQAPGKEREFVSA
		ISWSGGSTLYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQL
		GYESGYSLTYDYDWGQGTLVTVSS
7-21	2322	EVQLVESGGGLVQPGGSLRLSCAASGRTFSNYAMGWFRQAPGKERELVA
		AITSGGSTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGD
		WRYGWGQGTLVTVSS
7-22	2323	EVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKERELVAA
		ISTGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGDW
		RYGWGQGTLVTVSS
7-23	2324	EVQLVESGGGLVQPGGSLRLSCAASGRSFSSVGMGWFRQAPGKEREFVAV
		ISRSGASTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASAGP
		ADARNGERWAWGQGTLVTVSS
7-24	2325	EVQLVESGGGLVQPGGSLRLSCAASGRAFRRYTMGWFRQAPGKERELIAV
		INWSGDRRYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAATL
		AKGGGRWGQGTLVTVSS
7-25	2326	EVQLVESGGGLVQPGGSLRLSCAAMAWAGFARRRAKNAKWWRALPRGG
		PTYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGGMWYGSS
		LYVRFDLLEDGMDWGQGTLVTVSS
7-26	2327	EVQLVESGGGLVQPGGSLRLSCAASGSISSINGMGWFRQAPGKERELVALI
		SRSGGTTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASAGP
		ADARNGERWAWGQGTLVTVSS
7-27	2328	EVQLVESGGGLVQPGGSLRLSCAASGRTFSNNVMGWFRQAPGKERELVA
		AAISGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGD
		WRYGWGQGTLVTVSS
7-28	2329	EVQLVESGGGLVQPGGSLRLSCAASGRTFSISAMGWFRQAPGKEREFVAAI
		SRSGTTMYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQLGY
		ESGYSLTYDYDWGQGTLVTVSS
7-29	2330	EVQLVESGGGLVQPGGSLRLSCAASGGTFSYYDLAAMGWFRQAPGKEREF
		VAAISWSQYNTKYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCA
		ARVVVRTAHGFEDNWGQGTLVTVSS
7-30	2331	EVQLVESGGGLVQPGGSLRLSCAASGRTFNNYGMGWFRQAPGKEREFVA
		VISRSGSLKAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASDP
		TYGSGRWTWGQGTLVTVSS
7-31	2332	EVQLVESGGGLVQPGGSLRLNCAASGFTLDDYVMGWFRQTPGKEREFVA
		AISSSGALTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDAAVYYCAAKE
		YGGTRRYDRAYNWGQGTLVTVSS
7-32	2333	EVQLVESGGGLVQPGGSLRLSCAASGRTFNAMGWFRQAPGKEREFVAAIR
		WSGDMSVYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQDR
		RRGDYYTFDYHWGQGTLVTVSS
7-33	2334	EVQLVESGGGLVQPGGSLRLSCAASGLTFSTYAMGWFRQAPGKEREFVAA
		ITSGGSTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGDW
		RYGWGQGTLVTVSS
7-34	2335	EVQLVESGGGLVQPGGSLRLSCAASGSIFTINAMGWFRQAPGKEREGVAAI
		GSDGSTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVVRW
		GADWGQGTLVTVSS
7-35	2336	EVQLVESGGGLVQPGGSLRLSCAASGLTFSSYAMGWFRQAPGKERELVAA
		ITSSSGSTPAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGD
		WRYGWGQGTLVTVSS
7-36	2337	EVQLVESGGGLVQPGGSLRLSCAASGIPFSTRTMGWFRQAPGKEREFVAAI
		SWSQYNTKYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAARH
		WGMFSRSENDYNWGQGTLVTVSS
7-37	2338	EVQLVESGGGLVQPGGSLRLSCAASGRSRFSTYVMGWFRQAPGKEREFVA
		AISWSQYNTKYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAG
		NGGRNYGHSRARYDWGQGTLVTVSS
7-38	2339	EVQLVESGGGLVQPGGSLRLSCAASGLTLSSYGMGWFRQAPGKEREYVAV
		ISRSGSLKAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATRA
		DAEGWWDWGQGTLVTVSS
7-39	2340	EVQLVESGGGLVQPGGSLRLSCAASGSIFRVNVMGWFRQAPGKEREFVAA
		INNFGTTKYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADLP
		SRWGQGTLVTVSS
7-40	2341	EVQLVESGGGLVQPGGSLRLSCAASGRTFRNYAMGWFRQAPGKERELVA
		AISSGGSTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGD
		WRYGWGQGTLVTVSS
7-41	2342	EVQLVESGGGLVQPGGSLRLSCAASGRTFSSFAMGWFRQAPGKERELVAA
		ISSGGSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGDW
		RYGWGQGTLVTVSS
7-42	2343	EVQLVESGGGLVQPGGSLRLSCAASGTTFRINAMGWFRQAPGKEREFVAA
		MNWSGGSTKYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQ
		DRRRGDYYTFDYHWGQGTLVTVSS
7-43	2344	EVQLVESGGGLVQPGGSLRLSCAASGFTLGDYVMGWFRQAPGKEREFVA
		AIHSGGSTLYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAKE
		YGGTRRYDRTYNWGQGTLVTVSS
7-44	2345	EVQLVESGGGLVQPGGSLRLSCAASGFTFSRSAMGWFRQAPGKERELVAG
		ILSSGATVYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKAPR
		DWGQGTLVTVSS
7-45	2346	EVQLVESGGGLVQPGGSLRLSCAASGRTFNNYAMGWFRQAPGKERELVA
		AITSGGSTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGD
		WRYGWGQGTLVTVSS
7-46	2347	EVQLVESGGGLVQPGGSLRLSCAASGFTFRSYPMGWFRQAPGKEREFVAAI
		NNFGTTKYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAAKGI
		GVYGWGQGTLVTVSS
7-47	2348	EVQLVESGGGLVQPGGSLRLSCAASGNIFTRNVMGWFRQAPGKEREFVAA
		IHWNGDSTKYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGS
		NIGGSRWRYDWGQGTLVTVSS
7-48	2349	EVQLVESGGGLVQPGGSLRLSCAASGRTISRYTMGWFRQAPGKERELVAAI
		KWSGASTVYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAKGI
		WDYLGRRDFGDWGQGTLVTVSS
7-49	2350	EVQLVESGGGLVQPGGSLRLSCAASGFRFSSYGMGWFRQAPGKEREFVAII
		TSGGLTVYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAARKTF
		YFGTSSYPNDYAWGQGTLVTVSS
7-50	2351	EVQLVESGGGLVQPGGSLRLSCAASGRTFDNHAMGWFRQAPGKEREGVA
		AIGSDGSTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVVR
		WGVDWGQGTLVTVSS
7-51	2352	EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHAMGWFRQAPGKEREFVAG
		ISWSGESTLTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAD
		VNGDWGQGTLVTVSS
7-52	2353	EVQLVESGGGLVQPGGSLRLSCAASGMTFRLYAMGWFRQAPGKEREFVA
		AISWSQYNTKYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQ
		LGYESGYSLTYDYDWGQGTLVTVSS
7-53	2354	EVQLVESGGGLVQPGGSLRLSCAASGGTFRKLAMGWFRQAPGKEREFVA
		VISWTGGSSYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARL
		TSFATWGQGTLVTVSS
7-54	2355	EVQLVESGGGLVQPGGSLRLSCAASGRTFSANGMGWFRQAPGKEREFVAA
		ISASGTLRAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAARSP
		MSPTWDWGQGTLVTVSS
7-55	2356	EVQLVESGGGLVQPGGSLRLSCAASGSAFRSTVMGWFRQAPGKEREFVAA
		ISWTGESTLYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATGP
		YRSYFARSYLWGQGTLVTVSS
7-56	2357	EVQLVESGGGLVQPGGSLRLSCAASGGTFDYSGMGWFRQAPGKEREFVA
		VVSQSGRTTYYADSVKGLFTITADNSKNTAYLQMNLLKPEDTAVYYCPTA
		TRPGEWDGGQGTLVTVSR
7-57	2358	EVQLVESGGGLVQPGGSLRLSCAASGVFGPIRAMGWFRQAPGKERELVAL
		MGNDGSTYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIGW
		RWGQGTLVTVSS
7-58	2359	EVQLVESGGGLVQPGGSLRLSCAASGFNFNWYPMGWFRQAPGKEREFVA
		AIRWSGGITYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATG
		PYRSYFARSYLWGQGTLVTVSS
7-59	2360	EVQLVESGGGLVQPGGSLRLSCAASGMTFHRYVMGWFRQAPGKERELVA
		SITTGGTPNYADSVKGRFTIITDNNKNTAYLLMINLQPEDTAVYYCCKVPYI
		WGQGTLGTVGT
7-60	2361	EVQLVESGGGLVQPGGSLRLSCAASGISTMGWFRQAPGKEREFVAAINNFG
		TTKYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAASQSGSGY
		DWGQGTLVTVSS
7-61	2362	EVQLVESGGGLVQPGGSLRLSCAASGRAFNTRAMGWFRQAPGKERELVA
		LMGNDGSTYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIG
		WRWGQGTLVTVSS
7-62	2363	EVQLVESGGGLVQPGGSLRLSCAASGLTDRRYTMGWFRQAPGKEREFVA
		AINSGGSTLYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGN
		GGRTYGHSRARYEWGQGTLVTVSS
7-63	2364	EVQLVESGGGLVQPGGSLRLSCAASGRTFNVMGWFRQAPGKERELVALM
		GNDGSTYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVRWG
		VDWGQGTLVTVSS
7-64	2365	EVQLVESGGGLVQPGGSLRLSCAASGRAFNTRAMGWFRQAPGKERELVA
		LMGNDGSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIG
		WRWGQGTLVTVSS
7-65	2366	EVQVVESGGGVVHPGGSVRMRCAASGVTVDYSGMGWFGQAPGKEREFV
		AVVSQSARTTYYADSVKGRFTISADNSKNTEYLQMNSMKPEDTAVYYCAT
		ATRPGEWDWGQGTLVTVSS
7-66	2367	EVQLVESGGGLVQPGGSLRLSCAASGRTPRLGAMGWFRQAPGKEREFVAA
		ISRSGGLTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQLV
		GSNIGGSRWRYDWGQGTLVTVSS
7-67	2368	EVQLVESGGGLVQPGGSLRLSCAASGLTFRNYAMGWFRQAPGKEREFVA
		AITSGGSTLYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGD
		WRYGWGHGTLVTESS
8-1	2369	EVQLVESGGGLVQPGGSLRLSCAASGGRTFSDLAMGWFRQAPGKEREFVA
		LITRSGGTTFYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAIGR
		GSWGQGTLVTVSS
8-2	2370	EVQLVESGGGLVQPGGSLRLSCAASGFTFGEYAMGWFRQAPGKEREFVAA
		VSSLGPFTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVL
		DGYSGSWGQGTLVTVSS
8-3	2371	EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYGMGWFRQAPGKEREFVAA
		ISWSGVRSGVSAIYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCT
		TDLTGDLWYFDLWGQGTLVTVSS
8-4	2372	EVQLVESGGGLVQPGGSLRLSCAASGLTAGTYAMCWFRQAPGKEREGVA
		CASSTDGSTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAV
		RTYGSATYDWGQGTLVTVSS
8-5	2373	EVQLVESGGGLVQPGGSLRLSCAASGFTLDDYVMGWFRQAPGKERELVA
		AVSSLGPFTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAK
		EYGGTRRYDRAYNWGQGTLVTVSS
8-6	2374	EVQLVESGGGLVQPGGSLRLSCAASGPTLGSYVMGWFRQAPGKEREFVAA
		ISWSQYNTKYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQR
		WRGGSYEWGQGTLVTVSS
8-7	2375	EVQLVESGGGLVQPGGSLRLSCAASGPTFSSYVMGWFRQAPGKEREFVAA
		ISWSQYNTKYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAAS
		RSGSGYDWGQGTLVTVSS
8-8	2376	EVQLVESGGGLVQPGGSLRLSCAASGYLYSKDCMGWFRQAPGKEREGVA
		TICTGDGSTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVIA
		YEEGVYRWDWGQGTLVTVSS
8-9	2377	EVQLVESGGGLVQPGGSLRLSCAASGFTIDDYAMGWFRQAPGKEREGVAA
		ISGSGDDTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKLP
		YVSGDYWGQGTLVTVSS
8-10	2378	EVQLVESGGGLVQPGGSLRLSCAASGGRFSDYGMGWFRQAPGKERELVA
		LISRSGNLKSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAKT
		GTSFVWGQGTLVTVSS
8-11	2379	EVQLVESGGGLVQPGGSLRLSCAASGLSFSNYAMGWFRQAPGKERELVAA
		ITSGGSTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGDW
		RYGWGQGTLVTVSS
8-12	2380	EVQLVESGGGLVQPGGSLRLSCAASGIPSTLRAMGWFRQAPGKEREFVALI
		NRSGGSQFYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAIGRG
		SWGQGTLVTVSS

TABLE 29
Membrane Protein CDR Sequences
	SEQ		SEQ		SEQ
	ID		ID		ID
Variant	NO	CDRH1	NO	CDRH2	NO	CDRH3
9-1	2381	RTFSRLAM	2453	AAISRSGRSTS	2525	CAARRSQILFTSRTDYEW
		G		YA
9-2	2382	SFSIAAMG	2454	ATINYSGGGT	2526	CAAVNTFDESAYAAFAC
				YYA		YDVVW
9-3	2383	RTFSRYAM	2455	AAISRSGKSTY	2527	CAASSVFSDLRYRKNPK
		G		YA		W
9-4	2384	RTFSKYAM	2456	ALITPSSRTTY	2528	CAIAGRGRW
		G		YA
9-5	2385	RTFRRYAM	2457	ASINWGGGNT	2529	CAKTKRTGIFTTARMVD
		G		YYA		W
9-6	2386	RTFSRFAM	2458	AAIRWSGGRT	2530	CAIEPGTIRNWRNRVPFA
		G		VYA		RGNFGW
9-7	2387	LGIAFSRRT	2459	AAISWRGGNT	2531	CAARRWIPPGPIW
		AMG		YYA
9-8	2388	RTFRRYPM	2460	AAISRSGGSTY	2532	CAAKRLRSFASGGSYDW
		G		YA
9-9	2389	GTLRGYGM	2461	ASISRSGGSTY	2533	CAARRRVTLFTSRADYD
		G		YA		W
9-10	2390	RMFSSRSM	2462	ALINRSGGSQF	2534	CAARRWIPPGPIW
		G		YA
9-11	2391	RTFGRRAM	2463	AGISRGGGTN	2535	CAAKGIWDYLGRRDFGD
		G		YA		W
10-1	2392	LSSPPFDDF	2464	SSIYSDDGDS	2536	CARQTFDFWSASLGGNF
		PMG		MYA		WYFDLW
10-2	2393	GTFSSYSM	2465	SAISWIIGSGG	2537	CTAGAGDSW
		G		TTNYA
10-3	2394	SIFSTRTMG	2466	ASITKFGSTNY	2538	CTRGGGRFFDWLYLRW
				A
10-4	2395	RTLWRSNM	2467	ASISSFGSTKY	2539	CARGHGRYFDWLLFARP
		G		A		PDYW
10-5	2396	RSLGIYRM	2468	AAITSGGRKN	2540	CAKRTIFGVGRWLDPW
		G		YA
10-6	2397	TTLTFRIMG	2469	PAISSTGLASY	2541	CSKDRAPNCFACCPNGF
				T		DVW
10-7	2398	SRFSGRFNI	2470	ARIGYSGQSIS	2542	CARGRFLGGTEW
		LNMG		YA
10-8	2399	TLFKINAM	2471	AQINRHGVTY	2543	CARGRTIFFGGGRYFDY
		G		YA		W
10-9	2400	IPFRSRTMG	2472	AGITGSGRSQ	2544	CARGARIFGSVAPWRGG
				YYA		NYYGMDVW
10-10	2401	FTFSSFRMG	2473	AGISRGGSTN	2545	CARASGLWFRRPHVW
				YA
10-11	2402	RNFRRNSM	2474	AGISWSGART	2546	CARVSRRPRSPPGYYYG
		G		HYA		MDVW
10-12	2403	RNLRMYRM	2475	ATIRWSDGST	2547	CTRARLRYFDWLFPTNF
		G		YYA		DYW
10-13	2404	GLTFSSNTM	2476	ASISSSGRTSY	2548	CARRVRRLWFRSYFDLW
		G		A
10-14	2405	FTLAYYAM	2477	AAISWSGRNI	2549	CARERARWFGKFSVSW
		G		NYA
10-15	2406	RTFSSFPMG	2478	AAISWSGSTS	2550	SACGRLGFGAW
				YA
10-16	2407	ISSSKRNMG	2479	ATWTSRGITT	2551	CARGGPPRLWGSYRRKY
				YA		FDYW
10-17	2408	RTFSIYAMG	2480	ARITRGGITKY	2552	CARGLGWLLGYYW
				A
10-18	2409	RMYNSYSM	2481	ARISPGGTFYA	2553	CTTSARSGWFWRYFDSW
		G
10-19	2410	RTFRSYGM	2482	ASISRSGTTM	2554	CARRGLLQWFGAPNSWF
		G		YA		DPW
10-20	2411	RTIRTMG	2483	ATINSRGITNY	2555	CTTERDGLLWFRELFRPS
				A		W
10-21	2412	RSFSFNAM	2484	ARISRFGRTN	2556	CAKVHSYVWGGHSDYW
		G		YA
10-22	2413	RTYYAMG	2485	GAIDWSGRRI	2557	CARVRFSRLGGVIGRPID
				TYA		SW
10-23	2414	RAFRRYTM	2486	ASITKFGSTNY	2558	CAKDRGVLWFGELWYW
		G		A
10-24	2415	RTFSNYRM	2487	ASINRGGSTK	2559	CASGKGGSATIFHLSRRP
		G		YA		LYFDYW
10-25	2416	ITFSPYAMG	2488	ATINWSGGYT	2560	CAKRKNRGPLWFGGGG
				VYA		WGYW
10-26	2417	RTFSGFTMS	2489	AGIITNGSTNY	2561	CARRVAYSSFWSGLRKH
		STWMG		A		MD VW
10-27	2418	RTFRRYSM	2490	ASITPGGNTN	2562	CASRRRWLTPYIFW
		G		YA
10-28	2419	SIFSIGMG	2491	ARIWWRSGAT	2563	CAAISIFGRLKW
				YYA
10-29	2420	RTFTSYRM	2492	AEISSSGGYTY	2564	CARVGPLRFLAQRPRLRP
		G		YA		DYW
10-30	2421	RTFSSFRFR	2493	ALIFSGGSTYY	2565	CAREWGRWLQRGSYW
		AMG		A
10-31	2422	RTFGSYGM	2494	ATISIGGRTYY	2566	CARGSGSGFMWYHGNN
		G		A		NYDRWRYW
10-32	2423	RTFRSYPM	2495	ASINRGGSTN	2567	CARGRYDFWSGYYRWF
		G		YA		DPW
10-33	2424	RTFSRSDM	2496	AAISWSGGST	2568	CATVPPPRRFLEWLPRRL
		G		SYA		TYIW
10-34	2425	RTFRRYTM	2497	ASMRGSRSYY	2569	CARMSGFPFLDYW
		G		A
10-35	2426	SIFRLSTMG	2498	ASISSFGSTYY	2570	CARTRGIFLWFGESFDY
				A		W
10-36	2427	IAFRIRTMG	2499	ASITSGGSTNY	2571	CARGGPRFGGFRGYFDP
				A		W
10-37	2428	FTFTSYRM	2500	AGISRFFGTAY	2572	CARVTRWFGGLDVW
		G		YA
10-38	2429	RTFSRYVM	2501	ASISRFGRTNY	2573	CARHHGLGILWWGTMD
		G		A		VW
10-39	2430	RTFSMG	2502	ASISRFGRTNY	2574	CAKRSTWLPQHFDSW
				A
10-40	2431	RTFSTYTM	2503	ARIWRSGGNT	2575	CARGVRGVFRAYFDHW
		G		YYA
10-41	2432	RNLRMYRM	2504	ALISRVGVTS	2576	CARGTSFFNFWSGSLGRV
		G		YA		GFDSW
10-42	2433	ITIRTHAMG	2505	ATISRSGGNT	2577	CTTAGVLRYFDWFRRPY
				YYA		W
10-43	2434	RTFRRYHM	2506	AAITSGGRTN	2578	CTTDGLRYFDWFPWASA
		G		YA		FDIW
10-44	2435	RTFRRYTM	2507	AVISWSGGST	2579	CARKGRWSGMNVW
		G		KYA
10-45	2436	RTFSWYPM	2508	ASISWGGART	2580	CARSTGPRGSGRYRAHW
		G		YYA		FDSW
10-46	2437	RTFTSYRM	2509	AAITWNSGRT	2581	CSPSSWPFYFGAW
		G		RYA
10-47	2438	RPLRRYVM	2510	AAITNGGSTK	2582	CARGTPWRLLWFGTLGP
		G		YA		PPAFDYW
10-48	2439	RTFRRYAM	2511	AAINRSGSTE	2583	CARQHQDFWTGYYTVW
		G		YA
10-49	2440	RTFRRYTM	2512	ASISRSGTTYY	2584	CAKEGWRWLQLRGGFD
		G		A		YW
10-50	2441	RTLSTYNM	2513	ASISRFGRTNY	2585	CARRGKLSAAMHWFDP
		G		A		W
10-51	2442	RFFSTRVM	2514	ARIWPGGSTY	2586	CARDRGIFGVSRW
		G		YA
10-52	2443	RFFSICSMG	2515	AGINWRSGGS	2587	CARGSGWWEYW
				TYYA
10-53	2444	RMFSSRSN	2516	ASISSGGTTAY	2588	CARGFGRRFLEWLPRFD
		MG		A		YW
10-54	2445	RTFSSARM	2517	AGINMISSTKY	2589	CAHFRRFLPRGYVDYW
		G		A
10-55	2446	RTFRRYTM	2518	ARIAGGSTYY	2590	CARQQYYDFWSGYFRSG
		G		A		YFDLW
10-56	2447	HTFRNYGM	2519	AAITSSGSTNY	2591	CATVPPPRRFLEWLPRRL
		G		A		TYTW
10-57	2448	RTFSRYAM	2520	ASITKFGSTNY	2592	CAKERESRFLKWRKTDW
		G		A
10-58	2449	RNLRMYRM	2521	ASISRFGRTNY	2593	CARHDSIGLFRHGMDVW
		G		A
10-59	2450	RTFRRYAM	2522	ARISSGGSTSY	2594	C ARDRGFGFWS GLRGYF
		G		A		DLW
10-60	2451	IPASMYLG	2523	AAITSGGRTS	2595	CAKRKKRGPLWFGGGG
				YA		WGYW
10-61	2452	IPFRSRTFSA	2524	AQITRGGSTN	2596	CARRHWFGFDYW
		YAMG		YA

TABLE 30
Membrane Protein VH Sequences
	SEQ
	ID
Variant	NO	VH
9-1	2597	EVQLVESGGGLVQPGGSLRLSCAASGRTFSRLAMGWFRQAPGKEREFVAA
		ISRSGRSTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAARRS
		QILFTSRTDYEWGQGTLVTVSS
9-2	2598	EVQLVESGGGLVQPGGSLRLSCAASGSFSIAAMGWFRQAPGKEREFVATIN
		YSGGGTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVNTF
		DESAYAAFACYDVVWGQGTLVTVSS
9-3	2599	EVQLVESGGGLVQPGGSLRLSCAASGRTFSRYAMGWFRQAPGKEREFVAA
		ISRSGKSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASSV
		FSDLRYRKNPKWGQGTLVTVSS
9-4	2600	EVQLVESGGGLVQPGGSLRLSCAASGRTFSKYAMGWFRQAPGKEREFVSH
		ISRDGGRTFSSSTMGWFRQAPGKERELVALITPSSRTTYYADSVKGRFTISA
		DNSKNTAYLQMNSLKPEDTAVYYCAIAGRGRWGQGTLVTVSS
9-5	2601	EVQLVESGGGLVQPGGSLRLSCAASGRTFRRYAMGWFRQAPGKEREFVAS
		INWGGGNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKT
		KRTGIFTTARMVDWGQGTLVTVSS
9-6	2602	EVQLVESGGGLVQPGGSLRLSCAASGRTFSRFAMGWFRQAPGKEREFVAA
		IRWSGGRTVYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAIEP
		GTIRNWRNRVPFARGNFGWGQGTLVTVSS
9-7	2603	EVQLVESGGGLVQPGGSLRLSCAASGLGIAFSRRTAMGWFRQAPGKEREF
		VAAISWRGGNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYC
		AARRWIPPGPIWGQGTLVTVSS
9-8	2604	EVQLVESGGGLVQPGGSLRLSCAASGRTFRRYPMGWFRQAPGKEREFVAA
		ISRSGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAKR
		LRSFASGGSYDWGQGTLVTVSS
9-9	2605	EVQLVESGGGLVQPGGSLRLSCAASGGTLRGYGMGWFRQAPGKEREFVA
		SISRSGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAARR
		RVTLFTSRADYDWGQGTLVTVSS
9-10	2606	EVQLVESGGGLVQPGGSLRLSCAASGRMFSSRSMGWFRQAPGKEREFVAL
		INRSGGSQFYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAARR
		WIPPGPIWGQGTLVTVSS
9-11	2607	EVQLVESGGGLVQPGGSLRLSCAASGRTFGRRAMGWFRQAPGKEREFVA
		GISRGGGTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAKG
		IWDYLGRRDFGDWGQGTLVTVSS
10-1	2608	EVQLVESGGGLVQPGGSLRLSCAASGLSSPPFDDFPMGWFRQAPGKEREFV
		SSIYSDDGDSMYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAR
		QTFDFWSASLGGNFWYFDLWGQGTLVTVSS
10-2	2609	EVQLVESGGGLVQPGGSLRLSCAASGGTFSSYSMGWFRQAPGKEREFVSAI
		SWIIGSGGTTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCTA
		GAGDSWGQGTLVTVSS
10-3	2610	EVQLVESGGGLVQPGGSLRLSCAASGSIFSTRTMGWFRQAPGKEREFVASI
		TKFGSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCTRGGGR
		FFDWLYLRWGQGTLVTVSS
10-4	2611	EVQLVESGGGLVQPGGSLRLSCAASGRTLWRSNMGWFRQAPGKEREFVA
		SISSFGSTKYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGHG
		RYFDWLLFARPPDYWGQGTLVTVSS
10-5	2612	EVQLVESGGGLVQPGGSLRLSCAASGRSLGIYRMGWFRQAPGKEREFVAA
		ITSGGRKNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKRTIF
		GVGRWLDPWGQGTLVTVSS
10-6	2613	EVQLVESGGGLVQPGGSLRLSCAASGTTLTFRIMGWFRQAPGKEREFVPAI
		SSTGLASYTDSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCSKDRAP
		NCFACCPNGFDVWGQGTLVTVSS
10-7	2614	EVQLVESGGGLVQPGGSLRLSCAASGSRFSGRFNILNMGWFRQAPGKEREF
		VARIGYSGQSISYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAR
		GRFLGGTEWGQGTLVTVSS
10-8	2615	EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWFRQAPGKEREFVAQ
		INRHGVTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGRT
		IFFGGGRYFDYWGQGTLVTVSS
10-9	2616	EVQLVESGGGLVQPGGSLRLSCAASGIPFRSRTMGWFRQAPGKEREFVAGI
		TGSGRSQYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGAR
		IFGSVAPWRGGNYYGMDVWGQGTLVTVSS
10-10	2617	EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFRMGWFRQAPGKEREFVAGI
		SRGGSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARASGL
		WFRRPHVWGQGTLVTVSS
10-11	2618	EVQLVESGGGLVQPGGSLRLSCAASGRNFRRNSMGWFRQAPGKEREFVAG
		ISWSGARTHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARVS
		RRPRSPPGYYYGMDVWGQGTLVTVSS
10-12	2619	EVQLVESGGGLVQPGGSLRLSCAASGRNLRMYRMGWFRQAPGKEREFVA
		TIRWSDGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCTRA
		RLRYFDWLFPTNFDYWGQGTLVTVSS
10-13	2620	EVQLVESGGGLVQPGGSLRLSCAASGGLTFSSNTMGWFRQAPGKEREFVA
		SISSSGRTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARRVR
		RLWFRSYFDLWGQGTLVTVSS
10-14	2621	EVQLVESGGGLVQPGGSLRLSCAASGFTLAYYAMGWFRQAPGKEREFVA
		AISWSGRNINYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARE
		RARWFGKFSVSWGQGTLVTVSS
10-15	2622	EVQLVESGGGLVQPGGSLRLSCAASGRTFSSFPMGWFRQAPGKEREFVAAI
		SWSGSTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYSACGRLG
		FGAWGQGTLVTVSS
10-16	2623	EVQLVESGGGLVQPGGSLRLSCAASGISSSKRNMGWFRQAPGKEREFVAT
		WTSRGITTYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGGP
		PRLWGSYRRKYFDYWGQGTLVTVSS
10-17	2624	EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYAMGWFRQAPGKEREFVARI
		TRGGITKYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGLGW
		LLGYYWGQGTLVTVSS
10-18	2625	EVQLVESGGGLVQPGGSLRLSCAASGRMYNSYSMGWFRQAPGKEREFVA
		RISPGGTFYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCTTSARS
		GWFWRYFDSWGQGTLVTVSS
10-19	2626	EVQLVESGGGLVQPGGSLRLSCAASGRTFRSYGMGWFRQAPGKEREFVAS
		ISRSGTTMYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARRGL
		LQWFGAPNSWFDPWGQGTLVTVSS
10-20	2627	EVQLVESGGGLVQPGGSLRLSCAASGRTIRTMGWFRQAPGKEREFVATINS
		RGITNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCTTERDGLL
		WFRELFRPSWGQGTLVTVSS
10-21	2628	EVQLVESGGGLVQPGGSLRLSCAASGRSFSFNAMGWFRQAPGKEREFVAR
		ISRFGRTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKVHS
		YVWGGHSDYWGQGTLVTVSS
10-22	2629	EVQLVESGGGLVQPGGSLRLSCAASGRTYYAMGWFRQAPGKEREFVGAID
		WSGRRITYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARVRFS
		RLGGVIGRPIDSWGQGTLVTVSS
10-23	2630	EVQLVESGGGLVQPGGSLRLSCAASGRAFRRYTMGWFRQAPGKEREFVAS
		ITKFGSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKDRG
		VLWFGELWYWGQGTLVTVSS
10-24	2631	EVQLVESGGGLVQPGGSLRLSCAASGRTFSNYRMGWFRQAPGKEREFVAS
		INRGGSTKYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASGKG
		GSATIFHLSRRPLYFDYWGQGTLVTVSS
10-25	2632	EVQLVESGGGLVQPGGSLRLSCAASGITFSPYAMGWFRQAPGKEREFVATI
		NWSGGYTVYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKRK
		NRGPLWFGGGGWGYWGQGTLVTVSS
10-26	2633	EVQLVESGGGLVQPGGSLRLSCAASGRTFSGFTMSSTWMGWFRQAPGKER
		EFVAGIITNGSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCA
		RRVAYSSFWSGLRKHMDVWGQGTLVTVSS
10-27	2634	EVQLVESGGGLVQPGGSLRLSCAASGRTFRRYSMGWFRQAPGKEREFVAS
		ITPGGNTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASRRR
		WLTPYIFWGQGTLVTVSS
10-28	2635	EVQLVESGGGLVQPGGSLRLSCAASGSIFSIGMGWFRQAPGKEREFVARIW
		WRSGATYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAISIF
		GRLKWGQGTLVTVSS
10-29	2636	EVQLVESGGGLVQPGGSLRLSCAASGRTFTSYRMGWFRQAPGKEREFVAE
		ISSSGGYTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARVG
		PLRFLAQRPRLRPDYWGQGTLVTVSS
10-30	2637	EVQLVESGGGLVQPGGSLRLSCAASGRTFSSFRFRAMGWFRQAPGKEREF
		VALIFSGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARE
		WGRWLQRGSYWGQGTLVTVSS
10-31	2638	EVQLVESGGGLVQPGGSLRLSCAASGRTFGSYGMGWFRQAPGKEREFVAT
		ISIGGRTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGSGS
		GFMWYHGNNNYDRWRYWGQGTLVTVSS
10-32	2639	EVQLVESGGGLVQPGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASI
		NRGGSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGRY
		DFWSGYYRWFDPWGQGTLVTVSS
10-33	2640	EVQLVESGGGLVQPGGSLRLSCAASGRTFSRSDMGWFRQAPGKEREFVAA
		ISWSGGSTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATVPP
		PRRFLEWLPRRLTYIWGQGTLVTVSS
10-34	2641	EVQLVESGGGLVQPGGSLRLSCAASGRTFRRYTMGWFRQAPGKEREFVAS
		MRGSRSYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARMSG
		FPFLDYWGQGTLVTVSS
10-35	2642	EVQLVESGGGLVQPGGSLRLSCAASGSIFRLSTMGWFRQAPGKEREFVASI
		SSFGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARTRGIF
		LWFGESFDYWGQGTLVTVSS
10-36	2643	EVQLVESGGGLVQPGGSLRLSCAASGIAFRIRTMGWFRQAPGKEREFVASI
		TSGGSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGGPR
		FGGFRGYFDPWGQGTLVTVSS
10-37	2644	EVQLVESGGGLVQPGGSLRLSCAASGFTFTSYRMGWFRQAPGKEREFVAG
		ISRFFGTAYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARVTR
		WFGGLDVWGQGTLVTVSS
10-38	2645	EVQLVESGGGLVQPGGSLRLSCAASGRTFSRYVMGWFRQAPGKEREFVAS
		ISRFGRTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARHHG
		LGILWWGTMDVWGQGTLVTVSS
10-39	2646	EVQLVESGGGLVQPGGSLRLSCAASGRTFSMGWFRQAPGKEREFVASISRF
		GRTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKRSTWLPQ
		HFDSWGQGTLVTVSS
10-40	2647	EVQLVESGGGLVQPGGSLRLSCAASGRTFSTYTMGWFRQAPGKEREFVAR
		IWRSGGNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGV
		RGVFRAYFDHWGQGTLVTVSS
10-41	2648	EVQLVESGGGLVQPGGSLRLSCAASGRNLRMYRMGWFRQAPGKEREFVA
		LISRVGVTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGTS
		FFNFWSGSLGRVGFDSWGQGTLVTVSS
10-42	2649	EVQLVESGGGLVQPGGSLRLSCAASGITIRTHAMGWFRQAPGKEREFVATI
		SRSGGNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCTTAGV
		LRYFDWFRRPYWGQGTLVTVSS
10-43	2650	EVQLVESGGGLVQPGGSLRLSCAASGRTFRRYHMGWFRQAPGKEREFVA
		AITSGGRTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCTTDG
		LRYFDWFPWASAFDIWGQGTLVTVSS
10-44	2651	EVQLVESGGGLVQPGGSLRLSCAASGRTFRRYTMGWFRQAPGKEREFVAV
		ISWSGGSTKYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARKG
		RWSGMNVWGQGTLVTVSS
10-45	2652	EVQLVESGGGLVQPGGSLRLSCAASGRTFSWYPMGWFRQAPGKEREFVAS
		ISWGGARTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARST
		GPRGSGRYRAHWFDSWGQGTLVTVSS
10-46	2653	EVQLVESGGGLVQPGGSLRLSCAASGRTFTSYRMGWFRQAPGKEREFVAA
		ITWNSGRTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCSPSS
		WPFYFGAWGQGTLVTVSS
10-47	2654	EVQLVESGGGLVQPGGSLRLSCAASGRPLRRYVMGWFRQAPGKEREFVA
		AITNGGSTKYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGT
		PWRLLWFGTLGPPPAFDYWGQGTLVTVSS
10-48	2655	EVQLVESGGGLVQPGGSLRLSCAASGRTFRRYAMGWFRQAPGKEREFVA
		AINRSGSTEYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARQH
		QDFWTGYYTVWGQGTLVTVSS
10-49	2656	EVQLVESGGGLVQPGGSLRLSCAASGRTFRRYTMGWFRQAPGKEREFVAS
		ISRSGTTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKEGW
		RWLQLRGGFDYWGQGTLVTVSS
10-50	2657	EVQLVESGGGLVQPGGSLRLSCAASGRTLSTYNMGWFRQAPGKEREFVAS
		ISRFGRTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARRGK
		LSAAMHWFDPWGQGTLVTVSS
10-51	2658	EVQLVESGGGLVQPGGSLRLSCAASGRFFSTRVMGWFRQAPGKEREFVAR
		IWPGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARDRG
		IFGVSRWGQGTLVTVSS
10-52	2659	EVQLVESGGGLVQPGGSLRLSCAASGRFFSICSMGWFRQAPGKEREFVAGI
		NWRSGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARG
		SGWWEYWGQGTLVTVSS
10-53	2660	EVQLVESGGGLVQPGGSLRLSCAASGRMFSSRSNMGWFRQAPGKEREFVA
		SISSGGTTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARGFG
		RRFLEWLPRFDYWGQGTLVTVSS
10-54	2661	EVQLVESGGGLVQPGGSLRLSCAASGRTFSSARMGWFRQAPGKEREFVAG
		INMISSTKYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAHFRRF
		LPRGYVDYWGQGTLVTVSS
10-55	2662	EVQLVESGGGLVQPGGSLRLSCAASGRTFRRYTMGWFRQAPGKEREFVAR
		IAGGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARQQYY
		DFWSGYFRSGYFDLWGQGTLVTVSS
10-56	2663	EVQLVESGGGLVQPGGSLRLSCAASGHTFRNYGMGWFRQAPGKEREFVA
		AITSSGSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATVPP
		PRRFLEWLPRRLTYTWGQGTLVTVSS
10-57	2664	EVQLVESGGGLVQPGGSLRLSCAASGRTFSRYAMGWFRQAPGKEREFVAS
		ITKFGSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKERES
		RFLKWRKTDWGQGTLVTVSS
10-58	2665	EVQLVESGGGLVQPGGSLRLSCAASGRNLRMYRMGWFRQAPGKEREFVA
		SISRFGRTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARHDS
		IGLFRHGMDVWGQGTLVTVSS
10-59	2666	EVQLVESGGGLVQPGGSLRLSCAASGRTFRRYAMGWFRQAPGKEREFVAR
		ISSGGSTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCARDRGF
		GFWSGLRGYFDLWGQGTLVTVSS
10-60	2667	EVQLVESGGGLVQPGGSLRLSCAASGIPASMYLGWFRQAPGKEREFVAAIT
		SGGRTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKRKKRG
		PLWFGGGGWGYWGQGTLVTVSS
10-61	2668	EVQLVESGGGLVQPGGSLRLSCAASGIPFRSRTFSAYAMGWFRQAPGKERE
		FVAQITRGGSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCA
		RRHWFGFDYWGQGTLVTVSS

Example 12. SARS-CoV-2 S Protein Ectodomain in Complex with a Bispecific Antibody

This experiment evaluated bispecific antibodies for effector functions (e.g. Fc gamma receptor and C1q binding), neonatal Fc receptor binding, and inhibition of ACE2-SARS-CoV-2 spike protein interaction. The goal of this experiment was to assess the potential for effector function of antibody 493-004 using a panel of Fc receptor binding assays. The study also assessed the ability of antibody 493-004 to inhibit the ACE2/SARS-CoV-2 spike protein binding interaction.

Antibody 493-004 is a synthetic, humanized, anti-SARS-CoV-2 spike protein receptor-binding domain (RBD) bispecific monoclonal antibody comprised of two different variable heavy chains (VHH) linked together with the constant heavy chain 2 (CH2) and the constant heavy chain 3 (CH3) human IgG1 Fc regions. Antibody 493-004 does not contain a variable light chain (FIG. 23).

To identify and construct this bispecific antibody with binding regions capable of binding and neutralizing the SARS-CoV-2 virus and known variants of concern, an approach was deployed that included the screening and epitope binning of numerous VHH antibodies using high-throughput surface plasmon resonance (SPR). This is a real-time binding kinetics assay which calculates the apparent equilibrium dissociation constant (KD) based on increasing concentrations of SARS-CoV-2 S Trimer or S1 monomer protein injections into the assay. From this screen, two distinct VHH leads were identified: first, antibody 202-03 was selected and following the emergence of the delta variant, the second lead, antibody 339-031 was selected. The resulting traces of the first VHH, antibody 202-03 in the SPR assays demonstrating binding to the SARS-CoV-2 variants of concern (VOC) at that time, are presented in FIG. 28.

Following the completion of the binding assays and characterization of binding kinetics for the two VHH antibodies, a series of functional, cell-based assays were initiated. The mean fluorescence intensity (MFI) was determined by subjecting the antibodies to flow cytometry assays to measure inhibition of S1 binding to Vero E6 cells, which constitutively express African Green monkey ACE2. Vero E6 cells were aliquoted in 96-well plates at 1.5×10⁵ cells per well. Antibodies were diluted in PBS and serial diluted 1:3 from 100 nM. Antibody dilutions were then mixed with 1 μg/ml S1 RBD-mFc (Acro SPD-05259) equally, and incubated at 4° C. for 1 hr. The antibody and S1 RBD-mFc mixture then were added to Vero E6 cells, incubated at 4° C. for 1 hr, and washed 3× in PBS. APC-conjugated anti-mouse antibody was then aliquoted and incubated for 1 hr at 4° C. Cells were analyzed by flow by measuring the APC signal.

Interpretively, the assays are assessing the binding of S1 receptor binding domain (RBD) fused to mouse Fc (and variants) to Vero E6 cells expressing ACE2 and detection is via APC-conjugated secondary anti-mouse antibody. Increasing concentrations of inhibitory antibody leads to lower levels of binding. From these assays, the inhibitory concentration at 50% (IC50) was determined for each assay (Table 31).

TABLE 31
IC50 Values Calculated from Cross Reaction
Competition Assays.
	IC50 (nM)
SARS-CoV-2 Variant/	Antibody 202-03	Antibody 339-031
VHH Antibody
S1 RBD fusion (Wuhan/WT)	0.9043	1.248
Alpha	0.8912	0.9901
Beta	0.5868	>100
Epsilon, CA_L452R	5.141	1.401

During the VHH screening campaigns conducted, the alpha and beta variants were used in place of the original Wuhan spike protein. Additionally, with the emergence of the delta variant, both the variant (when available) and several surrogates with the L452R mutation, were integrated into the screening and functional assays. As shown in Table 31, antibody 339-031 had improved activity against Epsilon, which has the L452R mutation, but had reduced activity against Beta.

To better understand and assess the potential for the two selected VHH antibodies (202-03 and 339-031) to compete with each other for the same binding regions, epitope binning experiments on SPR were performed to determine whether binding of antibody 202-03 will block binding of antibody 339-031, and vice versa, to SARS-CoV-2 S Trimers. Epitope binning SPR experiments were performed on a Carterra LSA SPR biosensor equipped with a HC30M chip at 25° C. in HBS-TE. Briefly, antibodies were diluted to 10 pg/mL and amine-coupled to the sensor chip by EDC/NHS activation, followed by ethanolamine HCl quenching. Binding test and regeneration scouting showed reproducible binding to SARS-CoV-2 S Trimer at 10 nM using IgG elution buffer (Thermo). Premixes were then assembled with 150 nM antibody and 10 nM SARS-CoV-2 S Trimer. Data were analyzed in Carterra's Epitope Tool software. Competition assignments were determined relative to the binding responses for SARS-CoV-2 S1 alone (normalized to 1).

From this data, heatmaps representing the pair-wise epitope binning were generated using both WA1 S Trimer and Delta S Trimer and are presented in FIG. 31. The list of antibodies along the rows represent the immobilized antibody and those listed along the columns are the analyte antibody premixed with S Trimer. The specific VHH antibodies antibody 202-03 and antibody 339-031 are indicated by the arrows. The color coding represents red=competition, yellow=partial competition, and green=non-competitive. These data demonstrated that antibody 202-03 and antibody 339-031 have some partial overlap in their epitope bins. Both antibody 202-03 and antibody 339-031 show complete competition with themselves in both experiments.

Following the characterization of the VHH antibodies through competitive binding experiments and the generation of the heat maps, a series of experiments were conducted using pseudovirus to determine the neutralization ability of the individual antibodies. To perform these experiments, pseudovirus expressing the various SARS-CoV-2 spike protein mutations were utilized, representing the current variants of concern. Briefly, the ability to neutralize vesicular stomatitis virus (VSV) pseudotyped with the SARS-CoV-2 D614G spike glycoprotein variant (i.e., a VSV encoding the SARS-CoV-2 D614G spike variant) and all the other variants of concern (see FIG. 32) was tested. Two separate surrogates for the delta variant were used in these experiments, each expressing the L452R mutation.

The results of these experiments demonstrate and further substantiate the strong binding affinity and neutralization ability of both VHH antibodies, with calculated EC50 values at or below 0.1 ug/ml in most cases. There was reduced apparent binding affinity observed with the VHH antibody 202-03 to variants expressing the L452R mutation (e.g., Epsilon California strains; B.1.427 and B.1.429), however, this was mitigated by the affirmative apparent binding affinity and neutralization of the second VHH antibody 339-031 to these variants (with EC50 values at 0.2 and <0.1 ug/ml, respectively, for the two Epsilon/California strains. FIG. 33 and Table 32 provide a summary of the pseudovirus data as described.

TABLE 32
Results of pesudovirus Testing of VHH antibodies.
Antibody	D614G	Alpha	Beta	Gamma	Epsilon-427	Epsilon-429
202-03	0.049	0.049	0.003	0.002	>10	>10
339-031	0.036	<0.1	<0.1	<0.1	0.219	<0.1

Based on all the characterization and performance data for the two VHH antibodies, a decision was made to construct a single bispecific antibody using the 202-03 and 339-031 antibodies. This strategy was selected over a standard cocktail approach with individual VHH antibodies as a bispecific antibody may offer greater overall potency and therapeutic benefits for patients. A final bispecific construct named antibody 493-004 (see FIG. 23 for schematic) was made.

Following the characterization and determination of the binding kinetics for the constructed bispecific antibody 493-004, functional cell-based assays using pseudovirus for the L452R mutation were performed. As surrogates for this mutation, the surrogate Epsilon California variant was used with spike proteins B.1.427 and B.1.429. As shown in FIG. 34, the bispecific antibody neutralized the Epsilon variant for both spike proteins with EC50 values of 0.5443 and 0.5654 mg/ml, respectively.

In a separate pseudovirus assay using the Delta variant (B.1.617.2), a comparison between the individual VHH antibody 339-031 and the bispecific antibody 493-004 was performed. The results are presented in FIG. 35. As suspected from previous binding and neutralization data, both the VHH antibody and the bispecific antibody performed similarly in this assay. The calculated EC50 values were 0.08538 and 0.08136, respectively. In this assay, the VHH antibody 202-03 was not included as it has been shown in previous experiments to bind considerably less efficiently and therefore have limited neutralization potential against the delta variant.

Following the completion of pseudovirus testing, the ability of the bispecific antibody 493-004 to reduce infection with SARS-CoV-2 in a live virus model was tested. In addition to the bispecific antibody and similar to the approach taken with the pseudovirus testing, the VHH antibody 339-031 was also included in the live virus testing, along with a laboratory control (h2165).

An overview of the materials and methods used in the assay are presented below. For the viruses used in the assay and the cells, SARS-CoV-2 isolates were obtained from the Biodefense and Emerging Infections (BEI) Research Resources Repository or isolated at Saint Louis University. Virus stocks were generated by infecting Vero cells overexpressing human Ace2 and TMPRSS2 (VAT cells) at a multiplicity of infection of 0.005. Virus was harvested at 96 hours post infection, cellular debris was removed by centrifugation and virus was aliquoted and frozen at −80 C. Virus titer was determined by focus forming assay (FFA).

The assay deployed the use of Focus Reduction Neutralization Test or FRNT. Specifically, four-fold serial dilutions of the monoclonal antibodies were mixed with ˜100 focus-forming units (FFU) of virus, incubated at 37° C. for 1 h, and added to VAT cell monolayers in 96-well plates for 1 h at 37° C. to allow virus adsorption. Cells were overlaid with 2% methylcellulose mixed with DMEM containing 5% FBS and incubated for 24 hours at 37° C. Media was removed and the monolayers were fixed with 5% paraformaldehyde in PBS for 15 min at room temperature, rinsed, and permeabilized in Perm Wash (PBS, 0.05% Triton-X). Infected cell foci were stained by incubating cells with polyclonal anti-SARS Guinea Pig sera for 1 h at 37° C. and then washed three times with Perm Wash. Foci were detected after the cells were incubated with a 1:5000 dilution of horseradish peroxidase-conjugated goat anti-guinea pig IgG (Sigma) for 1 hour. After three washes with Perm Wash, staining was visualized by addition of TrueBlue detection reagent (KPL). Infected foci were then enumerated by CTL Elispot. FRNT curves were generated by log-transformation of the X axis followed by non-linear curve fit regression analysis using Graphpad Prism 8 (FIG. 36).

Consistent with the results obtained from the pseudovirus testing, the bispecific antibody 493-004 demonstrated superior performance and ability to reduce infection when compared to the individual VHH antibody 339-031. Of interest, when looking at the effect of these antibody constructs in cells infected with the Delta variant, the contributory effect of the second VHH antibody 202-03 (used in the construct of the bispecific) can be seen by the difference between the two curves from the VHH antibody 339-031 and the bispecific antibody TB493-04. This is not surprising because although the VHH antibody 202-03 has an apparent reduced binding affinity and neutralization potential against variants expressing the L452R mutation, there is a contributory effect observed when this VHH antibody is in the bispecific construct.

Comparing the neutralization potential of the bispecific antibody 493-004 to the individual VHH antibody 339-031 against the wild type (AZ1), Beta, and Delta variants of SARS-CoV-2, it is clear that the bispecific demonstrates improved neutralization potential as shown by reductions in FRNT₅₀ values across wild type and the two variants of concern tested. This is highlighted by the bar graph in FIG. 37.

Consistent with the results obtained from the pseudovirus testing, the bispecific antibody 493-004 demonstrated superior performance and ability to reduce infection when compared to the individual VHH antibody 339-031. Of interest, when looking at the effect of these antibody constructs in cells infected with the Delta variant, the contributory effect of the second VHH antibody 202-03 (used in the construct of the bispecific) can be seen by the difference between the two curves from the VHH antibody 339-031 and the bispecific antibody 493-004. This is not surprising because it has been shown previously that although the VHH antibody 202-03 has an apparent reduced binding affinity and neutralization potential against variants expressing the L452R mutation, there is a contributory effect observed when this VHH antibody is in the bispecific construct.

Comparing the neutralization potential of the bispecific antibody 493-004 to the individual VHH antibody 339-031 against the wild type (AZ1), Beta, and Delta variants of SARS-CoV-2, it is clear that the bispecific demonstrates improved neutralization potential as shown by reductions in FRNT50 values across wild type and the two variants of concern tested (FIG. 37).

Table 33 shows a Fc fusion bispecific antibody developed against SARS-CoV-2 spike protein for the treatment of COVID-19. Antibody 493-004 contains an unmodified human IgG1 Fc.

Results showed that antibody 493-004 showed binding to the neonatal Fc receptor (FcRn), Fcγ receptors and C1q similar to those of an isotype-matched positive control IgG1 antibody. Results also found that antibody 493-004 showed inhibition of the Ancestral spike RBD as well as SARS-CoV-2 spike trimers of Ancestral, Delta, and Omicron variants.

Wild-type forms of human IgG1 have the potential to bind various Fcγ receptors and elicit effector function. For example, Fcγ receptors on immune cells may mediate recruitment and activation of these cells toward cells or tissues where antibody is bound to antigen, resulting in antibody-dependent cell-mediated cytotoxicity (ADCC). Similarly, complement component 1q (C1q) can also recognize antibody-bound Fc regions and mediate a process called complement-dependent cytotoxicity (CDC). The lower hinge region (amino acid 233-239) of the human IgG1 Fc is known to be important for its Fcγ receptor (FcγR) binding and complement binding. In this in vitro study, the potential for antibody 493-004-mediated effector function was evaluated using a panel of Fc binding assays with recombinant proteins (FcγR, FcRn, and C1q). The ability of antibody 493-004 to inhibit the ACE2/SARS-CoV-2 spike binding interaction using Ancestral, Delta and Omicron spike variants was also evaluated.

TABLE 33
Sequences of Fc Fusion Bispecific Antibody 493-004
	SEQ
Variant	ID NO	Sequence
Antibody	2669	ATGGGATGGTCATGTATCATCCTTTTTCTGGTAGCAACTGCAAC
493-004		TGGAGTACATAGCGAGGTGCAGCTGGTCGAGTCTGGCGGTGGC
DNA		TTGGTGCAACCCGGCGGCAGCTTGAGACTGTCTTGCGCCGCCTC
Sequence		CGGGTTCACCTTCTCCCCAAGTTGGATGGGATGGTTTCGGCAAG
		CCCCAGGCAAGGAACGCGAATTCGTGGCCACTATCAATGAATA
		CGGCGGCCGGAACTACGCCGACTCCGTGAAAGGGCGATTTACA
		ATTTCCGCTGATAACTCCAAGAACACCGCATATCTGCAAATGAA
		CAGCCTCAAGCCTGAGGACACAGCCGTCTACTATTGTGCTAGAG
		TGGACCGGGACTTTGACTACTGGGGTCAGGGTACACTGGTTACG
		GTTTCCTCGGGAGGAGGCGGAAGCGAACCCAAGTCTTCTGACA
		AAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGG
		GGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCC
		TCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGAC
		GTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGG
		ACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGG
		AGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC
		CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGG
		TCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCC
		AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGC
		CCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGAC
		CTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGT
		GGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGC
		CTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAG
		CTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCT
		CATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAG
		AAGAGCCTCTCCCTGTCTCCGGGCGGCGGAGGTGGATCTGGGG
		GCGGCGGTTCCGCTTCTGAGGTTCAGCTCGTAGAATCCGGTGGA
		GGACTTGTTCAACCTGGAGGTAGTCTGAGGCTGAGCTGTGCTGC
		AAGTGGCAGCACATTTAGCATCAATGCTATGGGTTGGTTCCGAC
		AAGCTCCAGGGAAGGAGCGCGAGTTCGTGGCTGGGATCACCAG
		CTCTGGAGGCTATACCAACTACGCTGACTCTGTCAAAGGTCGCT
		TTACCATATCGGCCGACAATTCTAAGAATACTGCCTACCTGCAA
		ATGAACTCCCTGAAGCCTGAAGACACCGCCGTGTATTACTGCGC
		CGCTGATGGCGTGCCGGAGTACAGCGATTACGCGTCGGGACCA
		GTCTGGGGCCAAGGCACATGGTGACTGTATCGTCGTAATAG
Antibody	2670	MGWSCIILFLVATATGVHS EVQLVESGGGLVQPGGSLRLSCAAS
493-004		GFTFSPSWMGWFRQAPGKEREFVATINEYGGRNYADSVKGRF
AA		TISADNSKNTAYLQMNSLKPEDTAVYYCARVDRDFDYWGQGT
Sequence		LVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL
		MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
		YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
		GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG
		QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
		ALHNHYTQKSLSLSPGGGGGSGGGGSASEVQLVESGGGLVQPGG
		SLRLSCAASGSTFSINAMGWFRQAPGKEREFVAGITSSGGYTNY
		ADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADGVPE
		YSDYASGPVWGQGTLVTYSS**

A biosensor-based binding assay was carried out using surface plasmon resonance (SPR) detection, to quantitatively evaluate the binding affinities of Fc receptors (panel of FcγR proteins and FcRn) for antibody 493-004, an anti-SARS-CoV-2 Spike RBD quadrivalent bispecific VHH-Fc fusion (from human IgG1). An isotype-matched commercially sourced anti-RBD neutralizing monoclonal antibody (human IgG1) from Acro Biosystems (SAD-S35) was used as a positive control in these experiments.

Interaction analysis was conducted on a Biacore 8K biosensor equipped with CMS sensor chip at 25° C. in the standard run buffer of HBS-P, pH 7.4 with 0.2 g/L BSA (for FcγR panel) or PBS-P, pH 6.0 with 0.2 g/L of BSA (Dulbecco phosphate buffer saline with 0.01% Tween-20 adjusted to pH 6.0 using dilute phosphoric acid) for analysis of FcRn interaction, respectively. Neutravidin (ThermoFisher Scientific, MA, US, Cat #31000) was coated onto all flow cells of the chip at high levels (˜8000 RUs) using a standard amine-coupling procedure and then coated with high levels of biotinylated SARS-CoV-2 spike RBD (˜6000 RUs). The RBD-coated chip was utilized as a ‘capture surface’ to capture (tether) appropriate amounts of antibody 493-004 (˜80-500 RUs) on flow cell 2 (the active surface) with flow cell 1 left empty, representing the naked RBD-coated surface, to serve as a reference surface. Binding of Fc receptors (as analytes) to antibody 493-004 (as ligand) was evaluated by injecting Fc receptors in increasing concentrations over flow cells 1 and 2 at 30 μL/min using the ‘single cycle kinetics’ module. Analyte titrations used were 5-(or 6-)membered, 3-fold serial dilutions with top concentration of 30 nM (FcγR1), 300 nM (FcRn), 1000 nM (FcγR2a and 3a) or 3000 nM (FcγR2b/c). Within the same experiment, the binding of Fc receptors (as analytes) to flow cells tethered with a commercially sourced isotype-matched anti-RBD neutralizing antibody (as ligand), served as a positive control. Blank cycles using buffer (instead of Fc receptors) as analyte were used for double-referencing the binding data. After each binding cycle, the ligands (antibody 493-004 or the control antibody) were stripped from the RBD-coated surface by regenerating it with 10 mM glycine, pH2.0 for 30 s (for the FcγR interactions) or with PBS-P pH7.4 for 1 min (for the FcRn interactions).

Biacore data were processed and analyzed in the BiaEvaluation™ software. Biacore data for antibody 493-004 or the isotype-matched (human IgG1) control anti-RBD neutralizing antibody binding to the Fc receptors were fit globally to a simple 1:1 Langmuir binding model to calculate the kinetics parameters, including the association and dissociation kinetic rate constants (Ka and Kd) and the affinity constant (also known as the equilibrium dissociation constant, or KD) from their ratio, where KD=Kd/Ka. The binding data were also fitted, where appropriate, to a steady state affinity model to generate binding isotherms to obtain KD using this alternate equilibrium-based model. All interactions except those of the ‘high affinity’ FcγR1 met the criteria for steady state fitting, which requires that all sensorgrams attain equilibrium binding responses during the allowed association phase per analyte injection. All experiments were repeated for a total of 3 times and values are reported as mean±SD.

An ELISA-based binding assay was used to evaluate the ability of antibody 493-004 to bind complement C1q. Native human IgG1, IgG2 and IgG4 isolated from human plasma were used as positive controls in the ELISA. A purified human IgG1 isotype control recombinant monoclonal antibody (clone QA16A12) from Biolegend was also used as positive control in this experiment.

The binding of human C1q (Prospec, NJ, US) to antibody 493-004 and control antibodies (native human IgG1, native human IgG2, native human IgG4 and Biolegend recombinant human IgG1 clone QA16A12) was assessed by ELISA.

A Nunc Maxisorp flat bottom ELISA plate was absorption-coated overnight with antibody 493-004 and control antibodies (native human IgG1, native human IgG2, native human IgG4 and Biolegend human IgG1 clone QA16A12) at a concentration of 2 μg/ml (15 nM molecules) in PBS at 4° C. Subsequently, the wells were washed and blocked using START-Block buffer for 1h at room temperature. Dose titrated C1q (20 μg/mL, 2 fold dilution in START-Block buffer) was added to the appropriate wells and incubated at room temperature for 1h with gentle shaking. This was followed by addition of polyclonal sheep anti-human C1q antibody conjugated to horseradish peroxidase (0.5 μg/mL, 1h incubation) to detect C1q bound to the coated antibodies. The plate was developed by addition of TMB substrate. The reaction was stopped by the addition of ELISA stop solution and the OD was measured at 492 nm using Envision 2105 multimode plate reader (Perkin Elmer, CT, US). Experiments were repeated in triplicate and the binding data was fitted in GraphPad Prism™ using nonlinear regression-4PL.

A biochemical inhibition assay was carried out using AlphaLISA to quantitatively evaluate the inhibition of the binding interaction between ACE2 and SARS-CoV-2 spike protein by antibody 493-004, antibody 339-031, and antibody 202-03 (antibody 339-031 and antibody 202-03 are bivalent monospecific parent VHH-Fc fusions from which the quadrivalent bispecific antibody 493-004 is derived). The anti-RBD neutralizing monoclonal antibody from Acro Biosystems and ACE2-His were used as positive controls in these experiments.

The inhibition of the ACE2/SARS-CoV-2 spike binding interaction by antibody 493-004 was carried out using AlphaLISA.

Initial experiment was designed to identify optimal conditions of ACE2-muFc complex formation with biotinylated SARS-CoV-2 spike RBD recombinant protein. For this purpose, a cross-titration experiment was set up whereby different concentrations of ACE2-muFc (100-0.14 nM, 3-fold dilution) was cross-titrated against different concentrations of biotinylated SARS-CoV-2 spike RBD (100-0.14 nM, 3-fold dilution) in a checkerboard format. Each concentration combination of ACE2-muFc and biotin spike RBD were mixed in a 384-well Proxiplate™. Streptavidin donor beads (final concentration of 40 μg/mL) and anti-mouse IgG acceptor beads (final concentration of 10 μg/mL) were then added to the wells. The samples were incubated at room temperature, in the dark for 1h. AlphaLISA signal was read using Envision 2105 multimode plate reader equipped with AlphaLISA optical module (Perkin Elmer, CT, US).

Similar cross-titrations were carried out between ACE2-mu Fc and biotinylated SARS-CoV-2 spike trimers from Ancestral (D614G), Delta, and Omicron (B.1.1.529) variants to identify optimal concentrations of complex formation between ACE2 and the respective spike trimers.

A quantitative inhibition of ACE2-SARS-CoV2 spike protein interaction by antibody 493-004, antibody 339-031, and antibody 202-03 (antibody 339-031 and antibody 202-03 are parent Fc fusions from which the bispecific antibody 493-004 is derived) was assessed by AlphaLISA. The anti-RBD neutralizing monoclonal antibody from Acro Biosystems and ACE2-His were used as positive controls in these experiments.

Based on the cross-titration experiment described above, appropriate concentration of ACE2-muFc was allowed to complex with optimal concentrations of biotinylated SARS-CoV2 spike proteins [i.e. Ancestral RBD, and trimers from Ancestral (D614G), Delta, and Omicron (B.1.1.529)] by incubating them together (ACE2-muFc+each separate spike variant) in the assay buffer (PBS, 0.01% P20, 0.2 mg/mL BSA, pH7.4) at room temperature for 1h. The complex (5 μL) was then added to the 384-well Proxiplate™. This was followed by addition of 5 μL of the inhibitors (antibody 493-004, antibody 339-031, antibody 202-03, anti-RBD, ACE2-His) in a dose titration. Streptavidin donor beads (final concentration of 40 μg/mL) and anti-mouse IgG acceptor beads (final concentration of 10 μg/mL) were then added to the wells. The samples were incubated at room temperature, in the dark for 1h. AlphaLISA signal was read using Envision 2105 multimode plate reader (Perkin Elmer, CT, US) equipped with AlphaLISA optical module. Inhibition experiments were repeated in triplicate and dose dependent curves fitted using non-linear regression-4PL [GraphPad Prism™]. IC50 values are reported as mean±SD.

The SPR based binding interactions of antibody 493-004 and anti-RBD neutralizing antibody (isotype control) with ‘high affinity’ FcγR1 are shown in FIG. 13 and the deduced binding kinetics and KD values derived from the Langmuir 1:1 binding model (kinetic fit) are reported in Table 34. The results show that the parameter values for FcγR1 interactions with antibody 493-004 and the isotype control were identical within the error of the measurements (Table 34).

The SPR based binding interactions of antibody 493-004 and anti-RBD neutralizing antibody (isotype control) with ‘low affinity’ Fc receptors is shown in FIGS. 14-18 and the deduced KD values derived from the Langmuir 1:1 binding model (kinetic fit) and steady state affinity model (steady state fit) are summarized in Table 35. Both binding models estimate comparable KD values for each studied Fc receptor interaction. The only discrepancy observed was for FcγR3a (176V) binding and is likely due to the heterogeneous quality of the commercial protein as judged by the markedly heterogeneous sensorgrams (FIG. 18), resulting in a poor KD estimate from the kinetic fit. When comparing antibody 493-004 to the isotype control, Table 35 shows that the affinities of all ‘low affinity’ Fc receptors studied were within 2-fold or better. Taken together, Table 34 and Table 35 show that antibody 493-004 retains the Fc receptor engagement properties that are characteristic of a human IgG1, hence, are expected to function similarly in vivo in this regard.

The half-life of therapeutic antibodies can be prolonged by virtue of interactions with the neonatal receptor, FcRn, at acidic pH in serum. To probe this interaction, the binding of FcRn was tested (as analyte) to antibody 493-004 (as ligand) at pH6.0 (FIG. 19). antibody 493-004 shows similar binding kinetics and affinity for FcRn as those for an isotype-matched (human IgG1) anti-RBD neutralizing antibody (positive control) (Table 34), hence is expected to exhibit a serum half-life equivalent to that for a wild-type human IgG1. Table 34 and Table 35 also report ‘apparent’ % activity (ratio of experimental Rmax to theoretical Rmax) for all interactions tested. The reported % activity values were calculated using experimental Rmax obtained from kinetic fitted data (Table 34 and Table 35). Similar % activity values were also obtained when this ratio was calculated using experimental Rmax from steady state fits (data not shown). For all interactions tested, the fitted Rmax values were close to the theoretical ones (% activity was around 100%), thereby validating the assay set up and overall quality/reliability of the assay.

TABLE 34
Kinetic and Affinity Determination of the ‘High Affinity’ FcγR1
Binding to Antibody 493-004 and Anti-RBD Isotype Control.
Ligand	ka			%
(on Chip)	(M − 1 s − 1)	kd (s − 1)	KD (pM)	Activity
antibody	(4.50 ± 0.46) ×	(2.46 ± 0.01) ×	550 ± 7	118
493-004	106	10 − 4
Anti-RBD	(5.34 ± 0.15) ×	(2.45 ± 0.02) ×	460 ± 10	120
Isotype	106	10 − 4
Control

The parameter values represent the mean±SD of 3 independent measurements. The ‘apparent’ % Activity was calculated as the ratio of experimental Rmax (obtained from kinetic fitting) to the theoretical Rmax. Theoretical Rmax values were calculated according to the binding stoichiometries of the analyte/ligand interactions, which are (on a per molecule basis) 1:1 for FcγR1 (one analyte per whole homodimer ligand).

TABLE 35
Affinity Determination of Fc Receptor Binding to Antibody 493-004 and Isotype-
Matched Control Anti-RBD Neutralization Antibody by SPR.
	Antibody 493-004 KD (nM)	Anti-RBD Isotype Control KD (nM)
Fc receptor	Kinetic	Steady	%		Steady
(Analyte)	Fit	State Fit	Activity	Kinetic Fit	State Fit	% Activity
H167)	487 ± 2.1	544 ± 6.4	99	462 ± 24	537 ± 17	110
FcγR2a	539 ± 12.1	550 ± 6.5	103	596, 650	636, 640	ND
(R167)				(n = 2)	(n = 2)
FcγR2b/c	1950 ± 90	2010 ± 64	91	1360 ± 28	1590 ± 40	93
FcγR3a	399 ± 17.6	368 ± 15.5	75	1110 ± 45.7	796 ± 13.6	92
(176F)
FcγR3a	75.3 ± 1.5	147 ± 5	107	161 ± 3.7	210 ± 1	110
(176V)
Human FcRn	162 ± 10	195 ± 9.4	109	81.1 ± 1.5	86.5 ± 2.1	116
(at pH6.0)

The parameter values represent the mean±SD of 3 independent measurements, except for FcγR2a (R167) which was analyzed twice and both values are reported. n/a=not applicable. ‘Apparent” % Activity was calculated as the ratio of experimental Rmax (obtained from kinetic fitting) to the theoretical Rmax. Theoretical Rmax values were calculated according to the binding stoichiometries of the analyte/ligand interactions, which are (on a per molecule basis) 1:1 for FcγR1 (one analyte per whole homodimer ligand) and 2:1 for FcRn (two analytes per whole homodimer ligand, or one analyte per ligand monomer). ND=not determined.

Recombinant Clone QA16A12 (human IgG1) and native human IgG1 showed high dose dependent binding to C1q, while native human IgG2 and IgG4 showed low C1q binding (FIG. 20), in agreement with the literature. antibody 493-004 showed high, dose dependent binding to C1q similar to that for Clone QA16A12 human IgG1 (FIG. 20), suggesting comparable C1q engagement as a human IgG1 isotype. The difference in C1q binding between antibody 493-004 (and Clone QA16A12) and native human IgG1, despite having same Fc backbone is likely due to lower adherence of the latter to the ELISA plate during the different steps of the ELISA.

The ability of antibody 493-004 to inhibit the binding interaction between ACE2 and various forms of SARS-CoV-2 spike protein was determined quantitatively using AlphaLISA.

To develop the assay conditions, the optimal concentrations of ACE2-muFc and biotinylated SARS-CoV-2 spike RBD (Ancestral) for complex formation were determined using a cross-titration experiment in a matrix format (FIG. 21). Based on the results from this experiment, binding of ACE2-muFc (11 nM, highlighted by the vertical bar in the graph) with RBD (11 nM, highlighted in green in the legend) gave a 20-fold signal-to-noise (S/N) (FIG. 21) which was considered an optimal binding signal for setting up a subsequent inhibition assay. Similar cross-titration experiments between ACE2-muFc (11 nM) and biotinylated D614G SARS-CoV-2 spike trimer (11 nM) resulted in 30-fold S/N (data not shown). ACE2-mu Fc (11 nM) binding to SARS-CoV-2 spike trimers for Delta and Omicron (B.1.1.529) variants (33 nM) resulted in 17-fold and 25-fold S/N respectively (data not shown). These optimized binding conditions were utilized to prepare complexes of ACE2 with different variants of SARS-CoV-2 spike proteins to examine the inhibition of the interaction by antibody 493-004, parent VHH-Fc fusions (antibody 339-031 and antibody 202-03) and positive controls (Anti-RBD neutralizing antibody and ACE2-His) (FIG. 22).

Antibody 493-004 showed comparable potency to the commercially sourced control anti-RBD neutralizing antibody in inhibition of ancestral spike RBD and D614G spike trimer (Table 36). Unlike the control anti-RBD antibody, antibody 493-004 also exhibited inhibition of the interaction of ACE2 to Delta and Omicron spike trimers (FIG. 22) and exhibited similar potency in comparison to ACE2-His (positive control) (Table 36). Antibody 202-03 showed weak inhibition of the Delta spike trimer (FIG. 22, Panel C) at high concentrations, and antibody 339-031 showed no inhibition of Omicron spike trimer (FIG. 22, Panel D). In the case of inhibition of the Omicron variant, a residual 30% binding to ACE2 was observed even at the highest concentration of inhibitors (350 nM). This residual binding likely relates to heterogeneous quality of the commercially sourced protein.

TABLE 36
Inhibition of ACE2 Interaction with Different Variants of
SARS-CoV-2 Spike Proteins.
Antibody (or Control) IC50 (nM)
Inhibition of ACE2/SARS-CoV-2 Spike RBD (Ancestral)
Antibody 493-004      10.4 ± 1.9
Antibody 339-031      5.6 ± 0.84
Antibody 202-03       8.3 ± 0.25
Anti-RBD (Acro)       6.8 ± 0.50
ACE2-His              11.5 ± 0.82
Inhibition of ACE2/SARS-CoV-2 Spike Trimer (Ancestral)
Antibody 493-004      2.1 ± 0.4
Antibody 339-031      2.1 ± 0.34
Antibody 202-03       4.0 ± 0.22
Anti-RBD (Acro)       1.84 ± 0.35
ACE2-His              6.65 ± 0.46
Inhibition of ACE2/SARS-CoV-2 Spike Trimer (Delta)
Antibody 493-004      8.8 ± 1.3
Antibody 339-031      3.0 ± 0.5
Antibody 202-03       No inhibition
Anti-RBD (Acro)       No inhibition
ACE2-His              8.4 ± 1.16
Inhibition of ACE2/SARS-CoV-2 Spike Trimer (Omicron)
Antibody 493-004      9.04 ± 0.46
Antibody 339-031      No inhibition
Antibody 202-03       5.16 ± 0.32
Anti-RBD (Acro)       No inhibition
ACE2-His              7.91 ± 1.2

Using a variety of in-vitro binding assays (SPR, ELISA, and AlphaLISA), antibody 493-004 was shown to be a potent ACE2 inhibitor and retains intact Fc functionality consistent with that of a human IgG1 isotype. Antibody 493-004 is a potent bispecific inhibitor of SARS-CoV-2 spike recombinant proteins [Ancestral RBD and spike trimer (D614G)] that also inhibits Delta and Omicron variants of the spike trimer, with similar potency as ACE2-His (control). Antibody 493-004 showed similar binding to C1q as the human IgG1 isotype control, hence will likely activate the complement pathway similarly to a human IgG1. Antibody 493-004 also showed similar affinity for interaction with Fcγ receptors and FcRn as the isotype-matched control anti-RBD neutralizing antibody (human IgG1 Fc). This suggests that antibody 493-004 is likely to exhibit similar in vivo activity in terms of Fc effector function and exhibit the long serum half-life (via the FcRn recycle/rescue pathway) characteristic of a human IgG1.

Example 13. Humanized Anti-SARS-CoV-2 S Protein Receptor Binding Domain VHH Bispecific Antibody for Treatment of COVID-19

This experiment aims to treat COVID-19 with humanized anti-SARS-CoV-2 antibodies. Antibody 493-004 is a single dose, formulated in either an intravenous and/or subcutaneous dosage form for injection.

The bispecific monoclonal antibody 493-004 is constructed with two individual, single domain VHH antibodies: antibody 202-03 and antibody 339-031 linked together with the constant heavy chain 2 (CH2) and the constant heavy chain 3 (CH3) Fc region of the antibody (FIG. 23). The resulting bispecific monoclonal antibody is derived from humanized antibody VHH single domain sequences with specific affinity to distinct binding motifs on the SARS-CoV-2 S1 spike protein. The two individual VHHs used in the construct of the bispecific antibody contain llama-derived sequences in the CDR1 and CDR2 regions with a human CDR3 sequence. The framework for the bispecific antibody is >95% human. The CDRs fit into a VHH framework in the same manner that CDRs fit into human heavy chain frameworks and the FC regions of the bispecific are human IgG 1 Fc, which is the most common form of human IgG.

The upstream manufacturing process is depicted in FIG. 24. The drug substance is expressed in Chinese Hamster Ovary (CHO) cells using a fed batch process. Cell culture process development occurs in progressively larger bioreactor sizes, starting at 250 ml size, culminating in a 500 L GMP. The first step in the USP is vial thaw, where cells from the Master Cell Bank (MCB) are thawed and transferred into an appropriate cell culture flask containing inoculum medium. The cells are then incubated throughout the cell expansion stage for the upstream process. This is followed by the second step of inoculum scale-up, where a series of passages are performed to obtain a sufficient number and density of cells to inoculate the bioreactor. Inoculum steps progress from 250 mL shake flasks and move through 1 L, 2.8 L, 50 L and 100 L, to the 500 L bag bioreactor.

Growth medium is used for seed culture in the 100 L bioreactor and an initial cell density is targeted to be in the range of 0.25 to 0.45×10⁶ cells/mL. Cells from the 100 L bioreactor are transferred to the 500 L bioreactor to initiate the production culturing process, with a target cell density of 0.8 to 1.2×10⁶ cells/mL.

Pre-harvest samples are tested for sterility, mycoplasma, adventitious virus in vitro, detection of MVM DNA by qPCR using Taqman Technology, and quantitation of viral contaminants by negative stain electron microscopy. After 8 to 14 days of cultivation in the production phase, the cell culture fluid is harvested.

The downstream manufacturing process is depicted in FIG. 25. FIG. 26 depicts the drug product process. During bulk drug substance thawing, pooling, and mixing, frozen drug substance is thawed at room temperature, protected from light, and then pooled into a container where it is mixed to homogeneity. The mixed material is tested for pH, protein concentration, bioburden, and endotoxin. During sterile filtration, the pooled drug substance is aseptically filtered from Grade C area via a peristaltic pump through two in series connected 0.22 μm sterile filters into a sterile single use bag in the Grade B area. Prior to and after sterile filtration, filter integrity testing is performed on both filters. During aseptic filling, stoppering, and capping, aseptic filling is performed inside the open restricted access barrier system (ORABS) unit, which fully encloses the filler and provides a Grade A environment. The ORABS unit separates the operator from the aseptic interior. All filling components are performed inside the open restricted access barrier system (ORABS) unit, which fully encloses the filler and provides a Grade A environment. The ORABS unit separates the operator from the aseptic interior. All filling components are performed periodically during the filling process. Filled vials are automatically stoppered with sterilized rubber stoppers inside the ORABS unit. The stoppered vials are capped with sterilized plastic aluminum flip-off caps. During visual inspection, bulk packaging and storage, a manual 100% visual inspection is performed on the filled vials by production personnel, followed by a statistically based acceptable quality limit (AQL) inspection by Quality Assurance. Release and stability samples are taken after visual inspection. The filled drug product vials are bulk packaged, labelled and stored at 2-8° C. Drug substance specifications for the pharmaceutical formulation of monoclonal antibody 493-004 can be found in FIG. 47.

A pharmacokinetic (PK) study is performed of the bispecific monoclonal antibody 493-004 following single intravenous infusion and subcutaneous injection into Sprague Dawley rats. This experiment evaluates the serum pharmacokinetics (PK) and immunogenicity (anti-drug antibodies, ADA) of the bispecific monoclonal antibody following a single intravenous infusion (IV) and subcutaneous injection (SC) administration in male and female SD rats; the bispecific monoclonal antibody is determined in serum up to day 56 post-dosing. The experimental design of the PK study can be found in FIG. 48A. Dose volume is determined based on body weight of the rats, which are weighed prior to dose administration. During IV infusion, the dose formulation is administered via tail vein.

Each blood sample is collected via jugular vein puncture (right jugular vein cannulation from the animals in Group 1). Actual sample collection times is recorded in the study records. The acceptable deviation of blood collection is ±1 min for sample collected within 1 hour post-dose and 5% of the nominal time for other timepoints. A sample collection schedule is shown in FIG. 48B.

From each treatment group, about 0.3 mL blood sample is collected at sampling time points. The actual sample collection times is recorded. All blood samples are collected into commercially available BD tubes containing polymer silica activator. After blood is collected, the tubes containing blood samples are rested at room temperature for at least 30 minutes. Then centrifugation at 4° C. for 10 minutes at 3200×g occurs within 1 hour after collection. The clarified serum is then collected after centrifugation. The samples are then quickly frozen under dry ice and stored at −60° C. or lower in a freezer until being transferred in dry ice to Immunology Laboratory of Bioanalysis Department using a qualified Enzyme-Linked Immuno Sorbent Assay (ELISA) method for analysis.

From each treatment group, an about 0.45 mL blood sample is collected at sampling time points. The actual sample collection times will be recorded. All blood samples are collected into commercially available BD tubes containing polymer silica activator. After blood is collected, the tubes containing blood samples are rested at room temperature for at least 30 minutes. Then centrifugation at 4° C. for 10 minutes at 3200×g occurs within 2 hours after collection. The clarified serum is then collected after centrifugation. The samples are be quickly frozen under dry ice and stored at −60° C. or lower in a freezer until being transferred in dry ice to Immunology Laboratory of Bioanalysis Department using a validated method for analysis.

Neutralization effectiveness of the bispecific antibody was determined against ancestral and both the Delta and omicron (BA.1) variants of the SARS-CoV-2 virus, resulting in dose response curves and associated 50% effective concentration (EC50) and 90% effective concentration (EC90) determination for each variant (FIG. 42).

Based on an EC90 against the Omicron variant using an in vitro plaque reduction assay, the effective concentration of antibody 493-004 is 3000 ng/mL or 3 ug/mL. From the two-week rat PK data, the projected concentration-time profile for humans for 3 mg/kg antibody 493-004 dose indicate that the concentrations of antibody 493-004 will remain above 3 ug/mL at least for 10 days and therefore, can be therapeutically meaningful as an effective treatment. Most recently, the rat PK study has completed and the plasma exposures across the entire 42-day study are now available. Taking the same 3000 ng/mL or 3 ug/mL value calculated from the in vitro plaque reduction assay assessing the efficacy of antibody 493-004 against the Omicron variant, the concentration of antibody 493-004 will remain above 3 ug/mL for approximately 21-days or 3-weeks (FIG. 43).

The allometric modeling for all methods utilized PK data from a single species (e.g., the rat) and overall, all four methods projected FIH dose of 493-004 comparatively higher than typically observed with non-COVID-19 antibodies but consistent with what has been both demonstrated, as well as reviewed and granted EUA for antibodies against the SARS-CoV-2 virus. Using 70 kg as the average human weight, the modeling predicts the range of a single human IV dose of 493-004 to be between 329 mg (4.7 mg/kg) and 637 mg (9.1 mg/kg). This is aligned with the proposed Phase 1 dosing scheme of 1 mg/kg (70 mg), 3 mg/kg (210 mg), 6 mg/kg (420 mg), and 10 mg/kg (700 mg) single IV dose in healthy volunteers to establish human PK and safety, as well as the proposed Phase 2a dosing scheme of 6 mg/kg (420 mg) and 10 mg/kg (700 mg) single IV dose in non-hospitalized patients with SARS-CoV-2 experiencing mild to moderate disease.

A good laboratory practices (GLP), 15-day once weekly intravenous infusion or subcutaneous injection repeated dose toxicity and toxicokinetic study in rats is performed with a 28-day recovery period. This experiment determines the potential toxicity of the bispecific monoclonal antibody 493-004 when administered once weekly to Sprague Dawley rat for 3 doses (Days 1, 8 and 15) by intravenous infusion (IV) or subcutaneous injection (SC), and to assess the reversibility, persistence, or delayed occurrence of toxic effects following a 28-Day recovery period. In addition, the toxicokinetics (TK) and immunogenicity (anti-drug antibody, ADA) of the bispecific monoclonal antibody 493-004 are evaluated. Liquid chromatography with mass spectrometry (LC-MS) or liquid chromatography with tandem mass spectrometry (LC-MS/MS) methods are used for the detection and quantitation of the amount of bispecific mAb in plasma samples.

A GLP, tissue cross-reactivity (TCR) study was performed with frozen normal human and Sprague Dawley rat tissues. The objective of this study was to determine the cross-reactivity of the bispecific monoclonal antibody 493-004 with frozen normal human and Sprague Dawley rat tissues.

In the Sprague Dawley rat tissues, no Biotin-493-004 bispecific antibody staining was observed at 1 μg/mL. Positive staining was observed at 25 μg/mL in the cytoplasm only of the histiocytes from 3/3 of the lymph nodes. The staining intensity was weak, and the staining frequency was “rare” to “rare to occasional.” Positive staining was also observed in the cytoplasm of the thymic cells. The staining intensity was weak, and the staining frequency was “rare”. There was no Biotin-493-004 bispecific antibody staining in other Sprague-Dawley rat tissues.

Given that the staining as relegated to the cytoplasm only in the histiocytes, it was concluded that the staining observed presents insignificant toxicological risk factor since the mechanism of action would preclude accessibility of the bispecific antibody 493-004 to cytoplasmic structures in vivo. Furthermore, and importantly, there were no untoward findings in the histopathology for the low, medium, and high dosages of the 21-day repeat-dose, IV infusion or subcutaneous (SC) injection GLP toxicology study in the rat, with a 28-day recovery period. The clean toxicology study provides direct, supportive evidence that the cytoplasmic staining in the histocytes observed in the TCR study is of no toxicological significance.

The purpose of the toxicology study was to determine the potential toxicity of 493-004, a bispecific antibody targeting SARS-CoV-2 in order to prevent or treat coronavirus disease 19 (COVID-19), when administered once weekly to Sprague Dawley rats for 3 doses (Days 1, 8 and 15) by IV infusion or injection, to assess the reversibility, persistence, or delayed occurrence of toxic effects following a 28-Day recovery period. In addition, the toxicokinetics (TK) and immunogenicity (anti-drug antibody, ADA) of 493-004 was also evaluated. The study design is summarized in Table 37.

TABLE 37
Dosage, Volume, Concentration, and Route of 493-004 Administered in Rat
Toxicology Study
		Numbering of Animals
	WBP2495 (RBT-0813 DS) Dosesa	Dosing
Group/	Dose	Volume	Conc.		Phase	Recovery
Color	(mg/kg/dose)	(mL/kg)	(mg/mL)	Route	M	F	M	F
1/White	0	20	0	IV	1001-	1501-	1011-	1511-
					1010	1510	1015	1515
2/Green	30	20	1.5	IV	2001-	2501-	2011-	2511-
					2010	2510	2015	2515
3/Yellow	100	20	5	IV	3001-	3501-	3011-	3511-
					3010	3510	3015	3515
4/red	300	20	15	IV	4001-	4501-	4011-	4511-
					4010	4510	4015	4515
5/Cyan	0	10	0	SC	5001-	5501-	5011-	5511-
					5010	5510	5015	5515
6/Magenta	30	10	3	SC	6001-	6501-	6011-	6511-
					6010	6510	6015	6515
7/Blue	100	10	10	SC	7001-	7501-	7011-	7511-
					7010	7510	7015	7515
8/Dark	248	10	24.8	SC	8001-	8501-	8011-	8511-
Grey					8010	8510	8015	8515
In this protocol, “dose level” and “dosage” are used interchangeably.
aDoses represent active ingredient.
Conc. = Concentration; M = Male; F = Female.

Example 14. Non-GLP Studies of Antibody 493-004

This example is designed as a therapeutic, low-dose, early post-infection treatment study in hamsters infected with the Delta variant of the SARS-CoV-2 virus. The study has six groups of animals including a vehicle control and five treatment groups: single dose VHH antibody 202-03 at 1 and 5 mg/kg antibody administered by intraperitoneal injection, and the bispecific antibody 493-004 at 1 and 5 mg/kg administered by intraperitoneal injection, as well as 5 mg/kg administered nasally (50 μl). Two cohorts for each group of animals are assessed. Cohort A is monitored for weight and health Score for 4 days and terminated on day 4. Viral titer is determined in the lungs and trachea. Cohort B is monitored for weight and health score for 14 days (end of study). There are 5 time points for oral & nasopharyngeal swab and PFU determination in swabs. Additionally, the lungs from Cohort A and B terminal/animals are harvested at the end of the study for histopathology analysis with serum collected from Cohort B at timepoints: pre-challenge, +12h, +48h (2D), 72H (3D), +96H (4D), +144H (6D), Day 10, and Day 14.

Based on the data shown in FIG. 38, the bispecific antibody administered at 5 mg/kg by intraperitoneal injection and 1 mg/kg nasally, resulted in improved body weights in the animals starting 4-days after start of the challenge.

Another study is designed as a therapeutic, high-dose, late post-infection treatment study in both immunocompromised and non-immunocompromised Syrian hamsters infected with the Delta variant of the SARS-CoV-2 virus. The study has nine groups of animals including three control and six treatment groups: single dose bispecific antibody 493-004 administered at 10 mg/kg by intraperitoneal injection at days −1, +1, +2, +3, and +4 post-infection.

Based on the data shown in FIG. 39 and FIG. 40, the bispecific antibody appears to demonstrate a therapeutic response and animal weights increased after administration of the antibody on each of the days administered (e.g., day 1, 2, 3, or 4 post-infection).

Example 15. Human Clinical Studies with Antibody 493-004

This experiment described a first-in human combined Phase 1/2a, randomized, placebo-controlled clinical study.

FIG. 27A depicts a schematic design of a Phase 1 clinical trial in humans. The seven cohorts in the Phase 1 aspect of the study evaluate the safety and PK of the following doses and routes of administration for the bispecific monoclonal antibody: Cohort 1 is a single intravenous (IV) dose of 0.1 mg/kg anti-S1 mAb (n=3) or matching placebo (n=2); Cohort 2 is a single IV dose of 0.3 mg/kg anti-S1 mAb (n=3) or matching placebo (n=1); Cohort 3 is a single IV dose of 1 mg/kg anti-S1 mAb (n=3) or matching placebo (n=1); Cohort 4 is a single IV dose of 3 mg/kg anti-S1 mAb (n=6) or matching placebo (n=2); Cohort 5 is a single IV dose of 5 mg/kg anti-S1 mAb (n=6) or matching placebo (n=2); Cohort 6 is a single subcutaneous (SC) dose of 5 mg/kg anti-S1 mAb (n=8) or matching placebo (n=2); and Cohort 7 is a single SC dose of 7.5 mg/kg anti-S1 mAb (n=8) or matching placebo (n=2).

FIG. 27B depicts an schematic design of a Phase 2A clinical trial in humans. Dosing in the Phase 2A portion of the study includes only subcutaneous administration of the bispecific monoclonal antibody, in three cohorts: Cohort 1 is a single SC dose of 3 mg/kg anti-S1 mAb (n=30) or matching placebo (n=10); Cohort 2 is a single SC dose of 5 mg/kg anti-S1 mAb (n=30) or matching placebo (n=10); and Cohort 3 is a single SC dose of 7.5 mg/kg anti-S1 mAb (n=30) or matching placebo (n=10).

Example 16. Alanine Mutational Analysis

Alanine mutational analysis of the VHH antibodies 339-031 and 202-03 confirm that the two different VHHs target closely adjacent epitopes that share no critical contacts but are too close to bind simultaneously. Their collectively broader epitope coverage than either one of them individually compensate for one another and likely is responsible for the maintained binding and neutralization capabilities of the 493-004 bispecific against all known SARS-CoV-2 variants of concern (FIG. 44).

The yellow ribbon represents the RBD of a single spike protein from the SARS-CoV-2 virus; red modalities represent the critical contact points for the parent VHH antibody 202-03, and the specific amino acids are identified by their code and location; green modalities represent the critical contact points for the parent VHH antibody 339-031, and the specific amino acids are identified by their code and location.

Coupling together dual specificity and multi-valency gives the construct 493-004 a unique benefit over the use of traditional IgGs. Due to traditional IgG targeting of more than one specificity, the clinical therapeutic effects of bispecific antibodies are considered superior to those of monotherapies. Additionally, the construct's quadrivalent design allows for avidity-boosting effects beyond that of a traditional bivalent IgG format, such that binding is still retained even if one or both binding specificities reduce affinity for their target, in the context of an evolving mutational landscape for SARS-CoV-2. There are also significant structural differences between 493-004 and therapeutic IgG antibodies that may lead to not only acute advantages in neutralization but may also provide a cellular response and longevity of protection through the human IgG1 Fc effector function which is the scaffold to which the VHH antibodies of 493-004 are connected. It is plausible that a bispecific antibody constructs such as 493-004 may demonstrate greater resistance to ongoing mutational challenges from the SARS-CoV-2 virus and have greater longevity and offer a clinically meaningful therapeutic treatment for patients with COVID-19.

The assays are a triplicate 3-fold dilution series of the 493-004 antibody starting with 12,346 ng/mL and ending with 0.209 ng/mL (versus the initial series that assessed a range of 500,000 ng/mL to 2.8 ng/mL) to provide both greater definition and precision of the dose-response curves, as well as antibody concentrations exhibiting both 100% and 0% inhibition. The assays are for all three Omicron variant lineages BA.1, BA.2, and BA.3, as well as include the ancestral SARS-CoV-2 virus as a reference (preliminary results shown in FIG. 45). Calculations of the EC50 and EC90 values will also be determined for 493-004 against all three lineages.

Example 17. Cell Line Development

The Chinese Hamster Ovary (CHO) K1 cell line was used for generation of the 493-004 stable cell line. Expression plasmids were transfected into the CHO-K1 host cell line by electroporation. The transfected pools were cultured with selection pressure for two weeks. After pool recovery, the pools were used for cloning.

One round of fluorescence-activated cell sorting (FACS) couples with single cell imaging was used as the cloning method to obtain the production clonal cell line. From this, thirty clones were isolated and screened further in feed batch cultured in spin tubes, with fifteen clones emerging as promising. The highest clone titer from the fed-batch was 5.75 grams/Liter. Based on titer and growth profile, the top fifteen clones were selected for Size Exclusion Chromatography (SEC) analysis.

These top fifteen clones were subjected to fed-batch inoculation at a level of 0.4×106 cells/mL and feeding was in the range of 0-3% on days 3, 5, 7, 9, 11 and 14. Based on titer and SEC results, the top ten clones were selected for product quality attributes testing (clonality image quality, growth, and metabolic profiles and SEC-UPLC) and Ambr250 evaluation (FIG. 46A).

Clone screening was done by culturing each of the top ten clones in an Ambr250 bioreactor using a traditional fed batch process for screening and evaluation. Out of this, normally the top five clones are chosen for process optimization. Each top five clone is cultured 3× in Ambr250 specifically designed to reduce high molecular weight (HMW) species and separately in a 3 L bioreactor to evaluate process comparability between the 3 L bioreactor and Ambr250. The final clone is then chosen from this evaluation and produced also via traditional fed batch process in a 15 L bioreactor for process lock to mimic the future GMP run at 500 L being used to produce materials for clinical trials.

Evaluations for the clonal stages were as follows. The top five clones were selected based on cell culture, productivity and quality results of CLD spin tube study. The top two were selected based on cell culture, productivity and quality results of the Ambr250 clone screening. In addition, genetic stability was performed on these clones. For the final clone, the top priority was low levels of HMW species, with secondary criteria of promising productivity and good quality attributes.

Upon review of cell culture profiles of the top ten clones, the following attributes were evaluated: viable cell density over time, viability over time, lactate (g/mL) over time, and titer over time (starting at Day 10). From the top ten clones, clones 2495A-01-12 and 2495A-01-14 revealed lower lactate consumption rate compared to the other top clones. Clones 2495A-01-12 and 2495A-02-14 were ruled out as they showed insufficient productivity; all other clones reached promising productivity of greater than 4.0 g/L on harvest day.

When these five top clones were further subjected to quality parameter analysis as per the table above (FIG. 46C), only two clones proved suitable to take forward for screening: BV16-2495A-01-08 and BV21-2495A-02-08. These clones showed all-around performance in all categories, including favorable comparability to the 200 L batch generated for toxicology studies (FIG. 46B).

To make the final clone selection, the top two clones were each sub-cultured four times across two rounds of testing in Ambr250 and a 3 L bioreactor, varying temperatures, initial seed density and feeding schemes to optimize clonal performance. The clones were studied in the production medium and monitored for viability percentage. Key parameters evaluated included viable cell density (106 cells/mL) over time, viability (%) over time, lactate amount (g/L) over time and titer (g/L) over time (FIG. 46C). Clone 2495A-01-08 was chosen for creation of the Master Cell Bank (MCB).

Example 17. CyroEM Studies

In this experiment, CryoEM structure determination and epitope mapping was performed for SARS-CoV-2 S protein ectodomain in complex with bispecific antibody 493-004.

To prepare the target sample for analysis, 24 mg of lyophilized powder was dissolved in 260 μl of MilliQ water. After resting for 30 minutes at room temperature, the solution was transferred into a 200 μl dialysis button and covered by a dialysis membrane with 14 kDa cutoff. The solution was dialyzed at 4° C. overnight (17 hours) into PBS, pH 7.4 to remove the trehalose. To measure the concentration, the solution was transferred into an eppendorf tube and measured using NanoDrop with PBS as a reference. The solution was then immediately used for grid preparation.

To prepare the ligand for analysis, a stock solution stored at −80° C. was slowly thawed on ice and diluted sequentially first 7× into PBS pH 7.4, followed by 4.5× or 6× dilution also into PBS. 1 ul of either of these two diluted solutions was then used to prepare the spike/bsAb complex. A new aliquot was thawed before each preparation so that every sample used experienced at most one freeze/thaw cycle.

A cryogenic sample grid was made by taking the prepared spike target and bispecificAb solutions and mixing them to obtain a bispecificAb monomer to spike trimer ratio of 3:1 or 2:1, which was achieved by mixing 1 uL of bispecificAb solution with 9 uL of the spike solution. The mixed solution was incubated for 15 minutes at 4° C. and immediately vitrified in liquid ethane (FIG. 49).

Data collection was performed using a Titan Krios XFEG, 300 kV, Cs 2.7 mm, Gatan K3 DED microscope and movie properties were set at 5760×4092, 0.83 Å/pixel, 40 frames, 1.1e/Ų/frame. Collection mode was set to non-super resolution counting mode; compensated fringe free imaging in 3×3 or 5×5 beam shift pattern with 3 expositions per hole using custom serial EM scripts. The defocus range as −0.65 to −2.6 um.

Collected movies were subjected to a motion search algorithm and both motion-corrected and motion-corrected and dose-weighted micrographs were produced. Motion corrected-micrographs without dose-weighting were used for defocus estimation, while motion-corrected and dose-weighted micrographs were used for further processing (FIG. 50).

Particle picking was performed on denoised micrographs using deep learning-based approaches, selecting slightly over 7M potential particles. These potential particles were split into 71 sets of about 100 k particles each and each set was subjected to a “cleaning” 3D classification against a spike-only (i.e. without antibody) initial model created earlier in the screening phase of the project, leaving about 1.4M particles showing clear antibody density. These particles were then split into 6 sets, each about 233 k particles and each set was further subjected to two rounds of 2D classification (one standard, one suppressing low frequency CTF correction) to create a clean set of 588 k particles. A first unmasked consensus refinement was performed on this set, yielding a 3.5 Å map of the spike with strong densities for VHH in position 1 (VHH1) and VHH in position 2 (VHH2) and weak density for VHH in position 3 (VHH3). Following this initial refinement up with Bayesian polishing and per-particle defocus refinement improved the resolution to 3.2 Å. In further text this map is the “initial consensus map” (FIG. 51A).

Using the “initial consensus map”, a masked 3D classification to 2 classes with local searches was performed, where the mask encapsulated the locations of VHH1 and VHH2 and their respective RBDs. This classification separated remaining unbound spike particles (class 1) and particles with strong VHH1 and VHH2 densities (class 2). This class 2, containing 348 k particles, was then used for a masked 3D refinement with local searches, producing the 3.4 Å [M4.3] map used to build the majority of the VHH1 epitope/paratope (FIG. 51B). Since the density for VHH2 was still suboptimal, additional masked 3D refinement with local searches was performed but with mask specifically only around VHH2 and its corresponding RBD up location. This refinement produced the 3.3 Å [M4.5] map used to build the VHH2 epitope/paratope (FIG. 51D).

The VHH3 was clearly visible in the “initial consensus map” but too weak to interpret correctly. Thus the “initial consensus map” was used as a basis for no-align 3D classification to 6 classes. This 3D classification revealed 4 classes that represented either unbound, all RBD down spike or spike with very weak density at VHH position 3 and 2 classes with a stronger density around the VHH position 3. These 2 classes, comprising 274 k particles, were then combined and subjected to an unmasked 3D refinement that yielded the 3.3 Å [M4.1] map referred to as the “global consensus map”. Upon convergence, however, the density for the VHH3, was already misaligned due to the presence of the spike body. Indeed, 3.3 Å represents the resolution of the spike body, not the true resolution of the VHH3 part of the map. Most reliable fitting of the VHH3 density could be done using map from iteration 8 of this global consensus refinement, which yielded the 6 Å [M4.2] map used to assign the position of VHH3. Further attempts at improving the density of VHH3 using similar approaches as those used for VHH1 and VHH2 did not bring any improvement. The most likely reason being that while VHH1 and VHH2 are rigidly bound to their respective RBD domains, VHH3 appears to be only flexibly bound to its RBD domain and the mass of VHH3 itself is too small to refine properly on its own.

Finally, the “global consensus map” was used as a basis for multi-body refinement that encapsulated VHH1 and parts of its surrounding RBD domain and especially the N-term domain of the neighboring B chain as one body (with spike core being the second body). This multi-body refinement yielded the 3.7 Å [M4.4] map that resolved the N-term interface well and which was used to build the N-term B chain epitope of VHH in position 1 (FIG. 51C).

Initially, pdb:6×2b was used to rigid-body fit the map densities. Afterwards, all relevant residues were manually remodeled to correspond to the map density. The sequence of 6×2b was corrected to include all the amino acids present in the spike construct, which also aligned it such that the amino acids numbers correspond to the provided mutagenesis numbering. The model building then proceeded iteratively combining restrained molecular dynamics with manual intervention to build stereochemically valid models with best possible correspondence to the density.

A similar approach was adopted for building the VHH models, only here AlphaFold2 predictions of the N-term and C-term VHH domains of the bispecific construct were used as a starting point for the rigid body fitting and subsequent manual/molecular dynamics remodeling.

The structure reconstruction revealed densities for three out of the four VHHs present on the bispecific antibody, as well as a density corresponding to the constant fragment. The location or presence of the fourth VHH could not be confirmed (FIGS. 52A-52D).

Two of the revealed VHHs were confirmed to be the N-terminal VHHs and are bound to RBD down (position 1) and an RBD up domain (position 2). Identity of the third one could not be confirmed directly but stoichiometry of the bispecific antibody, connection with the constant fragment, and expected binding site all suggest it is the C-terminal VHH of the same bispecific antibody (FIGS. 52A-52D).

The two N-terminal VHHs are bound to RBDs in different positions. Their epitopes do overlap to a large extent but are not completely identical. Specifically (but not only) VHH in position 1 also interacts with neighboring chain B via the chain's N-terminal domain. This interaction is not present in VHH in position 2 epitope and the epitope in position 2 is limited solely to the respective RBD on chain B. The small change in the epitope/paratope between position 1 and 2 also suggests that the N-term VHH tolerates change/loss of several of its interface residues without losing capacity to bind (FIGS. 52A-52D).

The epitope of the third VHH bound to the second RBD up domain could not be determined in detail but the general position of the VHH with respect to the RBD suggests it is different from VHH1 and VHH2.

To determine the interacting epitope/paratope, three complementary strategies were used. In one strategy, the neighboring spike/VHH residues were manually inspected during model building; in the second approach residues were automatically verified using computational methods, which analyze residue interfacing based on solvent-accessible area, buried surface area, and solvation energy; and in the third one, residues were taken simply within 5 Å distance. The first two methods were used interchangeably, i.e. automatically determined residues were manually inspected for further undetected interactions and vice versa, manual residues were compared to the automatic list and if not present there, they were further examined in detail to confirm the interaction.

Additional results of the CryoEM experiments can be found in FIGS. 53-62.

While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. An antibody that binds to a region consisting of amino acids 380 to 430 of SARS-Cov-2 S receptor binding domain (RBD).

2. The antibody of claim 1, wherein the antibody binds one, two, three, or four residues of V382, S383, P384, or T430.

3. The antibody of claim 1, wherein the antibody binds to at least V382.

4. The antibody of claim 1, wherein the antibody binds to at least S383.

5. The antibody of claim 1, wherein the antibody binds to at least P384.

6. The antibody of claim 1, wherein the antibody binds to at least T430.

7. The antibody of claim 1, wherein the antibody binds to all residues of the following residues: V382, S383, P384, or T430.

8. The antibody of claim 1, wherein the antibody binds one or two residues K378 or P384.

9. The antibody of claim 1, wherein the antibody binds to at least K378.

10. The antibody of claim 1, wherein the antibody binds to at least P384.

11. (canceled)

12. An antibody that binds to a region consisting of amino acids 100 to 300 of SARS-Cov-2 S receptor binding domain (RBD).

13. The antibody of claim 12, wherein the antibody binds one, two, three, four, five, six, seven, or eight residues of R102, N125, F157, S172, F175, L176, R190, and Y265.

14-22. (canceled)

23. An antibody that binds to a region consisting of amino acids 400 to 500 of SARS-Cov-2 S receptor binding domain (RBD).

24. The antibody of claim 23, wherein the antibody binds to one, two, three, four, five, or six residues of K417, F456, G476, F486, N487, or Y489.

25-31. (canceled)

32. The antibody of claim 23, wherein the antibody binds to one, two, or three residues of N450, I472, or F490.

33-36. (canceled)

37. The antibody of claim 23, wherein the antibody binds to one, two, or three residues of L452, I468, or F490.

38-40. (canceled)

41. An antibody that binds to a region consisting of amino acids 300 to 600 of SARS-Cov-2 S receptor binding domain (RBD).

42. The antibody of claim 41, wherein the antibody binds to one, two, three, four, five, six, seven, eight, nine, or then residues of I326, R328, T531, N532, L533, F543, L552, S555, F559, or F562.

43-53. (canceled)

54. A bispecific antibody with at least 90% similarity to SEQ ID NO: 2670.

55. A method of treating SARS-CoV-2, the method comprising: a. administering an antibody to a subject wherein the antibody is at least 90% similar to SEQ ID NO: 2670.