CN117337301A

CN117337301A - Antibodies to

Info

Publication number: CN117337301A
Application number: CN202280025480.XA
Authority: CN
Inventors: G·斯克里顿; J·蒙哥沙巴亚
Original assignee: Ruiqiao Biotechnology Co ltd
Current assignee: Ruiqiao Biotechnology Co ltd
Priority date: 2021-02-04
Filing date: 2022-02-04
Publication date: 2024-01-02
Also published as: CN117715930A

Abstract

The present invention relates to antibodies capable of binding to the spike protein of coronavirus SARS-CoV-2, and methods and uses thereof in the prevention, treatment and/or diagnosis of coronavirus infection, diseases and/or complications associated with coronavirus infection, including COVID-19.

Description

Antibodies to

Technical Field

The present invention relates to antibodies useful in the prevention, treatment and/or diagnosis of coronavirus infection, diseases and/or complications associated with coronavirus infection, including covd-19.

Background

The pathogen SARS-CoV-2 of COVID-19 is a beta coronavirus, which is associated with SARS-CoV-1 and MERS coronavirus, both of which cause severe respiratory syndrome.

Coronaviruses have 4 structural proteins: nucleocapsid proteins, envelope proteins, membrane proteins, and spike (S) proteins. Spike proteins are the most important surface proteins. It has an elongated trimeric structure and is responsible for engaging the target cell and triggering fusion of the virus with the host membrane. Spike proteins from both SARS-CoV-2 and SARS-CoV-1 use angiotensin converting enzyme 2 (ACE 2) as a cell surface receptor. ACE2 is expressed in many tissues, including epithelial cells of the upper and lower respiratory tract.

The S protein consists of two subunits, S1, which mediate receptor binding, and S2, which is responsible for fusion of the viral and host cell membranes. It is a dynamic structure that can be converted to a post-fusion state by cleavage between S1 and S2 following receptor binding or trypsin treatment (Cai et al 2020). In some SARS-CoV-2 sequences, a furin cleavage site is inserted between the S1 and S2 subunits, and mutations in the cleavage site attenuate the disease in animal models (Johnson et al 2020). The S1 fragment occupies the membrane distal tip of S and can be subdivided into an N-terminal domain (NTD) and a Receptor Binding Domain (RBD). While both regions are immunogenic, RBD contains an interactive surface for ACE2 binding (Lan et al 2020). While the RBD is typically pressed down against the top of S2, it can swing up to engage ACE2 (Roy et al 2020). Monoclonal antibodies (mAbs) recognize one or both of an "up" and "down" conformation (Zhou et al, 2020; liu et al, 2020). The S protein is relatively conserved between SARS-CoV-2 and SARS-CoV-1 (76%), but the degree of conservation of RBD and NTD (74% and 50%, respectively) is lower than that of the S2 domain (90%) (Jaimes et al 2020). The degree of conservation for MERS-CoV and seasonal human coronaviruses is much lower (19-21%). Overall, the SARS-CoV-2 antibody shows limited cross-reactivity even with SARS-CoV-1 (Tian et al 2020).

The S protein has been intensively studied as a target for therapeutic antibodies. Previous studies on SARS-CoV-2 have shown that most potent antibodies bind near the ACE2 interacting surface on the Receptor Binding Domain (RBD) to block interactions with ACE2 expressed on target cells (Zost et al, 2020; liu et al, 2020) or disrupt pre-fusion conformations (Huo et al, 2020; yuan et al, 2020a; zhou et al, 2020). However, SARS-CoV-2 therapeutic antibodies have not been used clinically.

Variant b.1.1.7 now dominates the uk, with an increase in transmission rate. B.1.1.7 there are 9 amino acid changes in the spike, including N501Y in the ACE2 interacting surface. Irrelevant variants have been detected in South Africa (501y.v2, also known as b.1.351) and Brazil (p.1, 501y.v2), which have 10 and 12 amino acid changes in spike proteins, respectively. All of these contain mutations in the ACE2 receptor binding footprint of RBD, N501Y in b.1.1.7, K417N, E484K and N501Y in b.1.351, and K417T, E484K and N501Y in p.1, where the N501Y mutation is common to all. These mutations in the ACE2 receptor binding domain are believed to increase affinity for ACE2 (zahradadak et al 2021). These mutations also fall within the footprint of many potent neutralizing antibodies and several potentially therapeutic monoclonal antibodies that may provide vaccine-induced protection (Cheng et al, 2021; nelson et al, 2021), thus providing mutant viruses that are more suitable for infecting new hosts and that can evade pre-existing antibody responses.

SARS-CoV-2 assay kits using monoclonal antibodies have also been developed. Examples include lateral flow tests by, for example, innova (rapid qualitative test for SARS-CoV-2 antigen) and Quidel (Sofia 2SARS antigen FIA). However, these tests are reported to be very inaccurate.

It is an object of the present invention to identify further ameliorating antibodies useful in the prevention, treatment and/or diagnosis of coronavirus infections, diseases and/or complications associated with coronavirus infections including covd-19.

Disclosure of Invention

The inventors initially identified 42 human monoclonal antibodies (mabs) that recognized SARS-CoV-2 spike proteins (see table 1). These antibodies exhibit potent neutralizing activity against SARS-CoV-2, effectively blocking the interaction between spike protein and ACE2, and/or blocking high affinity binding to spike protein. Almost all highly potent neutralizing mabs (ICs) were found ₅₀ <0.1 μg/ml) will block the interaction with ACE2 but have a unique epitope that binds the N-terminal domain. Some of the antibodies in Table 1 show potent neutralization that is broadly effective against hCoV-19/Wuhan/WIV04/2019 strains as well as SARS-CoV-2 strains from various lineages, such as members of the B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.617 (Delta) and B.1.1.529 (Omicron) lineages.

Many mabs in table 1 use a common V gene (V gene common to most populations) and have few mutations relative to the germline. Several of the most potent inhibitory antibodies in table 1 were also found to bind unique epitopes compared to the antibodies previously described. Furthermore, N-glycosylation appears to increase antibody neutralization activity. The most potent mabs neutralized the virus in the low picomolar range and showed beneficial effects when administered either before or after infection in a murine model of covd-19, thus demonstrating prophylactic and therapeutic effects.

The inventors generated additional antibodies by exchanging the light and heavy chains of the antibodies of table 1. Antibodies derived from the same public V gene have been found to provide particularly useful mixed chain antibodies. For example, some of the resulting mixed chain antibodies exhibited potent neutralization that was broadly effective against the hCoV-19/Wuhan/WIV/2019 strain as well as SARS-CoV-2 strains from various lineages (such as B.1.1.7, B.1.351 and/or members of the P.1 lineages).

Accordingly, in one aspect the present invention provides an antibody capable of binding to the spike protein of coronavirus SARS-CoV-2, wherein the antibody: (a) At least three CDRs comprising any one of the 42 antibodies in table 1; and/or (b) binds to the same epitope as antibodies 159, 45 or 384 or competes with these antibodies.

The invention also provides an antibody combination comprising two or more antibodies according to the invention.

The invention also provides a polynucleotide encoding an antibody according to the invention, a vector comprising said polynucleotide or a host cell comprising said vector.

The invention also provides a method for producing an antibody capable of binding to the spike protein of coronavirus SARS-CoV-2, comprising culturing the host cell of the invention and isolating the antibody from said culturing.

The invention also provides a pharmaceutical composition comprising: (a) An antibody or combination of antibodies according to the invention, and (b) at least one pharmaceutically acceptable diluent or carrier.

The invention also provides an antibody, antibody combination or pharmaceutical composition according to the invention for use in a method of treatment of the human or animal body by therapy.

The invention also provides an antibody, antibody combination or pharmaceutical composition according to the invention for use in a method of treating or preventing a disease or complication associated with a coronavirus infection.

The invention also provides a method of treating a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody, antibody combination or pharmaceutical composition according to the invention.

The invention also provides the use of an antibody, antibody combination or pharmaceutical composition according to the invention in the manufacture of a medicament for treating a subject.

The invention also provides a method of identifying the presence of a coronavirus or a protein or protein fragment thereof in a sample, the method comprising: (i) Contacting the sample with an antibody or combination of antibodies according to the invention, and (ii) detecting the presence or absence of an antibody-antigen complex, wherein the presence of the antibody-antigen complex is indicative of the presence of coronavirus or a protein or protein fragment thereof in the sample.

The invention also provides a method of treating or preventing a coronavirus infection or a disease or complication associated therewith in a subject, the method comprising identifying the presence of a coronavirus according to the method of the invention and treating the subject with an antiviral or anti-inflammatory agent.

The invention also provides an antiviral or anti-inflammatory agent for use in a method of treating or preventing a coronavirus infection or a disease or complication associated therewith in a subject, wherein the method comprises identifying the presence of a coronavirus according to the method of the invention and treating the subject with a therapeutically effective amount of the antiviral or anti-inflammatory agent.

The invention also provides the use of an antibody, antibody combination or pharmaceutical composition according to the invention for the prevention, treatment and/or diagnosis of a coronavirus infection or a disease or complication associated therewith.

The invention also provides the use of an antibody, antibody combination or pharmaceutical composition according to the invention for identifying the presence of coronavirus or a protein or protein fragment thereof in a sample.

The invention also provides an antibody, combination or pharmaceutical composition of the invention for use in a method of preventing, treating or diagnosing a coronavirus infection caused by a SARS-CoV-2 strain, which SARS-CoV-2 strain comprises a substitution at positions 417, 484 and/or 501 in the spike protein relative to the spike protein of the hCoV-19/Wuhan/WIV/2019 strain, e.g. it is a member of lineage b.1.1.7, b.1.351 or p.1, or it is a member of lineage b.1.1.7, b.1.351, p.1 or b.1.1.529.

The invention also provides a method of preventing, treating or diagnosing a coronavirus infection by a strain of SARS-CoV-2 in a subject, wherein the method comprises administering to the subject an antibody, combination or pharmaceutical composition of the invention, wherein the strain of SARS-CoV-2 comprises a substitution at positions 417, 484 and/or 501 in the spike protein relative to the spike protein of the strain of hCoV-19/Wuhan/WIV04/2019, e.g., it is a member of lineage b.1.1.7, b.1.351 or p.1, or it is a member of lineage b.1.1.1.7, b.1.351, p.1 or b.1.1.529.

The invention also provides the use of an antibody, combination or pharmaceutical composition of the invention for the manufacture of a medicament for the prevention, treatment or diagnosis of a coronavirus infection caused by a SARS-CoV-2 strain comprising a substitution at positions 417, 484 and/or 501 in the spike protein relative to the spike protein of the hCoV-19/Wuhan/WIV04/2019 strain, e.g. it is a member of lineage b.1.1.7, b.1.351 or p.1, or it is a member of lineage b.1.1.7, b.1.351, p.1 or b.1.1.529.

Drawings

FIG. 1 shows characterization of monoclonal antibodies (mAbs) specific for SARS-CoV-2. (A) Cross-reactivity of 299 anti-spike (non-RBD) and 78 anti-RBD antibodies with trimeric spikes of human alpha and beta coronaviruses by capture ELISA. (B) Comparison of neutralizing efficacy (IC) between anti-spike (non-RBD) and anti-RBD antibodies against authentic SARS-CoV-2 using a reduced focal neutralization test (FRNT) ₅₀ ). Analysis was performed using the Mann-Whitney U test and double tail P values were calculated. (C) Correlation between SARS-CoV-2 neutralization of anti-RBD antibodies and RBD: ACE2 blockade. IC (integrated circuit) ₅₀ <Antibodies of 0.1. Mu.g/ml, 0.1-1. Mu.g/ml and 1-10. Mu.g/ml are highlighted in red, blue and orange, respectively. (D) With or without coating by RBD Ni-NTA beads deplete RBD-specific antibodies in plasma, and SARS-CoV-2 neutralization activity was then assessed by FRNT assay (n=8). Results are expressed as percent neutralization of the control without plasma. The percent of depleted neutralizing antibodies for each sample tested is indicated at the top of each panel.

Fig. 2 shows RBD anatomy and epitope definition based on mapping results. (A) The light gray RBD surface, depicted as a cartoon drawing with one single rainbow colored from blue (N-terminal) to red (C-terminal), alongside the gray surface depiction of RBD, labeled as corresponding to the adjacent Torso (Torso Gaddi, wikipedia, CC BY-SA 3.0, modified in Adobe Photoshop), is used BY analogy to effect definition of the epitope. (B) The cluster plots show the output of the mapping algorithm, with each dot corresponding to a "localized" antibody and color coded according to the epitope. (C) BLI antibody data from the cluster analysis competes with matrix (calculated) output showing clustering into 5 epitopes. (D) The attachment site of ACE2 (shown in purple) and RBD residues contacting ACE2 are shown green. (E) The mapping of the localization antibodies to RBD is shown as grey surface and ACE2 binding sites as green. Each antibody is depicted as a sphere and color coded as in (B), labeling the core antibodies herein. (F) As with (E), but the antibodies are color coded according to their ability to neutralize, see inset scale, red being the strongest neutralizing agent and blue being the weakest neutralizing agent.

FIG. 3 RBD complex. The Fab-RBD complexes reported herein are as determined by a combination of X-ray crystallography and cryo-EM, the depiction herein being based on the crystallography of the structure other than the complexes with Fab 40. Panel (a) shows a front view of the RBD surface shown in gray, panel (B) shows a rear view, and Fab is drawn as a cartoon, with heavy chains in red and light chains in blue. ACE2 footprint on RBD is green.

Fig. 4 spike morphology and Fab binding. (A) As an orthogonal view of the trimeric spikes of the light grey surface, one of the monomers is depicted as a cartoon and a rainbow colored from N-terminal to C-terminal (blue to red). (B) Surface depiction of the electron potential map of spike-mAb 159 complex by cryo-EM with resolution ofThe highlighting is shown tilted forward and colored in cyan except for RBD (gray), and the fragment of mAb 159 that can appear is shown orange. (C) The grey surface depiction of the RBD with blue spheres represents the position of Fab 45 as predicted using the mapping algorithm reported herein. (D) Gray surface depiction of the X-ray crystallographic structure of the observed RBD-Fab 45 complex. Fab 45 binds close to the predicted position but translates slightly. S309 Fab (closest structure in the competition matrix on which the mapping algorithm is based) is shown superimposed. Both Fab are depicted as cartoon figures, with the heavy chain being magenta and the light chain being blue. (E) Orthogonal gray surface depiction of RBD with bound Fab 384 and Fab CV07-270 superimposed onto the complex. These fabs use the same heavy chain V gene, but bind differently. They are depicted as cartoon diagrams, in which the heavy and light chains of Fab 384 are magenta and blue, respectively, while the heavy and light chains of CV07-270 are light pink and light blue, respectively.

FIG. 5 determining factors of binding, CDR length (A). Fab 384 interactions: the left panel outlines the interacting CDRs of the heavy (magenta) and light (cyan) chains with RBD (grey surface). The interactions of the H3, H2, and L1 and L3 rings are shown in the adjacent panels. (B) Distribution of IGHV, IGKV and IGLV gene usage of anti-RBD antibodies. Neutralizing IC according to antibody ₅₀ The values group and color them. (C) The left panel outlines CDR interactions of Fab150 (magenta), 158 (cyan) and 269 (orange). Adjacent panels (upper) show a close-up of H3 loop interactions retaining each of these antibodies encoded in the same color, and lower panels show the interactions of the L3 loops and the sequence alignment of loops (150H 3 loop (SEQ ID NO: 157), 158H3 loop (SEQ ID NO: 167), 269H3 loop (SEQ ID NO: 277), 150L3 loop (SEQ ID NO: 160), 158L3 loop (SEQ ID NO: 170) and 269L3 loop (SEQ ID NO: 280)). (D) Rear and side views of the complex of Fab40 and RBD (grey surface), wherein Fab is drawn as a cartoon, with the heavy chain being magenta and the light chain being blue. Fab 158 (grey cartoon) is superimposed. Note that although Fab40 was used with IGVH3-66 public V gene, whereas 158 uses IGVH3-53, but they bind almost identically. (E) RBD is plotted as a magenta cartoon and Fab is similarly depicted as Fab 75-RBD complex, with heavy chain orange and light chain gray. The antibodies used IGHV3-30 and were not potent neutralizers. It can be seen that the only re-link contact is via the extended H3 loop.

FIG. 6 determinants of binding, light chain exchange and glycosylation. (A) Tables of sequences for mAb253 (heavy chain AA linkage: SEQ ID NO:428; light chain AA linkage: SEQ ID NO: 431), 55 (heavy chain AA linkage: SEQ ID NO:429; light chain AA linkage: SEQ ID NO: 432) and 165 (heavy chain AA linkage: SEQ ID NO:430; light chain AA linkage: SEQ ID NO: 432). (B) The neutralizing activity of original mAb253, chimeric mAb253H55L and chimeric 253H165L on authentic SARS-CoV-2 (presented as IC ₅₀ Values). Immunoglobulin heavy and light chain gene alleles are presented in the table. Data were from 3 independent experiments, with two replicate wells per experiment, and data are shown as mean ± s.e.m. (C) The chimeric Fab 253H55L (mAb 253 (IGVH 1-58, IGVK 3-20) heavy chain complexed with RBD (shown here as a hydrophobic surface) is combined with the light chain of mAb 55 (IGVH 1-58, IGVK 3-20), but contains the IGKJ1 region in contrast to IGKJ2 in mAb 253). Fab is plotted as a ribbon with the heavy chain being magenta and the light chain being blue. The 10-fold increase in neutralization titer of this Fab compared to 253 appears to be due to the stable hydrophobic interaction created by the single substitution of tryptophan for tyrosine. (D) CDRs with saccharides bound in RBD complexes, where Fab 88 (upper panel) saccharides bind N35 in the H1 loop, 316 (middle panel) saccharides bind N59 in the H2 loop, and 253 (lower panel) saccharides bind N102 in the H3 loop. Note that Phe 486 is marked with diamonds to correlate various orientations.

FIG. 7. Determining factors for binding, RBD conformation, and potency of interaction. (A) cryo-EM spike-Fab complexes showing different RBD conformations. The density of spikes is shown as cyan, RBD as gray, and Fab as orange. The left "all RBD down" conformation has a bound Fab 316, the middle "one RBD up" conformation has a bound Fab158, and the right "all RBD up" conformation has 3 bound fabs 88. (B) Potent neutralizing Fab 159(cartoon representation with red heavy and blue light chain) is complexed with NTD (grey transparent surface) and the neighbors are depicted with another NTD binding Fab (4A 8) superimposed in grey bands, binding site spacing(C) Fab 159 (HC magenta, LC blue) is plotted as a cartoon in binding position on top of the NTD of the spike, which is plotted as a grey surface and viewed from the top (modeling intact IgG onto one monomer, indicating that it cannot cross over for bivalent binding). (D) ELISA binding (blue) and FRNT neutralization (red) curves for ten full-length antibodies against SARS-CoV-2 (solid line) and the corresponding Fab molecules (dashed line). Data from 2 independent experiments (mean ± s.e.m.)

Fig. 8. In vivo study. The neutralizing antibodies protect K18-hACE2 transgenic mice from SARS-CoV-2 infection. A-G. seven to eight week old male and female K18-hACE2 transgenic mice were vaccinated with 103 PFU SARS-CoV-2 by intranasal route. Mice were given a single 250 μg (10 mg/kg) dose of the indicated mAb by intraperitoneal injection 1 day (dpi) after infection. Weight change (mean ± SEM; n=5-10, two independent experiments: two-way ANOVA with Sidak post-test: ns, not significant, < P0.05, < P0.01, < P0.0001; compared to isotype control mAb treated group). The tissues were harvested 7 days post infection (dpi) and viral loads were determined in lung (B-C), heart (D), spleen (E), nasal wash (F) and brain (G) by plaque (B) or RT-qPCR (C-G) (n=7-11 mice per group; kruskal-Wallis test vs Dunn post test: ns, not significant, P <0.05, P <0.01, P <0.001, P < 0.0001). The dashed line indicates the detection limit.

FIG. 9 SARS-CoV-2 elicits binding and neutralizing antibodies against trimeric spike, RBD and NP proteins. (A) Plasma from donors diagnosed with SARS-CoV-2 infection was collected 1-2 months after onset of symptoms and tested for binding to SARS-CoV-2 spike, RBD and N proteins by capture ELISA. (B) neutralization titer against live, authentic virus. The data represent one experiment with 42 samples and are presented as mean ± s.e.m. (C) Comparison of the frequency of B cells expressing spike-reactive IgG in mild and severe cases as measured by FACS. The small horizontal lines represent the median. Data represent one experiment performed with 16 samples. Analysis was performed using the Mann-Whitney U test and two-tailed P values (in B and C) were calculated.

FIG. 10 SARS-CoV-2 antibody isolation strategy. Two different strategies were used to generate human monoclonal antibodies from memory B cells. (A) IgG-expressing B cells were isolated and cultured with IL-2, IL-21 and 3T3-msCD40L cells for 13-14 days. Supernatants were harvested and tested for responsiveness to spike proteins by ELISA. (B) Antigen-specific single B cells were isolated using labeled recombinant spike or RBD proteins as baits. IgG heavy and light chain variable genes from both strategies were amplified by nested PCR and cloned into expression vectors to generate full length IgG1 antibodies.

FIG. 11.377 specificity and sequence analysis of human antibodies. (A) Epitope mapping of SARS-CoV-2 specific antibodies against RBD, S1 subunit (aa 16-685) and S2 subunit (aa 686-1213) was assessed by ELISA and NTD binding agents were identified by cell-based fluorescent immunoassay. Antibodies that do not interact with any subdomain are defined as trimeric spikes. Numbers in the center indicate the total number of antibodies tested. (B) Frequency of amino acid substitutions from germline in SARS-CoV2 specific heavy and light chains (n=377). (C) Library analysis of anti-S (non-RBD) and anti-RBD antibodies heavy and light chain antibodies. Center is the number of antibodies. Each block represents a different clone and is proportional to the clone size. (D) Frequency of amino acid substitutions from germline in heavy and light chains of antibodies cross-reactive between SARS-CoV-2 and 4 seasonal coronaviruses (n=20).

Fig. 12. Crystal structure of ternary complex. (A) RBD-88-45, (B) RBD-253-75, (C) RBD-253H55L-75, and (D) RBD-384-S309 complex.

FIG. 13 resolution and image quality of cryo-EM data at RBD-Fab/IgG interface. (a-K) [ left ] the gold standard FSC curve (fsc=0.143 labeled) generated by crysparc for fab (or IgG in the case of 159) -spike structure, [ right ] shows the quality of the map at the antigen/antibody interface, 40, 88, 150, 158, 316, 384, 255H 55L RBD up, 255H 55L RBD down, 253H165L, 159RBD down, 159RBD up, respectively.

Fig. 14. Overrepresentation of binding patterns. (A) The sequence alignment of HC CDR3 using public V regions 3-53, antibodies were represented by numbers (from this study) or by PBD codes and designations (antibody 150:SEQ ID NO:433,CV30 (6 XE 1): SEQ ID NO:434, B38 (7 bZ 5): SEQ ID NO:435, p2C-1f11 (7 CDI): SEQ ID NO:436, BD604 (7 CH 4): SEQ ID NO:437, BD236 (7 CHB): SEQ ID NO:438, antibody 158:SEQ ID NO:439,COVA2-04 (7 JMO): SEQ ID NO:440, CC12.1 (6 XC 3): SEQ ID NO:441, antibody 175:SEQ ID NO:442,CC12.3 (6 XC 4): SEQ ID NO:443, BD629 (7 CHC): SEQ ID NO:444, CB6 (7C 01): SEQ ID NO:445,7CJF:SEQ ID NO:446, antibody 222:SEQ ID NO:447, antibody 269:SEQ ID NO:448). (B) Comparison of binding patterns of 150 (orange), 158 (cyan), 269 (magenta). (C) Superposition of RBD-Fab complexes available in PDB (by 10 months 21 in 2020). RBD is shown as gray surface, fab as ca trace, heavy chain as warm color, light chain as cool color. (D) Depending on the binding pattern of the bound Fab on RBD, they can be divided into four main clusters, neck (B38 (7 bZ 5), CB6 (7C 01), CV30 (6 XE 1), CC12.3 (6 XC 4), CC12.1 (6 XC 3), COV2-04 (7 JMO), BD629 (7 CHC), BD604 (7 CH 4), BD236 (7 CHB)), left shoulder (p 2B-2f6 (7 BWJ), BD368 (7 CHC), C07-270 (6 XKP)), left side (EY 6A (6 ZCZ), CR3022 (6 YLA), S304 (7 JX 3), COVA1-16 (7 JMW)) and right side (S309 (7 JX 3)). (E) Outliers included right shoulder binders (REGN 10987 (6 XDG), COVA2-39 (7 JMP), CV07-250 (6 XKQ), S2H14 (7 JX 3)). One Fab in the neck cluster is plotted as red and blue surfaces to show the relative position of outliers.

FIG. 15 importance of antibody glycosylation. (A-C) the effect of mutations in the glycosylated Asn residues in the heavy chains of antibodies 88, 253 and 316, respectively. (D-F) A graph of electron density of |2Fo-Fc| outlined at 1.2σ shows glycans at glycosylation sites at N102 of N35 of 88 (D), N59 of 316 (E) and N253 (F). (G) CDR-H3 and glycans are in relative binding position and orientation between 316 (green) and 88 (orange) and between (H) 316 and 253 (cyan). RBD is shown as a gray surface.

The classification of the cryo-EM dataset showed spike heterogeneity of 384 and 158. (A) Gaussian filtered reconstructed volumes (transparent grey) with refined spike (from two clusters 384 after local variability analysis using crysparc). At very low profile levels, and with gaussian filtering, there is little evidence that there is one (right) or two (left) additional binding fab. (B) 159 the reconstituted volumes in RBD up (left) and down (right) positions are colored by spike chains (blue, green, purple) and IgG (orange). The RBD in the up position is indicated by a red arrow.

Figure 17 prevention with mabs 40 and 88 prevented weight loss and reduced viral load. A-g. male and female K18-hACE2 transgenic mice seven to eight weeks old were administered a single 250g dose of the indicated mAb by intraperitoneal injection. One day later, mice were vaccinated with 103 PFU SARS-CoV-2 by intranasal route. (A) Weight change (mean ± SEM; n=6, two independent experiments: two-way ANOVA with Sidak post-test: ns, not significant, <0.05, <0.0001, < isotype control mAb-treated group). B-g. tissues were harvested 7 days after infection (dpi) and viral loads were determined in lung (B-C), heart (D), spleen (E), nasal wash (F) and brain (G) by plaque assay (B) or RT-qPCR (C-G) (n=6 mice per group; kruskal-Wallis assay vs Dunn post assay: ns, not significant, P <0.05, P <0.01, P < 0.001). The dashed line indicates the detection limit.

Fig. 18.B.1.1.7 (Kent) variant spike protein. SARS-CoV-2 spike trimer is depicted as a grey surface with mutations highlighted in yellow-green or symbolically. RBD N501Y and NTD 144 and 69-70 deletions are highlighted with green stars and red triangles, respectively. On the left, the protomer is highlighted as a colored band within the transparent gray spike surface, illustrating its topology and labeling the key domains.

Fig. 19. ACE2 binding comparison and effect on ACE binding for the n501y mutation. (A) RBD "" torso "" analogy. RBD is represented as grey surface and ACE2 receptor binding sites are dark green. The binding sites of the antibody sets plotted in this study are represented by spheres colored from red (strong) to blue (non-neutralizing) according to their neutralization. The position of the b.1.1.7n501y mutation in RBD is highlighted in light green toward the right shoulder. (B) proximity of ACE2 to N501Y. RBD is depicted as (a), with bound ACE2 (in yellow cartoon format), where glycosylation is plotted as a bar. (C) left panels: the interaction of N501 of WT RBD with residues Y41 and K353 (Lan et al 2020). When 501 is mutated to tyrosine with the conformation seen in the N501Y RBD-269Fab complex (right panel), Y501 undergoes T-ring stacking interactions with Y41 and more hydrophobic contacts with K353 of ACE2 (note that there is less conflict in the side chain of Y501 with the end of the K353 side chain, which has sufficient room to adjust to optimize the interactions). (D) BLI plot of WT (left) and N501Y (right) RBD combined with ACE 2. A series of titrations for each is shown (see methods). The dissociation rate of the mutant was noted to be much slower.

FIG. 20 mAb binds to WT and N501Y RBD. (A) Structural coverage of RBD-Fab complex in which Fab is in direct contact with N501. The overlay is performed by superimposing the RBDs. The structure of 38 antibody fabs complexed with RBD was analyzed. 18 are in direct contact with N501 (left side), including 14 IGHV3-53, 2 IGHV3-66, and two others. 20 Fab's were not in direct contact with N501 of RBD (right side), these included 3 IGHV3-53 or IGHV3-66 Fab (Table 13). RBD is shown as a gray surface with residue N501 highlighted in magenta. The C alpha backbone of Fab is depicted as a thin rod. (B) Examples of optimized binding of antibodies B38 and 158 to asparagine 501 side chain. (C) BLI results selected from a set of potent binders comparing antibodies to 501Y RBD and 501N RBD. (D) left pair: the BLI data is mapped onto the RBDs using the methods described herein. The front and rear views of the RBD are depicted as (A), but the spheres represent the antibody binding sites colored according to the ratio (KD 501Y/KD 501N). For white, the ratio is 1, for red the ratio <0.1 (i.e., reduced by at least a factor of 10). Right pair: as with the left pair, but colored according to the ratio of neutralization titers (IC 50501Y/IC 50501N). For white, the ratio is 1, for red the ratio <0.01 (i.e., reduced by at least a factor of 100). A strong agreement was noted between the two effects, of which 269 was the strongest one. The nearby pink antibodies were mainly IGHV3053 and IGHV3-66 antibodies.

FIG. 21 molecular mechanisms and comparisons of escape of N501Y RBD/269Fab and RBD/scFv269 complexes. (A) A set of V3-53 Fab positions CDR-L1 (slim rods) relative to N501 (surface, where N501 is highlighted in green) of RBD. (B) The side chain of N501 is widely contacted with residues from CDR-L1 in the RBD-158Fab complex (left). In the right panel, N501 did not make any contact with p2c-2f11 Fab (whose LC is most similar in sequence and has the same CDR-L1, L2 and L3 lengths as mAb 222 shown by a group of 222 LCs for PDB). The orientation and position of Y501 in the N501Y RBD-269Fab complex is shown in the two panels by overlapping RBDs. (C) the crystallographic structure of N501Y RBD/Fab 269. By superimposing the RBD of the two complexes, the Cα of N501Y RBD/Fab269 (blue) overlaps with the RBD/scFv269 (salmon red). (D) Structural changes in the 496-501 loop of RBD and the CDR-L1 loop contacting the mutation site. (E) structural differences in CDR-L3 loops between the two complexes.

FIG. 22 neutralization of SARS-CoV-2 strain Victoria and B.1.1.7 strain by mAb. (A) Neutralization curves for potent (FRNT 50<100 ng/ml) anti-RBD antibodies, including those expressing the common heavy chain VH 3-53. (B) a regenerator (regen) antibody REGN10933; REGN10987 and the Absiikan (AstraZeneca) antibodies AZD8895 and AZD7442 (AZD 1061 plus AZD 8895) were included for comparison. Neutralization of SARS-CoV-2 was measured using the Focus Reduction Neutralization Test (FRNT).

FIG. 23 neutralizing activity of convalescent plasma and vaccine serum. (A) The neutralization titers of 34 convalescent plasma collected 4-9 weeks after infection are shown with WHONBSC 20/130 reference serum. (B) Neutralizing titers of serum from volunteers vaccinated with the aslicon vaccine ADZ1222, samples were collected (i) 14 days after the second dose (n=10) and (ii) 28 days after the second dose (n=15). (C) Neutralization titers (n=25) of serum collected from volunteer caregivers enrolled after vaccination with Pfizer-BioNTech) BNT162b 2. Neutralization was measured by FRNT, analyzed using the Mann-Whitney U test, and two-tailed P values were calculated, with the average indicated above each column.

FIG. 24 neutralizing activity of serum collected from patients infected with B.1.1.7. (A) Neutralization titers in plasma from 13 patients infected with b.1.1.7 at different time points post infection. Days after infection are indicated in each panel. Neutralization was measured by FRNT. (B) FRNT50 titers of individual sera against Victoria and b.1.1.7 strains were compared, the numbers above each column being the average, analyzed using the Mann-Whitney U test, and the two-tailed P-value calculated.

FIG. 25 British contains the sequence of N5-1Y. (A) The proportion of the three subgroups of B.1.1.7 is expressed as a percentage of the total identifiable sequence containing 501Y. The black line shows the dominant form of 501Y and Δ69-70. Both blue and orange lines lack 69-70 and have either the wild-type or S982A mutation, respectively. (B) Relevant mutations for blue (left), orange (middle) and black (right) are plotted on the spike protein structure in which modeling was performed, with the modeled N-terminus (PDB encoding 6 ZWV) extended.

FIG. 26 electron density map of residue 501. The electron density map of residue 501 is refined to tyrosine in (a) and asparagine in (B). The 2Fo-Fc plot is outlined at 1.2σ and is colored in blue in both panels. (A) The negative density (red) in (a) is outlined at-3 sigma, and the positive density (green) in (B) is outlined at 3 sigma.

Fig. 27 evolution of variant b.1.351: (A-B) a sliding 7 day window for the ratio of (A) selected sequences containing UK, NTD deletions 69-70 and (B) South Africa, NTD deletions 241-243, depicted with wild type (grey), 501Y mutation only (green), NTD deletion only (purple) and double mutant variants (black). (C) A block diagram showing the distribution of mutations in the South African variant sequences as defined by 501Y and deletions 241-243. The structure map uses the spike protein structure in which modeling was performed (from the original framework of PDB code 6 ZWV), and the model is extended in Coot for the missing loop. (D) The location of the major changes in spike protein are highlighted in NTD and RBD, (E) the location of the K417N, E484K and N501Y (yellow) mutations within the ACE2 interaction surface of RBD (dark green).

FIG. 28 neutralization of Victoria and B.1.351 viruses by convalescent plasma. Plasma was collected in the uk prior to month 6 of 2020, during the first wave SARS-CoV-2, an early recovery period of 4-9 weeks after admission. (A) The neutralized FRNT assay (n=34) comparing Victoria (orange) and b.1.351 (green). (B) Victoria and b.1 were performed using plasma obtained from patients infected with b.1.1.7 at the indicated times after infection. 351 neutralization assay. (C-D) comparison of recovery period and FRNT in B.1.1.7 plasma between B.1.351 and Victoria strains, respectively ₅₀ Titers were analyzed using the Wilcoxon paired symbol rank test and double tail P values were calculated, with geometric mean indicated above each column. Each FRNT ₅₀ The values are shown in table 14.

Fig. 29 neutralization of b.1.351 by vaccine serum. Neutralization FRNT curves for Victoria and b.1.351 strains were as follows: (A) 25 parts of serum were collected 7-17 days after the second dose of the gabexate enniaceae vaccine. (B) 25 parts of serum were collected 14 days or 28 days after the second dose of Oxford-AstraZeneca (Oxford-AstraZeneca) vaccine. (C-D) comparison of FRNT50 titers between B.1.351 and Victoria strains for the Bulbophyceae and oxford-Alaskan vaccines, respectively, were analyzed using the Wilcoxon paired symbol rank test and double tail P values were calculated, with the geometric mean indicated above each column. The individual FRNT50 values are shown in table 15.

FIG. 30 neutralization of potent monoclonal antibodies. (A) Neutralization curves of Victoria and b.1.351 obtained using 22 human monoclonal antibodies. (B) Neutralization curves for the resulting Victoria and b.1.351 strains using monoclonal antibodies from the regenerator and the aslicon. The individual FRNT50 values are shown in table 16.

FIG. 31 interaction of mutation site residues with selected RBD binding mAbs. (a) interaction of Fab88 with K417 and E484 of RBD (PDB ID 7 BEL), (B) interaction of 150 with N501 and K417 (PDB ID 7 BEI), (C) 253 is not in contact with any of the three mutation sites (PDB ID 7 BEN), and (D) interaction of Fab 384 with E484 only (PDB ID 7 BEP). (E) IGHV3-51 and IGHV3-66 Fab structures obtained by overlapping the C.alpha.backbone of RBD. (F) Interaction of K417 with CB6 Fab (PDB ID 7C01 (Wajnberg et al 2020)). The (G) K417N mutation was modeled in the RBD/CB6 complex. In (A) to (G), the Fab light chain, heavy chain and RBD are blue, salmon red and grey, respectively. The cα backbone is depicted as thinner rods and the side chains are depicted as thicker rods, respectively. Contact withShown as yellow dashed lines, hydrogen bonds and salt bridges are shown as blue dashed lines. (H) B.1.351 changesMutations and deletions in the body's spike NTD relative to the positions of the bound antibodies 159 (PDB ID 7 NDC) and (I) 4A8 (PDB ID 7C 2L), the 242-244 deletion was predicted to disrupt 159 and 4A4 interactions. VH and VL domains of Fab are shown as salmon red and blue surfaces, respectively, NTD as gray bars. Mutation sites were plotted as green spheres and deletions as magenta spheres.

FIG. 32 antibody RBD interactions and structural modeling. BLI plots show a titration series (see methods) of (a) Wuhan RBD and (B) K417N, E484K, N y.1.351 RBD binding to ACE 2. Note that the dissociation rate of b.1.351 is much slower. (C and D) the RBD/mAb interactions of WT Wuhan RBD (left dot) and K417N, E484K, N Y501 B.1.351 RBD (right dot) measured by BLI. (E) Epitopes as defined by clusters of mabs on RBD (grey). (F) The BLI data is mapped onto the RBDs using the methods described herein. The front and rear views of the RBD are depicted as spheres representing antibody binding sites colored according to the ratio (kdb.1.351/KDWuhan). For white, the ratio is 1, for red the ratio <0.1 (i.e., reduced by at least a factor of 10). Black dots refer to mapped antibodies not included in the assay, dark green refers to RBD ACE2 binding surface, yellow refers to mutation K417N, E484K, N501Y. (G) As with the left pair, but colored according to the ratio of neutralization titers (ic50 b.1.351/IC50 Victoria), the ratio is 1 for white and <0.01 for red (i.e., reduced by a factor of at least 100). A strong agreement was noted between the two effects, of which 269 was the strongest one. The nearby pink antibodies were mainly IGHV3-53 and IGHV3-66 antibodies.

FIG. 33. Mutation case of P.1. The schematic shows the positions of amino acid substitutions in (A) P.1, (B) B.1.351 and (C) B.1.1.7 relative to the Wuhan SARS-CoV-2 sequence. Under the structural cartoon is a linear representation of S, on which the changes are marked. When there is a change in charge introduced by the mutation, the change is colored (red if the change makes the mutant more acidic/less basic, and blue if the basic/less acidic). (D) RBD is depicted as a grey surface with three positions of mutations K417T, E484K and N501Y (magenta), ACE2 binding surface of RBD is green. (E) The position of the N-linked glycans (red spheres) on the spike trimer is shown as a light blue surface representation, with two new sequence sub-markers found in p.1 being blue.

FIG. 34. Comparison of WT RBD/ACE2 and P.1 RBD/ACE2 complexes. (A) P.1 RBD/ACE2 (gray and salmon red) was compared with WT RBD/ACE2 (blue and cyan) by overlapping RBDs (PDB ID 6 LZG). Mutations in the P.1 RBD appear as rods. (B) An open book view of the electrostatic surface of (C) WT RBD/ACE2 complex, and an open book view of the electrostatic surface of (C), (D) p.1 RBD/ACE2 complex. Note the charge difference between WT and mutant RBD. The charge range shown is + -5 kJ/mol. (E) K417 of WT RBD forms a salt bridge with D30 of ACE 2. Effects of (F) and (G) E484K mutations on electrostatic surfaces. (H) Y501 of the p.1 RBD interacts with Y41 of ACE2 in a stacked manner. (I) The KD of RBD/mAb interactions of RBD measured by BLI for Victoria, b.1.1.7, p.1 and b.1.351 (left to right). (J) BLI data mapped onto RBDs using said method. A front view and a rear view of the RBD are shown. In the left hand pair, spheres represent antibody binding sites coloured according to the ratio (KDP.1/KWuuhan). For white, the ratio is 1, for red the ratio <0.1 (i.e., reduced by at least a factor of 10). Black dots refer to mapped antibodies not included in the assay, dark green refers to RBD ACE2 binding surface, yellow refers to mutation K417T, E484K, N501Y. For the right pair, the atoms were colored according to the ratio of neutralization titers (ic50 b.1.351/IC50 Victoria), for white, the ratio was 1, for red, the ratio <0.01 (i.e., reduced by at least a factor of 100). A strong agreement between KD and IC50 was noted. 269 is very strongly affected and approaches IGHV3-53 and IGHV3-66 antibodies (e.g., 222).

FIG. 35 neutralization of P.1 by monoclonal antibodies. (A) neutralization of P.1 by a panel of 20 potent human monoclonal antibodies. The curves for p.1 were superimposed on the curves for Victoria, b.1.1.7 and b.1.351 by neutralization of the FRNT measurements. FRNT50 titers are reported in table 18. Neutralization curves of monoclonal antibodies at different stages of development for commercial use. (B) Equivalent graphs of Vanilla (Vir), regenerator, aspirin, gift (Lilly) and Adagoo (Adagio) antibody therapeutic antibodies are shown.

FIG. 36 Structure of Fab 222 complexed with P.1 RBD. (A) The bands of the Fab 159/NTD complex are depicted with the P1 mutation in NTD highlighted as a cyan sphere. (B) The front and rear surfaces of the RBD that bind to typical VH 3-53. The P1 mutation in RBD is highlighted in dark green and marked. In this group, monoclonal antibody 222 has a slightly longer CDR3. Also shown are the sequences of the VH3-53 CDRs 1-3 heavy and light chains (150 CDR-H1 (SEQ ID NO: 449), 150 CDR-H2 (SEQ ID NO: 450), 150 CDR-H3 (SEQ ID NO: 451), 150 CDR-L1 (SEQ ID NO: 464), 150 CDR-L3 (SEQ ID NO: 465), 158 CDR-H1 (SEQ ID NO: 452), 158 CDR-H2 (SEQ ID NO: 453), 158 CDR-H3 (SEQ ID NO: 454), 158 CDR-L1 (SEQ ID NO: 466), 158 CDR-L3 (SEQ ID NO: 467), 222 CDR-H1 (SEQ ID NO: 455), 222 CDR-H2 (SEQ ID NO: 456), 222 CDR-H3 (SEQ ID NO: 457), 222 CDR-L1 (SEQ ID NO: 468), 222 CDR-L3 (SEQ ID NO: 469), 269 CDR-H1 (SEQ ID NO: 458), 269 CDR-H2 (SEQ ID NO: 460), 158 CDR-L3 (SEQ ID NO: 460), 158 CDR-L1 (SEQ ID NO: 463), 158 CDR-L1 (SEQ ID NO: 175), 222 CDR-H2 (SEQ ID NO: 463), and 175. (C) Crystal structures of P1 RBD, 222Fab and EY6A Fab (Zhou et al 2020). (D) The close-up of 222 CDRs interacting with RBD (grey) mutations is highlighted in yellow on the green ACE2 interface. (E) interaction of K417 with Fab 222. (F) interaction of N501 with Fab 222. (G), (H) Fab 222 chimeric model.

FIG. 37 neutralization curves of VH3-53 chimeric antibodies. Neutralization curves for Victoria, b.1.1.7, b.1.351 and p.1. Left hand column; neutralization curves using natural antibodies 222, 150, 158, 175 and 269. A right hand column; neutralization curves of chimeric antibodies, combining the heavy chains of 150, 158, 175, and 269 with the light chain of 222, natural 222 was used as a control. FRNT50 titers are given in table 18.

Fig. 38 neutralization of p.1 by convalescent plasma. Plasma (n=34) was collected from volunteers 4-9 weeks after SARS-CoV-2 infection, all samples were collected before month 6 in 2020, thus representing infection before the appearance of uk b.1.1.7. (A) Neutralization of p.1 by FRNT measurements was compared to neutralization curves of Victoria, b.1.1.7 and b.1.351 we have previously generated. (B) Neutralization of p.1 from plasma collected from volunteers infected with b.1.1.7 as evidenced by sequencing or by diagnostic PCR determination of S gene deletion. Samples were taken at different times after infection. (C-D) comparing FRNT50 titers between Victoria and P.1, data including B.1.1.7 and B.1.351 were used for comparison, and analyzed using the Wilcoxon paired symbol rank test, and double tail P values were calculated, with geometric averages indicated above each column.

FIG. 39 neutralization of P.1 by vaccine serum. (A) The serum of the psilosis was collected 7-17 days after the second dose of the psilosis-bayesian vaccine (n=25). FRNT titration curves are shown with Victoria, b.1.1.7 and b.1.351 as comparisons. (B) The serum of the aliskiren vaccine was collected 14 days or 28 days after the second dose of oxford-aliskiren vaccine (n=25). (C-D) comparing FRNT50 titers of individual samples of the Victoria, B.1.1.7, B.1.351 and P.1, the Wilcoxon paired symbol rank test was used for analysis and double tail P values were calculated, with the geometric mean indicated above each column.

FIG. 40 depicts a sliding 7-day window containing the proportion of sequences of K417T.

Figure 41. BLI titration of ace2 from p.1 RBD attachment and detachment attached to tip.

FIG. 42 cross-reactivity of a panel of mAbs identified from recovered COVID-19 patients. Neutralization assays were performed with selected mabs against Victoria, alpha (N501Y), beta (K417N, E484K, N501Y), gamma (K417T, E484K, N501Y), delta (L452R, T478K) and Omicron (G339D, S371L, S373P, S375F, K417N, N440K, G446S, S N, T478K, E484A, Q493R, G S, Q498R, N501Y, Y H) live virus isolates. The titration curves are shown as FRNT50 values reported in table 26.

FIG. 43 (A) is a table defining mutations in spike proteins from different strains when compared to the Wuhan SARS-CoV-2 spike protein sequence. (B) Shows the IC of selected antibodies against a panel of pseudoviral constructs with mutations when compared to the Wuhan SARS-CoV-2 spike protein sequence in Table 28 ₅₀ Graph of curve.

Detailed Description

Antibodies of the invention

The antibodies of the invention specifically bind to the spike protein of SAR-CoV-2. In particular, it specifically binds to the S1 subunit of spike proteins, such as the Receptor Binding Domain (RBD) or the N-terminal domain (NTD).

The antibodies of the invention may comprise at least three CDRs of an antibody in table 1. Table 1 lists 42 individual antibodies identified from recovered COVID-19 patients. Table 1 also lists the heavy and light chain variable region nucleotide and amino acid sequences and the Complementarity Determining Regions (CDRs) of the variable chains of each of the antibodies. CDRs of the heavy Chain (CDRH) and light chain variable domain (CDRL) are located at residues 27-38 (CDR 1), residues 56-65 (CDR 2) and residues 105-117 (CDR 3) of each chain according to the IMGT numbering system (http:// www.imgt.org; lefranc MP,1997,J,Immunol.Today [ J.Immunol., today ],18,509). This numbering system is used throughout this specification unless otherwise indicated.

The antibody in table 1 may be any antibody selected from the group consisting of: 253H55L, 253H165L, 253, 222, 318, 55, 165, 384, 159, 88, 40, and 316. The antibody in table 1 may be any antibody selected from the group consisting of: 253H55L, 253H165L, 253, 222, 318, 55, and 165. The antibody in table 1 may be any antibody selected from the group consisting of: 384. 159, 253H55L, 253H165L, 253, 88, 40, and 316.

Antibodies 253H/55L, 253H/165L, 253, 222, 318, 55 and 165 are all highly potent neutralizing mAbs, which have been shown to neutralize Victoria, kent (B.1.1.7), south Africa (B.1.351) and Brazilian (P.1) strains without loss of potency.

The antibody in table 1 may be any antibody selected from the group consisting of: 40. 88, 159, 222, 281, 316, 384 and 398. These antibodies were found to have potent cross-lineage neutralization, e.g., they were effective against Victoria and b.1.1.7 strains (e.g., b.1.1.7: victoria ratio less than 2 and/or IC50 less than 0.1 μg/ml as shown in table 11).

The antibody in table 1 may be any antibody selected from the group consisting of: 40. 55, 58, 150, 165, 222, 253, 278, 318, 253H55L and 253H165L. These antibodies were found to have potent cross-lineage neutralization, e.g., they were effective against Victoria and b.1.351 strains (e.g., b.1.1.7: victoria ratio less than 3 and/or IC50 less than 0.1 μg/ml, see tables 11 and 16A).

The antibody in table 1 may be any antibody selected from the group consisting of: 222. 318, 253H55L and 253H165. These antibodies were found to have potent cross-lineage neutralization, e.g., they were effective against Victoria and B.1.351 strains (e.g., B.1.1.7: victoria ratio less than 3 and/or IC50 less than 0.1 μg/ml, see tables 11 and 16A) and bind spike proteins with high affinity, e.g., with KD.ltoreq.4 nM (see Table 16A).

The antibodies in table 1 may be 222 or 253H165L. These antibodies were found to have potent cross-lineage neutralization, e.g., they have an IC50 of 0.02 μg/ml or less against Victoria strain, B.1.1.7 strain, B.1.351 strain, and P.1 strain, and bind with high affinity to spike proteins (see Table 18).

The antibody in table 1 may be any antibody selected from the group consisting of: 58. 222, 253 and 253H/55L. These antibodies were found to neutralize omicron (B.1.1.529) strains with an IC50 of 5. Mu.g/ml or less. The antibodies in table 1 may be 58 or 222. These antibodies were found to strongly neutralize omicron strains at an IC50 of 0.25. Mu.g/ml or less.

253H55L antibody was produced from a combination of antibody 253 and antibody 55. These antibodies alone are not the most potent antibodies identified. However, once the heavy chain from antibody 253 and the light chain from antibody 55 are combined, the resulting antibody unexpectedly has improved neutralization and antigen binding. For example, 253H55L can minimize viral RNA levels in an in vivo model of SARS-CoV-2 infection. Thus, in a preferred embodiment, the antibody in table 1 is 253H55L.

Thus, an antibody of the invention may comprise at least three CDRs of antibody 253H 55L. In one embodiment, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs 265, 266 and 267, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs 68, 69 and 70, respectively.

Antibodies 253H/165L were identified in a manner similar to 253H 165L. Surprisingly 253H/165L was found to bind SARS-CoV-2 more strongly than either antibody 253 or antibody 165 alone (Table 3).

Thus, an antibody of the invention may comprise at least three CDRs of antibody 253H 165L. In one embodiment, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs 265, 266 and 267, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs 188, 189 and 190, respectively.

In addition, antibodies 253, 165 and 55 were identified to unexpectedly retain potent neutralization of SARS-CoV-2 variants b.1.1.7 and b.1.351.

Thus, an antibody of the invention may comprise at least three CDRs of antibody 253, 165 or 55. In one embodiment, an antibody may comprise the heavy chain CDRs of antibody 253, 165, or 55 and the light chain CDRs of antibody 253, 165, or 55. In one embodiment, the antibody may comprise a light chain CDR of a first antibody and a heavy chain CDR of a second antibody, wherein both antibodies are derived from the same public V region. In one embodiment, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs 265, 266 and 267, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs 268, 269 and 270, respectively. In one embodiment, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs 185, 186 and 187, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs 188, 189 and 190, respectively. In one embodiment, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs 65, 66 and 67, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs 68, 69 and 70, respectively.

It was surprisingly found that antibody 222 retained potent neutralization of the SARS-CoV-2 variant Victoria, B.1.1.7, B.1.351 and P.1 strains, e.g.IC 50 for Victoria, B.1.1.7, B.1.351 and P.1 strains was ∈0.02 μg/ml (see Table 18). Thus, in one embodiment, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs 255, 256 and 257, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs 258, 259 and 260, respectively.

Interestingly, antibodies comprising the light chain of antibody 222 exhibited potent cross-lineage neutralization, e.g., they were effective against all of the SARS-CoV-2 strains tested in the examples (as shown in table 18 and fig. 37). Such mixed chain antibodies are discussed further below. Thus, antibodies of the invention may comprise CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOS 258, 259 and 260, respectively. The antibody may further comprise CDRH1, CDRH2 and CDRH3 from antibodies derived from IGHV3-53 or IGHV3-66 (such as IGHV 3-53).

It was surprisingly found that antibody 318 retains strong neutralization of the SARS-CoV-2 variant Victoria, B.1.1.7 and B.1.351. Thus, in one embodiment, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs 335, 336 and 337, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs 338, 339 and 340, respectively.

Antibody 316 is one of the most potent neutralizing antibodies identified and was surprisingly found to retain strong neutralization of the b.1.1.7 strain. Thus, in one embodiment, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs 325, 326 and 32, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs 328, 329 and 330, respectively.

Antibodies 159, 384, 88, 40 and 253H55L are all highly potent neutralizing mabs that have been shown to be prophylactically or therapeutically protective in animal models.

Antibody 159 binds to the NTD of spike protein and does not block ACE2 binding, but is unexpectedly one of the strongest neutralizing antibodies observed. In contrast, prior art antibodies co-localized to NTD do not show significant neutralization in the assays used herein. Antibody 159 has also been shown to have beneficial properties in animal models. Thus, in a preferred embodiment, the antibody in table 1 is 159.

Thus, an antibody of the invention may comprise at least three CDRs of antibody 159. For example, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOS 175, 176 and 177, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOS 178, 179 and 180, respectively. In one embodiment, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs 175, 176 and 177, respectively.

Antibody 384 binds to a unique epitope of RBD and is different from all previously reported binding patterns and is the most potent neutralizing mAb described herein. The increased potency of antibody 384 when compared to other antibodies derived from the same v-region is believed to be due to the 18 residue long CDRH3, which forms an extended interaction across the ACE2 binding site of RBD. Antibodies 384 have also been shown to have beneficial properties in animal models. Thus, in a preferred embodiment, the antibodies in table 1 are 384.

Thus, an antibody of the invention may comprise at least three CDRs of antibody 384. For example, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOS 375, 376 and 377, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOS 378, 379 and 380, respectively.

In one embodiment, an antibody of the invention may comprise CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOS 376 and 377, respectively, and CDRL1 and CDRL3 shown in SEQ ID NOS 378 and 380, respectively.

Antibody 88 is one of the most potent neutralizing antibodies found herein. Antibody 99 contains an N-glycosylation site in CDRH1 that is not necessary for RBD binding but is necessary for neutralization. Antibodies 88 have also been shown to have beneficial properties in animal models. Thus, in a preferred embodiment, the antibody in table 1 is 88.

Thus, an antibody of the invention may comprise at least three CDRs of antibody 88. For example, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 105, 106 and 107, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 108, 109 and 110, respectively.

Antibody 40 comprises a heavy chain derived from the IGHV3-66v region and is one of the potent neutralizing agents identified herein. Antibody 40 has also been shown to have beneficial properties in animal models. Thus, in a preferred embodiment, the antibody in table 1 is 40.

Thus, an antibody of the invention may comprise at least three CDRs of antibody 40. For example, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs 25, 26 and 27, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs 28, 29 and 30, respectively.

Antibodies 58, 222, 253 and 253H/55L are mAbs that have been shown to neutralize omicron strains.

Antibody 58 is unexpectedly one of the most potent neutralizing antibodies observed against omacron strains. Thus, in a preferred embodiment, the antibody in table 1 is 58.

Thus, an antibody of the invention may comprise at least three CDRs of antibody 58. For example, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOS 75, 76 and 77, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOS 78, 79 and 80, respectively.

Antibody 222 is one of the most potent neutralizing antibodies observed against the omacron strain. Antibody 222 also strongly neutralized all other strains tested in table 26. Thus, in a preferred embodiment, the antibody in table 1 is 222.

Thus, an antibody of the invention may comprise at least three CDRs of antibody 222. For example, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs 255, 256 and 257, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs 258, 259 and 260, respectively.

Antibody 253 was found to neutralize omicron strains. Antibody 253 also strongly neutralized all other strains tested in table 26. Thus, in a preferred embodiment, the antibody in table 1 is 253.

Thus, an antibody of the invention may comprise at least three CDRs of antibody 253. For example, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOS 265, 266 and 267, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOS 268, 269 and 270, respectively.

Antibody 253H/55L was found to neutralize omicron strains. Antibody 253H/55L also strongly neutralized all other strains tested in Table 26. Thus, in a preferred embodiment, the antibodies in Table 1 are 253H/55L.

Thus, an antibody of the invention may comprise at least three CDRs of antibody 253h/55L. For example, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOS 265, 266 and 267, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOS 68, 69 and 70, respectively.

The antibodies of the invention may comprise at least four, five or all six CDRs of an antibody in table 1. The antibody may comprise at least one, at least two or all three heavy chain CDRs (CDRH). The antibody may comprise at least one, at least two or all three light chain CDRs (CDRL). The antibody typically comprises all six (i.e., three heavy and three light chain) CDRs.

The antibodies of the invention may comprise a heavy chain variable domain having 80% >, > 90% >, > 95% >, 96% >, > 97% >, > 98% >, 99% or 100% sequence identity to the heavy chain variable domain amino acid sequence of an antibody in table 1 (e.g., 253H/55L, 253H/165L, 222, 318, 165, 55, 159, 384, 88, 318 or 40).

In one embodiment, an antibody of the invention may comprise a heavy chain variable domain that has 80% > or 90% > or 95% > 96% > or 97% > 98% > 99% or 100% sequence identity to the heavy chain variable domain of antibody 150 (i.e., SEQ ID NO: 152).

In one embodiment, an antibody of the invention may comprise a heavy chain variable domain that has 80% > or 90% > or 95% > 96% > or 97% > 98% > 99% or 100% sequence identity to the heavy chain variable domain of antibody 158 (i.e., SEQ ID NO: 162).

In one embodiment, an antibody of the invention may comprise a heavy chain variable domain that has 80% > or 90% > 95% > 96% > 97% > 98% > 99% or 100% sequence identity to the heavy chain variable domain of antibody 253H55L (i.e., SEQ ID NO: 262).

In one embodiment, an antibody of the invention may comprise a heavy chain variable domain that has 80% >, > 90% >, > 95% >, > 96% >, > 97% >, 98% >, > 99%, or 100% sequence identity with the heavy chain variable domain of antibody 222 (i.e., SEQ ID NO: 252).

In one embodiment, an antibody of the invention may comprise a heavy chain variable domain that has 80% >, > 90% >, > 95% >, > 96% >, > 97% >, 98% >, > 99%, or 100% sequence identity with the heavy chain variable domain of antibody 58 (i.e., SEQ ID NO: 72). In one embodiment, an antibody of the invention may comprise a heavy chain variable domain that has 80% >, > 90% >, > 95% >, > 96% >, > 97% >, 98% >, > 99%, or 100% sequence identity with the heavy chain variable domain of antibody 318 (i.e., SEQ ID NO: 332).

In one embodiment, an antibody of the invention may comprise a heavy chain variable domain that has 80% >, > 90% >, > 95% >, > 96% >, > 97% >, 98% >, > 99%, or 100% sequence identity with the heavy chain variable domain of antibody 165 (i.e., SEQ ID NO: 182).

In one embodiment, an antibody of the invention may comprise a heavy chain variable domain that has 80% >, > 90% >, > 95% >, > 96% >, > 97% >, 98% >, > 99%, or 100% sequence identity with the heavy chain variable domain of antibody 55 (i.e., SEQ ID NO: 62).

In this embodiment, the antibodies of the invention may comprise a heavy chain variable domain that has 80%,. Gtoreq.90%,. Gtoreq.95%,. Gtoreq.96%,. Gtoreq.97%,. Gtoreq.98%,. Gtoreq.99% or 100% sequence identity to the heavy chain variable domain of antibody 159 (i.e., SEQ ID NO: 172).

In one embodiment, an antibody of the invention may comprise a heavy chain variable domain that has 80% > or 90% > or 95% > 96% > or 97% > 98% > 99% or 100% sequence identity to the heavy chain variable domain of antibody 384 (i.e., SEQ ID NO: 372).

In one embodiment, an antibody of the invention may comprise a heavy chain variable domain that has 80% >, > 90% >, > 95% >, > 96% >, > 97% >, 98% >, > 99%, or 100% sequence identity with the heavy chain variable domain of antibody 88 (i.e., SEQ ID NO: 102).

In one embodiment, an antibody of the invention may comprise a heavy chain variable domain that has 80% >, > 90% >, > 95% >, > 96% >, > 97% >, 98% >, > 99%, or 100% sequence identity with the heavy chain variable domain of antibody 40 (i.e., SEQ ID NO: 22).

In one embodiment, an antibody of the invention may comprise a heavy chain variable domain that has 80% > or 90% > or 95% > 96% > or 97% > 98% > 99% or 100% sequence identity to the heavy chain variable domain of antibody 316 (i.e., SEQ ID NO: 322).

The antibodies of the invention may comprise a light chain variable domain having ≡80%,. Gtoreq.90%,. Gtoreq.95%,. Gtoreq.96%,. Gtoreq.97%,. Gtoreq.98%,. Gtoreq.99% or 100% sequence identity to the light chain variable domain amino acid sequence of an antibody (e.g., 253H/55L, 253H/165L, 222, 318, 253, 165, 55, 159, 384, 88, 40 or 316) in table 1.

In one embodiment, an antibody of the invention may comprise a light chain variable domain that has 80% > or 90% > or 95% > 96% > or 97% > 98% > 99% or 100% sequence identity to the light chain variable domain of antibody 150 (i.e., SEQ ID NO: 154).

In one embodiment, an antibody of the invention may comprise a light chain variable domain that has 80% > or 90% > or 95% > 96% > or 97% > 98% > 99% or 100% sequence identity to the light chain variable domain of antibody 158 (i.e., SEQ ID NO: 164).

In one embodiment, an antibody of the invention may comprise a light chain variable domain that has 80% > or 90% > 95% > 96% > 97% > 98% > 99% or 100% sequence identity to the light chain variable domain of antibody 253H55L (i.e., SEQ ID NO: 64).

In one embodiment, an antibody of the invention may comprise a light chain variable domain that has 80% > or 90% > 95% > 96% > 97% > 98% > 99% or 100% sequence identity to the light chain variable domain of antibody 253H165L (i.e., SEQ ID NO: 184).

In one embodiment, an antibody of the invention may comprise a light chain variable domain that has 80% >, > 90% >, > 95% >, > 96% >, > 97% >, 98% >, > 99%, or 100% sequence identity with the light chain variable domain of antibody 222 (i.e., SEQ ID NO: 254).

In one embodiment, an antibody of the invention may comprise a light chain variable domain that has 80% > or 90% > 95% > 96% > 97% > 98% > 99% or 100% sequence identity to the light chain variable domain of antibody 253 (i.e., SEQ ID NO: 264).

In one embodiment, an antibody of the invention may comprise a light chain variable domain that has 80% >, > 90% >, > 95% >, > 96% >, > 97% >, 98% >, > 99%, or 100% sequence identity with the light chain variable domain of antibody 58 (i.e., SEQ ID NO: 74).

In one embodiment, an antibody of the invention may comprise a light chain variable domain that has 80% >, > 90% >, > 95% >, > 96% >, > 97% >, 98% >, > 99%, or 100% sequence identity with the light chain variable domain of antibody 318 (i.e., SEQ ID NO: 334).

In one embodiment, an antibody of the invention may comprise a light chain variable domain that has 80% > or 90% > 95% > 96% > 97% > 98% > 99% or 100% sequence identity to the light chain variable domain of antibody 159 (i.e., SEQ ID NO: 174).

In one embodiment, an antibody of the invention may comprise a light chain variable domain that has 80% > or 90% > or 95% > 96% > or 97% > 98% > 99% or 100% sequence identity to the light chain variable domain of antibody 384 (i.e., SEQ ID NO: 374).

In one embodiment, an antibody of the invention may comprise a light chain variable domain that has 80% >, > 90% >, > 95% >, > 96% >, > 97% >, 98% >, > 99%, or 100% sequence identity with the light chain variable domain of antibody 88 (i.e., SEQ ID NO: 104).

In one embodiment, an antibody of the invention may comprise a light chain variable domain that has 80% > or 90% > or 95% > 96% > or 97% > 98% > 99% or 100% sequence identity to the light chain variable domain of antibody 40 (i.e., SEQ ID NO: 24).

In one embodiment, an antibody of the invention may comprise a light chain variable domain that has 80% >, > 90% >, > 95% >, > 96% >, > 97% >, 98% >, > 99%, or 100% sequence identity with the light chain variable domain of antibody 316 (i.e., SEQ ID NO: 324).

The antibodies of the invention may comprise a heavy chain variable domain and a light chain variable domain having greater than or equal to 80%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99%, 100% sequence identity to the heavy chain variable domain amino acid sequences and light chain variable domain amino acid sequences of the antibodies in table 1 (e.g., 253H/55L, 253H/165L, 222, 318, 253, 165, 55, 159, 384, 88, 40, or 316), respectively.

In one embodiment, an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain having 80% >, > 90% >, 95% >, > 96% >, 97% >, 98% >, 99%, 100% sequence identity, respectively, to the heavy chain variable domain and the light chain variable domain of antibody 253H55L (SEQ ID NOS: 262 and 64, respectively).

In one embodiment, an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain having 80% >, > 90% >, 95% >, > 96% >, 97% >, 98% >, 99%, 100% sequence identity, respectively, to the heavy chain variable domain and the light chain variable domain of antibody 253H165L (SEQ ID NOS: 262 and 184, respectively).

In one embodiment, an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain having 80% >, > 90% >, 95% >, 96% >, 97% >, 98% >, 99%, 100% sequence identity, respectively, to the heavy chain variable domain and the light chain variable domain of antibody 222 (SEQ ID NOS: 252 and 254, respectively).

In one embodiment, an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain having 80% >, > 90% >, 95% >, 96% >, 97% >, 98% >, 99%, 100% sequence identity, respectively, to the heavy chain variable domain and the light chain variable domain of antibody 58 (SEQ ID NOS: 72 and 74), respectively.

In one embodiment, an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain having 80% >, > 90% >, 95% >, 96% >, 97% >, 98% >, 99%, 100% sequence identity, respectively, to the heavy chain variable domain and the light chain variable domain of antibody 253 (SEQ ID NOS: 262 and 264, respectively).

In one embodiment, an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain having 80% >, > 90% >, 95% >, 96% >, 97% >, 98% >, 99%, 100% sequence identity, respectively, to the heavy chain variable domain and the light chain variable domain of antibody 318 (SEQ ID NOS: 332 and 334, respectively).

In one embodiment, an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain having 80% >, > 90% >, 95% >, 96% >, 97% >, 98% >, 99%, 100% sequence identity, respectively, to the heavy chain variable domain and the light chain variable domain of antibody 165 (SEQ ID NOS: 182 and 184, respectively).

In one embodiment, an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain having 80% >, > 90% >, 95% >, 96% >, 97% >, 98% >, 99%, 100% sequence identity, respectively, to the heavy chain variable domain and the light chain variable domain of antibody 55 (SEQ ID NOS: 62 and 64, respectively).

In one embodiment, an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain having 80% >, > 90% >, 95% >, 96% >, 97% >, 98% >, 99%, 100% sequence identity, respectively, to the heavy chain variable domain and the light chain variable domain of antibody 159 (SEQ ID NOS: 172 and 174), respectively.

In one embodiment, an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain having 80% >, > 90% >, 95% >, 96% >, 97% >, 98% >, 99%, 100% sequence identity, respectively, to the heavy chain variable domain and the light chain variable domain of antibody 384 (SEQ ID NOS: 372 and 374), respectively.

In one embodiment, an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain having 80% >, > 90% >, 95% >, 96% >, 97% >, 98% >, 99%, 100% sequence identity, respectively, to the heavy chain variable domain and the light chain variable domain of antibody 88 (SEQ ID NOS: 102 and 104, respectively).

In one embodiment, an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain having 80% >, > 90% >, 95% >, 96% >, 97% >, 98% >, 99%, 100% sequence identity, respectively, to the heavy chain variable domain and the light chain variable domain of antibody 40 (SEQ ID NOS: 22 and 24), respectively.

In one embodiment, an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain having 80% >, > 90% >, 95% >, 96% >, 97% >, 98% >, 99%, 100% sequence identity, respectively, to the heavy chain variable domain and the light chain variable domain of antibody 316 (SEQ ID NOS: 322 and 324, respectively).

Alternatively, an antibody of the invention may comprise a light chain variable domain amino acid sequence from one antibody in table 1 and a heavy chain variable domain amino acid sequence from another antibody in table 1. Thus, an antibody of the invention may comprise: (a) A heavy chain variable domain having 80% > or more, 90% > or more, 95% > or more, 96% > or more, 97% > or more, 98% > or more, 99% or more, 100% sequence identity to the heavy chain variable domain of the first antibody in table 1; and (b) a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to the light chain variable domain of the second antibody in table 1.

In one embodiment, the first antibody in table 1 is 253 and the second antibody in table 1 is 55, resulting in antibody 253H55L. In another embodiment, the first antibody in table 1 is 253 and the second antibody in table 1 is 165, resulting in antibody 253H165L.

The primary and/or secondary antibodies in table 1 may be derived from a primary public v region. The first and second antibodies in table 1 may be derived from the same germline heavy chain v region. The heavy chain v region may be IGHV3-53, IGHV1-58, or IGHV3-66 (described further below).

For example, an antibody of the invention may comprise a heavy chain variable domain amino acid sequence having ≡80% ≡90% ≡95% >,. Gtoreq.96% >, ≡97%. Gtoreq.98% >, 99%, 100% sequence identity with a heavy chain variable domain from a second antibody of table 1 having ≡80% >,. Gtoreq.90% >,. Gtoreq.95% >,. Gtoreq.96% >,. Gtoreq.97% >,. Gtoreq.98% >, ≡99%, 100% sequence identity with a heavy chain variable domain from a first antibody of table 1, wherein the first and second antibodies are derived from the same species of heavy chain v region, optionally wherein the heavy chain v region is IGHV3-53, IGHV1-58 or IGHV3-66.

In one embodiment, the invention provides any one of the antibodies listed in table 1, 21, 22, 23, 24 or 25.

The antibodies of the invention may be or may comprise modifications of the amino acid sequences of the antibodies from table 1 while maintaining the activity and/or function of the antibodies. The modification may be a substitution, deletion and/or addition. For example, the modification may comprise 1, 2, 3, 4, 5, up to 10, up to 20, up to 30 or more amino acid substitutions and/or deletions from the amino acid sequence of the antibody in table 1. For example, the modification may include amino acids substituted with alternative amino acids having similar properties. Some characteristics of the 20 main amino acids that can be used to select suitable substitutions are as follows:

The modification may include derivatized amino acids, such as labeled or unnatural amino acids, provided that the function of the antibody is not significantly adversely affected.

The modification of the antibodies of the invention as described above may be made during the course of synthesis of the antibody or by post-production modification, or when the antibody is in recombinant form, using known site-directed mutagenesis, random mutagenesis or enzymatic cleavage and/or ligation techniques of nucleic acids.

The antibodies of the invention can be modified (e.g., as described above) to increase the potency of the antibodies or to adapt the antibodies to new SARS-CoV-2 variants. These modifications may be amino acid substitutions to adapt the antibody to substitutions in the viral variants. For example, the known binding pattern of an antibody to a spike protein (e.g., by crystal structure measurement or modeling) can be used to identify amino acids of the antibody that interact with substitutions in a viral variant. This information can then be used to identify possible substitutions of the antibody that will compensate for the change in epitope characteristics. For example, substitution of hydrophobic amino acids in the spike protein with negatively charged amino acids can be compensated by substituting amino acids from antibodies that interact with the amino acids in the spike protein with positively charged amino acids. The present disclosure includes methods for identifying residues of an antibody that may be substituted, for example, by determining the structure of an antibody-antigen complex as described herein.

Antibodies of the invention may contain one or more modifications to increase their cross-lineage neutralization properties. For example, spike protein E484, a key residue that mediates interaction with ACE2, was mutated in some SARS-CoV-2 strains (e.g., victoria strain containing E484, but strain P.1 and strain B.1.351 contain E484K), resulting in a different neutralization of the antibodies (see example 24). Thus, antibodies that bind to E484 can be modified to compensate for changes in E484 of the spike protein. For example, E484 is mutated from a positively charged amino acid to a negatively charged amino acid in a SAR-CoV-2 strain of either the B.1.351 or P.1 lineages. The binding of the antibody or the amino acid residues near E484 may be mutated to compensate for the change in charge. Examples of such amino acid residues may be G104 and/or K108 in SEQ ID NO:102 of antibody 88 or R52 in SEQ ID NO:372 of antibody 384 (see example 24).

The antibodies of the invention may be isolated antibodies. An isolated antibody is an antibody that is substantially free of other antibodies having different antigen specificities.

The term "antibody" as used herein may refer to whole antibodies (i.e., elements comprising two heavy and two light chains interconnected by disulfide bonds) as well as antigen binding fragments thereof. Antibodies typically comprise an immunologically active portion of an immunoglobulin (Ig) molecule (i.e., a molecule that contains an antigen binding site that specifically binds (immunoreacts with) an antigen. By "specifically bind" or "immunoreact with … …" is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as HCVR or VH) and at least one heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The variable regions of the heavy and light chains contain binding domains that interact with antigens. VH and VL regions can be further subdivided into regions of higher variability termed Complementarity Determining Regions (CDRs) with more conserved regions termed Framework Regions (FR) interposed therebetween. Antibodies can include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibodies), single chain, fab 'and F (ab') 2 fragments, scFv, and Fab expression libraries.

The antibodies of the invention may be monoclonal antibodies. Monoclonal antibodies (mabs) of the invention can be produced by a variety of techniques, including conventional monoclonal antibody methods, such as "Monoclonal Antibodies: a manual of techniques [ monoclonal antibodies: technical manual ] "(Zola H,1987,CRC Press[CRC press ]) and" Monoclonal Hybridoma Antibodies: techniques and applications [ monoclonal hybridoma antibodies: techniques and applications ] "(Hurrell JGR,1982CRC Press[CRC publishers ]).

The antibodies of the invention may be multispecific, such as bispecific, i.e., one "arm" of the subject binds to the spike protein of SARS-CoV-2, while the other "arm" binds to a different antigen. In one embodiment, the bispecific antibodies of the invention can bind to two separate epitopes on a spike protein. In one embodiment, the bispecific antibodies of the invention bind to the NTD of a spike protein with one "arm" and to the RBD of a spike protein with the other "arm". In one embodiment, the bispecific antibodies of the invention bind to two different antibodies on the RBD of the spike protein. In one embodiment, the bispecific antibodies of the present invention bind to different proteins with each "arm". For example, one or more (e.g., two) antibodies of the invention can be coupled to form a multi-specific (e.g., bispecific) antibody.

The antibody may be selected from the group consisting of: single chain antibodies, single chain variable fragments (scFv), variable fragments (Fv), fragment antigen binding regions (Fab), recombinant antibodies, monoclonal antibodies, fusion proteins comprising an antigen binding domain or an aptamer of a natural antibody, single domain antibodies (sdabs) (also known as VHH antibodies), nanobodies (single domain antibodies of Camelid origin), single domain antibody fragments of shark IgNAR origin (known as VNARs), diabodies, triabodies, statins, aptamers (DNA or RNA), and active components or fragments thereof.

The constant region domains (if present) of the antibody molecules of the invention may be selected taking into account the proposed function of the antibody molecule, in particular the effector function that may be required. For example, the constant region domain may be a human IgA, igD, igE, igG or IgM domain. Typically, the constant region is of human origin. In particular, human IgG (i.e., igG1, igG2, igG3, or IgG 4) constant region domains can be used. Typically, human IgG1 constant regions.

The light chain constant region may be lambda or kappa.

Antibodies of the invention may be monospecific or multispecific (e.g., bispecific). The multispecific antibody comprises at least two different variable domains, wherein each variable domain is capable of binding to a separate antigen or a different epitope on the same antigen.

The antibody of the present invention may be a chimeric antibody, a CDR-grafted antibody, a nanobody, a human antibody or a humanized antibody. Typically, the antibody is a human antibody. Fully human antibodies are those in which the variable and constant regions of the heavy and light chains, if present, are of human origin or are substantially identical to sequences of human origin but not necessarily from the same antibody.

The antibodies of the invention may be full length antibodies. Accordingly, the present invention also provides an antibody which is a full length antibody of any one of the antibodies in tables 1 and 21 to 25. In other words, the antibodies of the invention comprise heavy chain variable domains and light chain variable domains consisting of the heavy chain variable domains and light chain variable domains, respectively, of any of the antibodies in tables 1 and 21-25, and an IgG (e.g., igG 1) constant region. For example, the full length antibody can be 222, 253H55L, 253H165L, 318, 253, 55, 165, 384, 159, 88, 40, 316, or 58.

The antibodies of the invention may be antigen binding fragments. The antigen-binding fragments of the invention bind to the same epitope of the parent antibody (i.e., the antibody from which the antigen-binding fragment is derived). The antigen binding fragments of the invention typically retain the epitope-interacting portion of the parent antibody. Antigen binding fragments typically comprise Complementarity Determining Regions (CDRs) that interact with an antigen, such as one, two, three, four, five, or six CDRs. In some embodiments, the antigen binding fragment further comprises a structural scaffold surrounding CDRs of the parent antibody, such as a variable region domain of a heavy chain and/or a light chain. Typically, the antigen binding fragment retains the same or similar binding affinity for the antigen as the parent antibody.

The antigen binding fragment does not necessarily have the same sequence as the parent antibody. In one embodiment, the antigen binding fragment may have a sequence identity of ≡70%,. Gtoreq.80%,. Gtoreq.90%,. Gtoreq.95%,. Gtoreq.96%,. Gtoreq.97%,. Gtoreq.98%,. Gtoreq.99%, 100% with the corresponding CDR of the parent antibody. In one embodiment, the antigen binding fragment may have greater than or equal to 70%, > or equal to 80%, > or equal to 90%, > or equal to 95%, > or equal to 96%, > or equal to 97%, > or equal to 98%, > or equal to 99%, 100% sequence identity to the corresponding variable region domain of the parent antibody. Typically, the non-identical amino acids of the variable region are not in the CDRs.

The antigen binding fragments of the antibodies of the invention retain the ability to selectively bind to an antigen. Antigen binding fragments of antibodies include single chain antibodies (i.e., full length heavy and light chains); fab, modified Fab, fab ', modified Fab ', F (ab ') 2, fv, fab-dsFv, single domain antibodies (e.g., VH or VL or VHH), scFv.

The antigen binding function of an antibody may be achieved by fragments of full length antibodies. Methods for producing and manufacturing these antibody fragments are well known in the art (see, e.g., verma R et al, 1998, J. Immunol. Methods [ J. Immunol. Methods ],216, 165-181).

Methods for screening antibodies of the invention that do not have 100% amino acid sequence identity to one of the antibodies disclosed herein having the desired specificity, affinity, and functional activity include the methods described herein, such as enzyme-linked immunosorbent assays, biacore, focus reduction neutralization assays (FRNTs), and other techniques known in the art.

Regarding function, the antibodies of the invention may be capable of neutralizing at least one biological activity of SAR-CoV-2 (neutralizing antibodies), in particular neutralizing viral infectivity.

Neutralization may also use ICs ₅₀ Or IC (integrated circuit) ₉₀ The value is determined. For example, the antibody may have an IC of 0.1. Mu.g/ml, 0.05. Mu.g/ml, 0.01. Mu.g/ml or 0.005. Mu.g/ml or less ₅₀ Values. In some cases, antibodies of the invention may have an IC of between 0.005 μg/ml and 0.1 μg/ml, sometimes between 0.005 μg/ml and 0.05 μg/ml, or even between 0.01 μg/ml and 0.05 μg/ml ₅₀ Values.

For example, the IC of some of the antibodies of Table 1 ₅₀ Values are provided in tables 3 and 9.

The ability of an antibody to neutralize viral infectivity can be measured using an appropriate assay, particularly using a cell-based neutralization assay, as shown in the examples. For example, neutralization capacity can be measured in a focal reduction neutralization assay (FRNT), wherein the reduction in the number of cells (e.g., human cells) infected with a virus (e.g., infected at 37 ℃ for 2 hours) in the presence of an antibody is compared to a negative control without antibody addition.

Examples show that neutralization activity may be affected by N-glycosylated sequences in the heavy chain variable region (having a consensus sequence of N-X-S/T; X is any amino acid other than proline). In particular, some of the antibodies of Table 1 were shown to have neutralizing IC less than 0.1 μg/ml ₅₀ And the N-glycan-removing mutations of these antibodies have a negative impact on neutralization, even though they may be deglycosylated without denaturing or losing RBD affinity.

In one embodiment, the antibodies of the invention comprise an N-glycosylated sequence (with a consensus sequence of N-X-S/T) starting at position 35 of the heavy chain variable region (KABAT numbering, using the EU index). In one embodiment, the antibody of the invention comprises CDRH1 of antibody 88 as defined in SEQ ID NO. 105. In one embodiment, the antibodies of the invention comprise CDRH1, CDRH2 and CDRH3 of antibody 88 as specified in SEQ ID NOs 105, 106 and 107, respectively. In one embodiment, the antibody of the invention comprises the VH domain of antibody 88 as specified in SEQ ID NO. 102.

In one embodiment, the antibodies of the invention comprise an N-glycosylated sequence (with a consensus sequence of N-X-S/T) starting at position 59 (KABAT numbering, using the EU index) of the heavy chain variable region. In one embodiment, the antibodies of the invention comprise CDRH1 of antibody 316 as specified in SEQ ID NO:326 and extended to include an N-glycosylated sequence. In one embodiment, the antibodies of the invention comprise CDRH1, CDRH2 and CDRH3 of antibody 316 as specified in SEQ ID NOs 325, 326 and 327, respectively. In one embodiment, an antibody of the invention comprises the VH domain of antibody 316 as specified in SEQ ID NO. 322.

In one embodiment, the antibodies of the invention comprise an N-glycosylated sequence (with a consensus sequence of N-X-S/T) starting at position 102 of the heavy chain variable region. In one embodiment, the antibody of the invention comprises CDRH1 of antibody 253 as defined in SEQ ID NO: 267. In one embodiment, the antibodies of the invention comprise CDRH1, CDRH2 and CDRH3 of antibody 253 as defined in SEQ ID NOs 265, 266 and 267, respectively. In one embodiment, an antibody of the invention comprises the VH domain of antibody 253 as specified in SEQ ID NO: 262.

The antibodies of the invention can block the interaction between the spike protein of SAR-CoV-2 and the cell surface receptor angiotensin converting enzyme 2 (ACE 2) of the target cell, for example, by blocking directly or by disrupting the pre-fusion conformation of the spike protein.

The blocking of the interaction between the spike protein and ACE2 may be all or part. For example, the antibodies of the invention may reduce spike protein-ACE 2 formation by ≡50%,. Gtoreq.60%,. Gtoreq.70%,. Gtoreq.80%,. Gtoreq.90%,. Gtoreq.95%,. Gtoreq.99% or 100%. The blocking of spike protein-ACE 2 formation may be measured by any suitable means known in the art, for example by ELISA.

Most antibodies that showed neutralization were also shown to block the interaction between spike protein and ACE 2. (see FIG. 1C). In addition, many non-neutralizing antibodies are good ACE2 blockers.

In terms of binding kinetics, the antibodies of the invention can have an affinity constant (K) for the spike protein of SARS-CoV-2 of < 5nM, < 4nM, < 3nM, < 2nM, < 1nM, < 0.5nM, < 0.4nM, < 0.3nM, < 0.2nM or < 0.1nM _D ) Values. K of some antibodies of Table 1 _D Values are provided in tables 3 and 9.

KD values can be measured by any suitable means known in the art, for example, by ELISA or surface plasmon resonance (Biacore) at 25 ℃.

Binding affinity (K) _D ) Can be determined by determining the dissociation constant (K _d ) And association constant (K) _a ) To quantify. For example, the antibody may have a length of 10000M or more ^-1 s ^-1 、≥50000M ^-1 s ^-1 、≥100000M ^-1 s ^-1 、≥200000M ^-1 s ^-1 Or greater than or equal to 500000M ^-1 s ^-1 Association constant (K) _a ) And/or be less than or equal to 0.001s ^-1 、≤0.0005s ^-1 、≤0.004s ^-1 、≤0.003s ^-1 、≤0.002s ^-1 Or less than or equal to 0.0001s ^-1 Dissociation constant (K) _d ). See, for example, table 3.

The antibodies of the invention are preferably capable of providing in vivo protection in animals infected with coronavirus (e.g., SARS-CoV-2). For example, administration of an antibody of the invention to an animal infected with a coronavirus (e.g., SARS-CoV-2) can result in a survival rate of 30%. Gtoreq.40%,. Gtoreq.50%,. Gtoreq.60%,. Gtoreq.70%,. Gtoreq.80%,. Gtoreq.90%,. Gtoreq.95%, or 100%. Survival rates may be determined using conventional methods.

The antibodies of the invention may have any combination of one or more of the above properties.

The antibodies of the invention can bind to the same epitope as any of the antibodies described herein (i.e., particularly with antibodies having the heavy and light chain variable regions described above), or compete with any of the antibodies described herein for binding to SARS-CoV-2 spike protein. Methods for identifying antibodies that bind to the same epitope or cross-compete with each other are used in the examples and are discussed further below.

The antibody may bind to the same epitope as antibody 159 or compete with the antibody. Antibody 159 binds to the NTD of spike protein. In one embodiment, the antibody of the invention binds NTD such that it does not block ACE2 binding. In one embodiment, the antibodies of the invention bind to epitopes comprising residues 144-147, 155-158, and 250-253 of NTD (numbering of NTD and RBD is based on the entire spike protein as used herein, unless otherwise indicated). All 3 CDRs of antibody 159 contributed to the binding footprint, while the light chain had little contact. Thus, in one embodiment, the antibodies of the invention comprise CDRH1, CDRH2 and CDRH3 of antibody 159 as shown in SEQ ID NOs 175 to 177, respectively. In one embodiment, the antibody of the invention comprises the heavy chain variable region of antibody 159 as set forth in SEQ ID NO. 172.

The antibodies of the invention may bind to the same epitope as antibody 45 or compete with the antibody. In one embodiment, the antibody does not compete for binding with the potent neutralizing agent S309 (piccola et al 2020). In one embodiment, the antibodies of the invention compete with antibody 45 for binding to SARS-CoV-2 spike protein.

In one aspect, the antibody binds to the same epitope as antibody 384 or competes with the antibody. The binding epitope of antibody 384 is unique among the SARS-CoV-2 antibodies reported to date. The epitope comprises residues F104, L105, L455, F456, and G482 to F486 of the RBD domain, which residues are bound by CDRH3 of antibody 384. In one embodiment, the antibodies of the invention bind to this epitope using only interactions from CDRH3. In one embodiment, the antibody of the invention comprises CDRH3 of antibody 384 as set forth in SEQ ID NO: 377. In another embodiment, the antibodies of the invention comprise CDRH2 and CDRH3 of antibody 384 as set forth in SEQ ID NOS 376 and 377. The antibody 384 interacts with the spike protein only through CDRH2 and CDRH3 of the heavy chain. In one embodiment, an antibody of the invention comprises CDRL1 of antibody 384 as set forth in SEQ ID NO:378 and CDRL3 of antibody 384 as set forth in SEQ ID NO: 380. In one embodiment, the antibodies of the invention comprise CDRH2, CDRH3, CDRL1 and CDRL3 of antibody 384 as set forth in SEQ ID NOS 376-378 and 380, respectively. The antibody 384 interacts with the spike protein only through CDRH2, CDRH3, CDRL1 and CDRL3 of the antibody. In another aspect, the antibody binds to the same epitope as CDRH2 and CDRH3 of antibody 384. In another embodiment, the antibodies of the invention do not contact the right chest of the RBD domain of the spike protein. In one embodiment, the antibodies of the invention comprise the heavy chain CDRs shown in SEQ ID NOS 375, 376 and 377, and optionally the light chain CDRs shown in SEQ ID NOS 378, 379 and 380. In another aspect, W107 of the antibody CDRH3 of the invention produces a strong pi-interaction with G485 of RBD, Y59 of CDRH2 contacts V483 and forms a bifurcated H bond with the carbonyl oxygen of G482 and the amino nitrogen of E484 of RBD, which in turn forms a salt bridge with R52 and H bonds with the side chains of T57 and Y59. The E484-F486 of RBD also forms a double stranded antiparallel beta sheet with residues A92-A94 of CDRL3 and creates a stacking interaction from F486 to Y32 of CDRL 1. The advantage of backbone RBD interactions may confer resistance to escape of mutations.

The skilled artisan can readily determine the binding site (epitope) of an antibody using standard techniques such as those described in the examples of the present application. The skilled artisan can also readily determine whether an antibody binds to the same epitope as an antibody described herein or competes for binding with an antibody described herein by using conventional methods known in the art.

For example, to determine whether the antibody being tested (i.e., where it is not known whether the test antibody competes with other antibodies for binding to the antigen) binds to the same epitope as the antibody described herein (referred to as a "reference antibody" in the following paragraphs), the reference antibody is allowed to bind to a protein or peptide under saturated conditions. Next, the ability of the test antibodies to bind to the protein or peptide is assessed. If the test antibody is capable of binding to a protein or peptide after saturation binding to the reference antibody, it can be concluded that: the test antibody binds to a different epitope than the reference antibody. In another aspect, if the test antibody is unable to bind to a protein or peptide after saturation binding to the reference antibody, the test antibody may bind to the same epitope as the reference antibody of the invention.

To determine whether an antibody competes for binding with a reference antibody, the above-described binding method is performed in two orientations. In the first orientation, the reference antibody is allowed to bind to the protein/peptide under saturated conditions, followed by assessment of the binding of the test antibody to the protein/peptide molecule. In the second orientation, the test antibody is allowed to bind to the protein/peptide under saturated conditions, followed by assessment of binding of the reference antibody to the protein/peptide. If only the first (saturated) antibody is able to bind the protein/peptide in both orientations, then it can be concluded that: the test antibody and the reference antibody compete for binding to the protein/peptide. As the skilled artisan will appreciate, an antibody that competes for binding with a reference antibody may not necessarily bind the same epitope as the reference antibody, but may spatially block binding of the reference antibody by binding overlapping or adjacent epitopes.

Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) the binding of the other antibody to the antigen. Alternatively, two antibodies have the same epitope if substantially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other antibody.

Additional routine experimentation (e.g., peptide mutation and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is in fact due to binding or steric blocking (or another phenomenon) of the same epitope as the reference antibody is the cause of the observed lack of binding. Such experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

In addition to sequences defined by percent identity or the number of sequence alterations, the present invention further provides an antibody defined by its ability to cross-compete with one of the specific antibodies shown herein. The antibody may also have one of the recited levels of sequence identity or number of sequence alterations.

The cross-competing antibodies can be identified using any suitable method in the art, for example, by using a competition ELISA or BIAcore assay, wherein binding of the cross-competing antibodies to a specific epitope on the spike protein prevents binding of the antibodies of the invention, and vice versa. In one embodiment, the antibody produces ≡50%,. Gtoreq.60%,. Gtoreq.70%,. Gtoreq.80%,. Gtoreq.90% or 100% reduction in binding of the specific antibodies disclosed herein.

The antibodies described below in the examples can be used as reference antibodies.

Other techniques that can be used to determine antibody epitopes include hydrogen/deuterium exchange, X-ray crystallography and peptide display libraries (as described in the examples). Combinations of these techniques can be used to determine the epitope of the test antibody.

The methods used herein are equally applicable to other data, such as surface plasmon resonance or ELISA, and provide a general method for rapidly determining position from highly redundant competition experiments.

Fc region

The antibodies of the invention may or may not comprise an Fc domain.

The antibodies of the invention may be modified in the Fc region in order to improve their stability. Such are known in the art. Modification may improve the stability of the antibody during storage of the antibody. The in vivo half-life of antibodies can be improved by modification of the Fc region.

For example, cysteine residues may be introduced into the Fc region, allowing for inter-chain disulfide bond formation in that region. Homodimeric antibodies thus produced may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). (see Caron et al, J.exp Med. [ journal of Experimental medicine ],176:1191-1195 (1992) and Shopes, J.Immunol. [ journal of immunology ],148:2918-2922 (1992)). Alternatively, antibodies with dual Fc regions may be engineered, whereby complement lysis and ADCC capacity may be enhanced. (see Stevenson et al, anti-Cancer Drug Design [ anticancer drug design ],3:219-230 (1989)).

For example, the antibodies of the invention may be modified to facilitate the interaction of the Fc domain with FcRn. The Fc domain may be modified to improve the stability of the antibody by affecting Fc and FcRn interactions at low pH (such as in endosomes). The M252Y/S254T/T256E (YTE) mutation can be used to improve the half-life of IgG1 antibodies.

Antibodies can be modified to affect the interaction of the antibody with other receptors, such as fcyri, fcyriia, fcyriib, fcyriii, and fcαr. Such modifications may be used to affect effector functions of the antibody.

In one embodiment, the antibodies of the invention comprise an altered Fc domain as described below. In another preferred embodiment, the antibodies of the invention comprise an Fc domain, but the sequence of the Fc domain has been altered to modify one or more Fc effector functions.

In one embodiment, an antibody of the invention comprises a "silent" Fc region. For example, in one embodiment, the antibodies of the invention do not exhibit effector functions or functions associated with a normal Fc region. The Fc region of the antibodies of the invention does not bind to one or more Fc receptors.

In one embodiment, the antibodies of the invention do not comprise CH ₂ A domain. In one embodiment, the antibodies of the invention do not comprise CH ₃ A domain. In one embodiment, the antibodies of the invention comprise an additional CH ₂ And/or CH ₃ A domain.

In one embodiment, the antibodies of the invention do not bind to Fc receptors. In one embodiment, the antibodies of the invention do not bind complement. In alternative embodiments, the antibodies of the invention do not bind fcγr, but bind complement.

In one embodiment, the antibodies of the invention may generally comprise modifications that alter the serum half-life of the antibody. Thus, in another embodiment, the antibodies of the invention have an Fc region modification that alters the half-life of the antibody. Such modifications may exist as those that alter Fc function. In a preferred embodiment, the antibodies of the invention have modifications that alter the serum half-life of the antibody.

In one embodiment, an antibody of the invention may comprise a human constant region, such as IgA, igD, igE, igG or IgM domain. In particular, when the antibody molecule is intended for therapeutic use where antibody effector function is desired, human IgG constant region domains, particularly of the IgG1 and IgG3 isotype, may be used. Alternatively, igG2 and IgG4 isotypes may be used when the antibody molecule is used for therapeutic purposes and antibody effector function is not required.

In one embodiment, the antibody heavy chain comprises CH ₁ Domain, and antibody light chain comprises CL domain (kappa or lambda). In one embodiment, the antibody heavy chain comprises CH ₁ Domain, CH ₂ Domain and CH ₃ Domain, and antibody light chain comprises CL domain (kappa or lambda).

The four human IgG isotypes bind with different affinities to the activated fcγ receptor (fcγri, fcγriia, fcγriic, fcγriiia), the inhibitory fcγriib receptor and the first component of complement (C1 q), thereby generating distinct effector functions (Bruhns p. Et al, 2009.Specificity and affinity of human Fc γ receptors and their polymorphic variants for human IgG subclasses [ the specificity and affinity of human fcγ receptor and polymorphic variants thereof for the human IgG subclass ]. Blood [ Blood ].113 (16): 3716-25), see Jeffrey b.stavenhagen et al, cancer Research [ Cancer Research ]2007, 9-15; 67 (18):8882-90. In one embodiment, the antibodies of the invention do not bind to Fc receptors. In another embodiment of the invention, the antibody does bind to one or more types of Fc receptors.

In one embodiment, the Fc region employed is mutated, particularly as described herein. In one embodiment, the Fc mutation is selected from the group comprising: mutations that remove or enhance binding of the Fc region to Fc receptors, mutations that increase or remove effector function, mutations that increase or decrease half-life of antibodies, and combinations thereof. In one embodiment, when referring to the effect of a modification, it can be demonstrated by comparison with an equivalent antibody lacking the modification.

Some antibodies that selectively bind FcRn at pH 6.0 but not pH 7.4 exhibit longer half-lives in various animal models. Located at CH ₂ And CH (CH) ₃ Several mutations at the interface between domains such as T250Q/M428L (Hinton PR. et al, 2004.Engineered human IgG antibodies with longer serum half-lives in primates [ engineered human IgG antibodies with longer serum half-life in primates ]]J Biol Chem journal of biochemistry]279 (8) 6213-6) and M252Y/S254T/T256 E+H2433K/N434F (Vaccaro C. Et al, 2005.Engineering the Fc region of immunoglobulin G to modulate in vivo antibody levels [ engineering the Fc region of immunoglobulin G to modulate antibody levels in vivo) ]Nat Biotechnol. [ Nat Biotechnology ]]23 (10) 1283-8) has been shown to increase binding affinity for FcRn and the in vivo half-life of IgG 1. Thus, there may be modifications at M252/S254/T256+H244/N434 that alter serum half-life, and in particular there may be M252Y/S254T/T256 E+H2433K/N434F. In one embodiment, it is desirable to increase half-life. In another embodiment, it may actually be desirable to reduce the serum half-life of the antibody, so modifications that reduce serum half-life may be present.

Already in CH of human IgG1 ₂ Numerous mutations were made in the domains and their effect on ADCC and CDC was tested in vitro (Idusogenie EE et al, 2001.Engineered antibodies with increased activity to recruit complement [ engineered antibodies with enhanced complement recruitment activity ]]J Immunol journal of immunology].166 (4):2571-5). Notably, alanine substitutions at position 333 have been reported to increase ADCC and CDC. Thus, in one embodiment, there may be a modification at position 333, particularlyIs a modification that alters the ability to recruit complement. Lazar et al describe a triple mutant (S239D/I332E/a 330L) with higher affinity for fcyriiia and lower affinity for fcyriib, thereby enhancing ADCC (Lazar GA. et al, 2006). Thus, modifications may be present at S239/I332/A330, particularly those that alter the affinity for Fc receptors, particularly S239D/I332E/A330L. An engineered antibody Fc variant with enhanced effector function. PNAS [ Prop of national academy of sciences of the United states ] ]103 (11):4005-4010). Antibody production with increased ADCC using the same mutations (Ryan MC. et al 2007.Antibody targeting of B-cell maturation antigen on malignant plasma cells [ antibody targeting of B cell maturation antigen on malignant plasma cells ]]Mol. Cancer Ther. [ molecular cancer treatment],6:3009-3018). Richards et al studied a slightly different triple mutant (S239D/I332E/G236A) with increased FcgammaRIIIa affinity and FcgammaRIIIb ratio, mediating enhanced phagocytosis of target cells by macrophages (Richards JO et al 2008.Optimization of antibody binding to Fcgamma RIIa enhances macrophage phagocytosis of tumor cells [ optimization of antibody binding to FcgammaRIIa enhanced phagocytosis of tumor cells by macrophages)]Mol Cancer Ther [ molecular Cancer treatment].7 (8):2517-27). In one embodiment, there may thus be an S239D/I332E/G236A modification.

In another embodiment, the antibodies of the invention may have modified hinge and/or CH1 regions. Alternatively, the employed isoform may be selected because it has a specific hinge region.

SARS-CoV-2 variants

The variant b.1.1.7 was first identified in a sequence extracted from a patient at the end of 9 months in 2020 (Rambaut et al 2020). This variant is rapidly dominant in many regions of the united kingdom, while the number of infections increases rapidly during the second pandemic, with cases and hospitalizations exceeding the first stage. The b.1.1.7 variant was estimated to be 30-60% more infectious than the strain encountered in the first wave (Walker et al 2021) and was able to defeat the public health department's efforts to control infection. B.1.1.7 contains a total of 9 changes in spike protein: residues 69-70 deleted, 144 deleted, N501Y, A570D, D614G, P681H, T716I, S982A and D1118H, where N501Y is probably the most interesting because it has the potential to increase RBD/ACE2 affinity while also disrupting binding of the potent neutralizing antibodies (figure 18).

The b.1.351 variant obtained mutations in the ACE2 interacting surface of RBD at positions K417N, E484K and N501Y. B.1.351 has 10 changes relative to the martial sequence: L18F, D80A, D G, L-244 deleted, R246I, K417N, E484K, N501Y, D614G, A V701V. 501y.v2 variant obtained mutations in the ACE2 interacting surface of RBD at positions K417T, E484K and N501Y.

B.1.617.2 The (delta) variant obtained the mutation L452R, T478K in RBD relative to the Wuhan sequence. B.1.1.529 The (omicron) variant obtained the mutation G339D, S371L, S373P, S375F, K417N, N K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H relative to the wuhan sequence.

Where not otherwise indicated herein, the strain referred to is SARS-CoV-2/human/AUS/VIC01/2020 (see example 13, FRNT assay). This strain is an early strain associated with the original Wuhan strain hCoV-19/Wuhan/WIV04/2019 (WIV 04) (GISAID accession number epi_isl_ 402124) and differs in a single amino acid.

Serum recovered from convalescence samples from first wave covd 19 patients has been found to be less effective at neutralizing variant strains. For example, recovery serum was found to be 3-fold less effective against the b.1.1.7 variant when compared to Victoria strain as used herein, and 13.3-fold less effective against the b.1.351 variant when compared to Victoria strain. Serum from subjects vaccinated with both the psilon and the aslicon strains was found to be 3-fold less effective for the b.1.1.7 variant and 7.6-fold and 9-fold less effective for the b.1.351 variant when compared to the Victoria strain. Thus, antibodies are expected to be less effective than these variants.

However, the inventors surprisingly found that many of the antibodies described herein retain their neutralizing potency against UK Kent (b.1.1.7) variants and South Africa (b.1.351) variants. The neutralizing IC50 of the highly potent mabs identified herein against variant strains are shown in fig. 22 and 30 and tables 11, 12 and 16.

Thus, in one embodiment, an antibody of the invention comprises at least 3, 4, 5, or all 6 CDRs of an antibody shown in table 12 or 16A. In one embodiment, the antibodies of the invention retain strong neutralization (such as less than a 10-fold decrease in IC 50) against b.1.1.7 and/or b.1.351 strains. In one embodiment, the antibodies of the invention retain strong neutralization (such as less than a 10-fold decrease in IC 50) against b.1.1.7, b.1.351 and/or p.1 strains. Fold reduction in IC50 can be calculated by comparison with the IC50 of a reference strain, such as the test Victoria strain used herein.

Primary public V-zone

The public V region (also described herein as a public V gene) is the V region of the germline heavy and light chain regions found in most populations. That is, many individuals share the same common v-region in their germline v-region pool.

As used herein, an antibody "derived from" a particular V region refers to an antibody produced by V (D) J recombination using such germline V region sequences. For example, germline IGHV3-53 v region sequences can undergo somatic recombination and somatic mutation to produce antibodies that specifically bind to the spike protein of SARS-CoV-2. The nucleotide sequence encoding the antibody may no longer comprise the same sequence as the IGHV3-53 germline sequence, but nevertheless the antibody is derived from the v region. The antibodies of the invention typically comprise no more than 20 non-silent mutations, such as no more than 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 non-silent mutations in the v region when compared to the germline sequence. Germline v-region sequences are well known in the art, and methods for identifying whether a region of an antibody is derived from a particular germline v-region sequence are also well known in the art.

In one embodiment, the antibodies of the invention are derived from the v-region selected from IGHV3-53, IGHV1-58, and IGHV 3-66. The inventors found that the potent neutralizing antibodies identified herein contained relatively few mutations in the CDRs of the v regions. Thus, in one embodiment, the antibodies of the invention are encoded by a v region selected from IGHV3-53, IGHV1-58, and IGHV3-66, and have 3-10 non-silent amino acid mutations or 2-5 non-silent mutations, such as 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 non-silent mutations when compared to a naturally occurring germline sequence.

In one embodiment, the antibodies of the invention comprise CDRs of heavy chain variable domains derived from antibodies selected from the primary public v regions of IGHV3-53, IGHV1-58, and IGHV3-66, such as antibodies 150, 158, 175, 222, and 269 for IGHV3-53, antibodies 55, 165, 253, and 318 for IGHV1-58, and antibodies 40 and 282 for IGHV 3-66. The SEQ ID NOs corresponding to the CDRs of each of these antibodies are shown in Table 1.

In one embodiment, the antibodies of the invention comprise heavy chain variable domains derived from antibodies selected from the primary public v regions of IGHV3-53, IGHV1-58, and IGHV3-66, such as antibodies 150, 158, 175, 222, and 269 for IGHV3-53, antibodies 55, 165, 253, and 318 for IGHV1-58, and antibodies 40 and 282 for IGHV 3-66. The SEQ ID NOs corresponding to the heavy chain variable domains of each of these antibodies are shown in Table 1.

In a preferred embodiment, the antibodies of the invention comprise heavy chain CDRs 1-3 shown in SEQ ID NOS 265 through 267, respectively. In another embodiment, the antibody comprises the heavy chain variable domain of antibody 253 shown in SEQ ID NO. 262.

There is a close association between the potent neutralising agent and the public V gene, indicating that the vaccination response should be strong (Yuan et al 2020 b). Three common V region genes occur at least twice in a group of 21: i) IGHV3-53: mabs 150, 158, 175, 222 and 269, ii) IGHV1-58: 55. 165, 253 and 318, and iii) IGHV3-66:282 and 40. In all cases, strong binders concentrated around the neck clusters, and the binding posture was generally determined by the H1 and H2 loops. By exchanging light chains within these groups, antibody 253 can be functionally enhanced by an order of magnitude by using alternative light chains to achieve better hydrophobic interactions with the identified critical bridging regions E484-F486. The most highly potent mAb 384 adopts a unique pose, with the footprint extending from the left shoulder epitope to the neck epitope via extended H3.

Five of the potent monoclonal antibodies used herein (150, 158, 175, 222 and 269) belong to the VH3-53 family, and the other 2 (282 and 40) belong to nearly identical VH3-66. Thus, embodiments related to the VH3-53 family may be equally applicable to the VH3-66 family.

As shown in fig. 5B, other common v regions are over represented in the highly potent antibodies identified herein. Thus, in one aspect, an antibody comprises a variable domain sequence derived from a V region selected from the list of: IGHV1-2, IGHV1-58, IGHV3-66, IGHV7-4-1, IGkappa V1-33, IGkappa V1-9, IGkappa V3-20, IGLV2-14, IGLV2-8, and/or IGLV3-21.

In one embodiment, the antibody comprises a heavy chain variable domain sequence derived from a V region selected from the group consisting of: IGHV1-58, IGHV1-18 or IGHV3-9; and/or a light chain variable domain sequence derived from a V region selected from the group consisting of: IG kappa V3-20, IG lambda 3-21 or IG kappa 1-39 or kappa 1D-39. Antibodies derived from these regions (e.g., antibodies 55, 58, 165, 253, 278, and 318) have proven to be particularly effective in cross-lineage neutralization (e.g., against both Victoria and b.1.351 strains) and have good binding affinity for spike proteins (see table 16A).

Furthermore, and as described in the examples, it has surprisingly been shown that antibodies derived from a specific public V region are able to maintain or improve neutralization against b.1.1.7 and/or b.1.351 strains when compared to Victoria strains. In particular, the antibodies of the invention are derived from IGHV1-58 v region (antibodies 55, 165, 253 and 318). In one embodiment, the light chain of an antibody having a heavy chain derived from IGHV1-58 may be exchanged with a light chain of a second antibody also derived from the same heavy chain V region. When exchanging the chains of antibodies, the light and heavy chains of each antibody are preferably derived from the same V region. For example, antibodies 55, 165 and 253 all have a heavy chain derived from IGHV1-58 v region and a light chain derived from Kappa 3-20. It is shown herein that combining the light chain of 55 or 165 with the heavy chain of 253 increases the neutralization titer by >1log. Other combinations are contemplated, as the structures with 253 and 253/55 and 253/165 of RBD or spike show that they bind almost identically to the same epitope and do not contact any of the three mutation site residues in the b.1.351 variant.

Thus, in one embodiment, the invention provides a method of producing an antibody that specifically binds to spike protein of SARS-CoV-2 (e.g., a strain of SARS-CoV-2 of the B.1.351 lineage), the method comprising identifying two or more antibodies derived from the same light chain and/or heavy chain v region, replacing the light chain of the first antibody with the light chain of the second antibody, thereby producing a mixed chain antibody comprising the heavy chain of the first antibody and the light chain of the second antibody. In one embodiment, the method further comprises determining the affinity and/or neutralization of SARS-CoV-2 by the mixed chain antibody. The method may further comprise comparing the affinity of the mixed chain antibody to the affinity of the first antibody and/or the second antibody. The method may further comprise selecting mixed chain antibodies having the same affinity as or greater affinity than the first antibody and/or the second antibody. In some embodiments, the heavy chain v region is IGHV 1-58 and/or the light chain v region is IGLV Kappa3-20.

In one embodiment, an antibody of the invention comprises at least three CDRs of antibody 222. Many of the antibodies identified herein use the public HC V region IGHV3-53. Four of these, 150, 158, 175 and 269, were severely compromised or abolished in terms of neutralizing and binding capacity for the b.1.351 variant. However, antibody 222 is an exception in that its binding is not affected by the b.1.351 variant. The IGHV3-53 antibody family binds at the same epitope in the posterior neck of RBD in a very similar proximity orientation as IGHV3-66 Fab also shares. Most of them are in direct contact with K417 and N501, but none with E484. The rather short HC CDR3 of these Fab is typically located directly above K417, forming hydrogen bonds or salt bridges and hydrophobic interactions, while N501 interacts with the LC CDR-1 loop. mAb 150 is slightly different, forming a salt bridge between K417 and LC CDR3D92 and an H bond between N501 and S30 in LC CDR1 (fig. 31B), while 158 more typically forms a hydrogen bond from the carbonyl oxygen of G100 and K417 of HC CDR3 and a hydrophobic contact from S30 to N501 of LC CDR 1. Thus, the combined effect of the K417N and N501Y mutations is expected to severely impair the binding of most IGHV3-53 and IGHV3-66 mAbs. However, antibody 222 was not affected by either the b.1.1.7 or b.1.351 variants. In addition, antibody 222 was one of the most potent neutralizing antibodies against the b.1.1.529 (omacron) variant tested.

Surprisingly, neutralization of antibodies 150, 158, 175 and 269 to b.1.1.7, b.1.351 and/or p.1 variants can be restored by exchanging the original light chain of antibodies 150, 158, 175 and 269 with the light chain of 222. As described in the examples, CDRH3 of IGHV3-53 derived antibodies was in relatively weak contact with RBD.

Thus, the antibodies of the invention comprise: (i) CDRL1, CDRL2 and CDRL3 of antibody 222 having the amino acid sequences specified in SEQ ID NOs 258, 259 and 260, respectively; and (ii) CDRH1 and CDRH2 independently selected from any one of the antibodies consisting of: 150. 158, 175, 269, 40 and 398. The antibody may optionally further comprise CDRH3 selected from any one of the following antibodies: 150. 158, 175, 269, 40 and 398. In one embodiment, CDRH1 and CDRH2 are independently selected from: 150. 158, 175 and 269, most preferably 150, 158.

Based on the sequence similarity between antibodies 150, 158, 175, 269, 40 and 398, the consensus sequences of CDRH1 and CDRH2 of the antibodies can be obtained as follows:

CDRH1：G-X ¹ -T-C-X ² -X ³ -N-Y(SEQ ID NO:407)

CDRH2：I-Y-X ⁴ -G-G-X ⁵ -T(SEQ ID NO:408)

X ⁿ any amino acid is possible. X is X ¹ Preferably non-polar, more preferably L, V or F, most preferably L or V. X is X ² Preferably a polar side chain, more preferably S or N, most preferably S. X is X ³ Preferably polar or charged side chains, more preferably S or R, most preferably S. X is X ⁴ Preferably polar or nonpolar side chains, more preferably S or P. X is X ⁵ Preferably a nonpolar side chain, more preferably S or T. Thus, an antibody of the invention can comprise CDRH1 and CDRH2 according to a consensus sequence, e.g., in combination with CDRL1, CDRL2 and CDRL3 of antibody 222.

Based on the known CDR sequences of antibodies derived from the same common v region, and structural data showing the interaction between the antibodies and viral spike proteins, consensus sequences of CDRs from antibodies 55, 165 and 253 can be obtained as follows:

CDRH1：G-F-T-F-T-X1-S-A(SEQ ID NO:401)

CDRH2：I-V-V-G-S-G-N-T(SEQ ID NO:402)

CDRH3：A-A-P-X2-C-X3-X4-S/T-C-X5-D-X6-F-D-I(SEQ ID NO:403)

CDRL1：Q-S-V-X7-S-S-Y(SEQ ID NO:404)

CDRL2：G-A-S(SEQ ID NO:405)

CDRL3：Q-Q-Y-G-S-S-P-X8-T(SEQ ID NO:406)

X1-X8 may be any amino acid. X1 is preferably a polar amino acid or S or T. The structural data provided in fig. 6B indicate that the invariant side chains of S105, D108 and F110 interact with spike proteins. Disulfide bonds are also formed between the two cysteines in CDRH 3. Based on the variation of the CDRH3 sequence in combination with structural data, it is reasonable that the variable amino acid can be any amino acid. X2 is preferably A or H. Furthermore, it was shown by structural analysis and by biochemical characterization that glycans of antibody 253 did not interact directly with spike proteins. Thus, X3 may be any amino acid and X4 may be any one or any two amino acids. If X4 is a single amino acid, one of X3 and X4 is preferably glycine, so that a disulfide bond between cysteine residues of CDRH3 can be formed. X3 is preferably any nonpolar or polar amino acid, or G/I/N. X4 is preferably T/GG/ST/S. X5 is preferably any polar or charged amino acid, or S/H/Y. X6 is preferably A. X7 is preferably any polar/charged amino acid, or more preferably R/S. X8 is preferably a hydrophobic amino acid such as an aromatic amino acid, W/Y/F or W/Y.

Thus, an antibody of the invention may comprise at least three CDRs of the consensus sequence as defined in the previous paragraph (i.e.as selected from SEQ ID NOS: 401 to 406). For example, the antibody may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 401, 402 and 403, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 404, 405 and 406, respectively. Such antibodies have potent cross-lineage neutralization, for example, against Victoria, b.1.1.7 and b.1.351 strains described herein.

Furthermore, it is contemplated that although it is reasonable that any antibody comprising shared CDRs be effective against SARS-CoV-2, the skilled artisan can readily screen for antibodies having the desired effect. Thus, the invention also includes methods of screening for such antibodies using any method known to the skilled artisan, such as those described herein.

Mixed chain antibodies

An antibody of the invention may comprise a light chain variable domain comprising CDRL1, CDRL2 and CDRL3 from a first antibody in table 1 and a heavy chain variable domain comprising CDRH1, CDRH2 and CDRH3 from a second antibody in table 1. Such antibodies are referred to herein as mixed chain antibodies or chimeric antibodies. Examples of mixed chain antibodies are provided in tables 21 to 25. Mixed chain antibodies have particularly potent cross-lineage neutralization as demonstrated in the examples.

Thus, in one embodiment, an antibody of the invention comprises a heavy chain variable domain comprising CDRH1, CDRH2 and CDRH3 from a first antibody in table 1 and a light chain variable domain comprising CDRL1, CDRL2 and CDRL3 from a second antibody in table 1. The antibody may comprise a heavy chain variable domain amino acid sequence having at least 80% sequence identity to a heavy chain variable domain from a first antibody in table 1 and a light chain variable domain amino acid sequence having at least 80% sequence identity to a light chain variable domain from a second antibody in table 1.

The first and second antibodies in table 1 may be derived from the same germline heavy or light chain v-region. For example, the heavy chain v region may be IGHV3-53, IGHV1-58, or IGHV3-66. The light chain V region may be IG kappa V3-20 or IG kappa V1-9. In one embodiment, the first antibody is 150 and the second antibody is 222. In another embodiment, the first antibody is 253 and the second antibody is 55. In another embodiment, the first antibody is 253 and the second antibody is 165.

In one embodiment, the second antibody is 222. Thus, in one embodiment, CDRL1, CDRL2 and CDRL3 have the amino acid sequences set forth in SEQ ID NOS 258, 259 and 260, respectively.

It was unexpectedly found that the light chain of antibody 222 can act as a "universal" light chain when combined with the heavy chain of another antibody in table 1, such as an antibody derived from IGHV3-53 or IGHV 3-66. 222 light chain enables the resulting mixed chain antibody to bind to and neutralize SARS-CoV-2 strain that would otherwise not be bound or neutralized by the parent antibody of the heavy chain.

In particular, it was found that by combining the light chain of antibody 222 with the heavy chain of another antibody derived from IGHV3-53 (e.g., antibodies 150, 158, 175, and 269), the resulting mixed chain antibody shows increased neutralization when compared to the parent antibody. For example, by combining the 222 light chain with 175 or 269 heavy chain, the resulting mixed chain antibodies have increased neutralization of the b.1.1.7 variant (see table 18 and fig. 37). Furthermore, by combining the 222 light chain with the 150 or 158 heavy chain, the resulting mixed chain antibodies had increased neutralization of the b.1.1.7, b.1.351 and p.1 variants (see table 18 and fig. 37). Thus, antibodies 222, 150H222L, 158H222L, 175H222L, and 260H222L are particularly useful for the present invention, particularly antibodies 222, 150H222L, and 158H222L, because they have potent cross-lineage neutralization.

Due to the similarity between IGHV3-53 and IGHV3-66, it is expected that similar results will be achieved by combining the light chain of antibody 222 with the heavy chain from antibodies derived from IGHV3-53 or IGHV 3-66.

However, it appears that the heavy chain of 222 may not be useful as the universal heavy chain for IGH3-53 antibodies, as modeling studies in example 33 show that there may be some steric clash when the light chain of VH3-53 mabs (e.g., 348, 150, 158, 175, and 269) is docked to the heavy chain of antibody 222 (see fig. 36H).

The antibody may comprise a heavy chain variable domain comprising CDRH1, CDRH2 and CDRH3 from the first antibody in table 1 and a light chain variable domain comprising CDRL1, CDRL2 and CDRL3 from the second antibody in table 1, wherein the first antibody and the second antibody are derived from the same germline heavy chain IGHV3-53. Thus, the first or second table 1 antibody may be selected from 150, 158, 175, 222, and 269.

In one embodiment, the first antibody may be 150 from table 1 and the second antibody may be 222 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs 155, 156 and 157, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs 258, 259 and 260, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 152 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 254. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 152 and a light chain variable domain consisting of SEQ ID NO. 254, i.e.the antibody is 150H222L. The antibody has potent cross-lineage neutralization, e.g., it is effective against all of the SARS-CoV-2 strains tested in the examples (as shown in Table 18 and FIG. 37).

In one embodiment, the first antibody may be 158 from table 1 and the second antibody may be 222 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 165, 166 and 167, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 258, 259 and 260, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity with SEQ ID NO. 162, and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity with SEQ ID NO. 254. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 162 and a light chain variable domain consisting of SEQ ID NO. 254, i.e.the antibody is 158H222L. The antibody has potent cross-lineage neutralization, e.g., it is effective against all of the SARS-CoV-2 strains tested in the examples (as shown in Table 18 and FIG. 37).

In one embodiment, the first antibody may be 175 from table 1 and the second antibody may be 222 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 205, 206 and 207, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 258, 259 and 260, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity with SEQ ID NO 202 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity with SEQ ID NO 254. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 202 and a light chain variable domain consisting of SEQ ID NO. 254, i.e., the antibody is 175H222L. The antibody is capable of exhibiting potent cross-lineage neutralization, e.g., it is effective against Victoria strain and b.1.1.7 strain (see fig. 37).

In one embodiment, the first antibody may be 269 from table 1 and the second antibody may be 222 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs 275, 276 and 277, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs 258, 259 and 260, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 272, and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 254. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO:272 and a light chain variable domain consisting of SEQ ID NO:254, i.e., the antibody is 269H222L. The antibody is capable of exhibiting potent cross-lineage neutralization, e.g., it is effective against Victoria strain and b.1.1.7 strain (see fig. 37).

In one embodiment, the first antibody may be 150 from table 1 and the second antibody may be 158 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 155, 156 and 157, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 168, 169 and 170, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 152 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 164. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 152 and a light chain variable domain consisting of SEQ ID NO. 164, i.e.the antibody is 150H158L.

In one embodiment, the first antibody may be 150 from table 1 and the second antibody may be 175 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 155, 156 and 157, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 208, 209 and 210, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 152 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 204. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 152 and a light chain variable domain consisting of SEQ ID NO. 204, i.e.the antibody is 150H175L.

In one embodiment, the first antibody may be 150 from table 1 and the second antibody may be 269 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 155, 156 and 157, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 278, 279 and 280, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 152 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 274. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 152 and a light chain variable domain consisting of SEQ ID NO. 274, i.e.the antibody is 150H269L.

In one embodiment, the first antibody may be 158 from table 1 and the second antibody may be 150 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 165, 166 and 167, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 158, 159 and 160, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 162, and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 154. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 162 and a light chain variable domain consisting of SEQ ID NO. 154, i.e.the antibody is 158H150L.

In one embodiment, the first antibody may be 158 from table 1 and the second antibody may be 175 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 165, 166 and 167, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 208, 209 and 210, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity with SEQ ID NO. 162, and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity with SEQ ID NO. 204. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 162 and a light chain variable domain consisting of SEQ ID NO. 204, i.e., the antibody is 158H175L.

In one embodiment, the first antibody may be 158 from table 1 and the second antibody may be 269 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 165, 166 and 167, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 278, 279 and 280, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 162, and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 274. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO:162 and a light chain variable domain consisting of SEQ ID NO:274, i.e., the antibody is 158H269L.

In one embodiment, the first antibody may be 175 from table 1 and the second antibody may be 150 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOS 205, 206 and 207, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOS 158, 159 and 160, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity with SEQ ID NO 202 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity with SEQ ID NO 154. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 202 and a light chain variable domain consisting of SEQ ID NO. 154, i.e.the antibody is 175H150L.

In one embodiment, the first antibody may be 175 from table 1 and the second antibody may be 158 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 205, 206 and 207, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 168, 169 and 170, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity with SEQ ID NO 202 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity with SEQ ID NO 164. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 202 and a light chain variable domain consisting of SEQ ID NO. 164, i.e.the antibody is 175H158L.

In one embodiment, the first antibody may be 175 from table 1 and the second antibody may be 269 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 205, 206 and 207, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 278, 279 and 280, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 202 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 274. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 202 and a light chain variable domain consisting of SEQ ID NO. 274, i.e.the antibody is 175H269L.

In one embodiment, the first antibody may be 222 from table 1 and the second antibody may be 150 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 255, 256 and 257, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 158, 159 and 160, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 252 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 154. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 252 and a light chain variable domain consisting of SEQ ID NO. 154, i.e.the antibody is 222H150L.

In one embodiment, the first antibody may be 222 from table 1 and the second antibody may be 158 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 255, 256 and 257, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 168, 169 and 170, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 252 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 164. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 252 and a light chain variable domain consisting of SEQ ID NO. 164, i.e.the antibody is 222H158L.

In one embodiment, the first antibody may be 222 from table 1 and the second antibody may be 175 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 255, 256 and 257, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 208, 209 and 210, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 252 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 204. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 252 and a light chain variable domain consisting of SEQ ID NO. 204, i.e.the antibody is 222H175L.

In one embodiment, the first antibody may be 222 from table 1 and the second antibody may be 269 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 255, 256 and 257, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 278, 279 and 280, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 252 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 274. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 252 and a light chain variable domain consisting of SEQ ID NO. 274, i.e.the antibody is 222H269L.

In one embodiment, the first antibody may be 269 from table 1 and the second antibody may be 150 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOS 275, 276 and 277, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOS 158, 159 and 160, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 272, and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 154. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO:272 and a light chain variable domain consisting of SEQ ID NO:154, i.e., the antibody is 269H150L.

In one embodiment, the first antibody may be 269 from table 1 and the second antibody may be 158 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs 275, 276 and 277, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs 168, 169 and 170, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 272, and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 164. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO:272 and a light chain variable domain consisting of SEQ ID NO:164, i.e., the antibody is 269H158L.

In one embodiment, the first antibody may be 269 from table 1 and the second antibody may be 175 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs 275, 276 and 277, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs 208, 209 and 210, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 272, and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 204. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO:272 and a light chain variable domain consisting of SEQ ID NO:204, i.e., the antibody is 269H175L.

Furthermore, because of the similarity between IGHV3-53 and IGHV3-66, the exchange of the light and heavy chains of these antibodies may result in antibodies useful in the present invention. Thus, an antibody of the invention may comprise a heavy chain variable domain comprising CDRH1, CDRH2 and CDRH3 from a first antibody in table 1 and a light chain variable domain comprising CDRL1, CDRL2 and CDRL3 from a second antibody in table 1, wherein the first and second antibodies are derived from germline heavy chain IGHV3-53 or IGHV3-66. Thus, the first or second table 1 antibody may be selected from 150, 158, 175, 222, 269, 40, 398.

In one embodiment, the first antibody may be 150 from table 1 and the second antibody may be 40 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 155, 156 and 157, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 28, 29 and 30, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 152 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 24. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 152 and a light chain variable domain consisting of SEQ ID NO. 24, i.e.the antibody is 150H40L.

In one embodiment, the first antibody may be 150 from table 1 and the second antibody may be 398 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs 155, 156 and 157, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs 398, 399 and 400, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 152 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 394. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 152 and a light chain variable domain consisting of SEQ ID NO. 394, i.e., the antibody is 150H398L.

In one embodiment, the first antibody may be 40 from table 1 and the second antibody may be 150 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOS 25, 26 and 27, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOS 158, 159 and 160, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity with SEQ ID NO. 22, and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity with SEQ ID NO. 154. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 22 and a light chain variable domain consisting of SEQ ID NO. 154, i.e.the antibody is 40H150L.

In one embodiment, the first antibody may be 40 from table 1 and the second antibody may be 158 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs 25, 26 and 27, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs 168, 169 and 170, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity with SEQ ID NO. 22, and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity with SEQ ID NO. 164. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 22 and a light chain variable domain consisting of SEQ ID NO. 164, i.e.the antibody is 40H158L.

In one embodiment, the first antibody may be 40 from table 1 and the second antibody may be 175 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 25, 26 and 27, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 208, 209 and 210, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity with SEQ ID NO. 22, and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity with SEQ ID NO. 204. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 22 and a light chain variable domain consisting of SEQ ID NO. 204, i.e.the antibody is 40H175L.

In one embodiment, the first antibody may be 40 from table 1 and the second antibody may be 222 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs 25, 26 and 27, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs 258, 259 and 260, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity with SEQ ID NO. 22, and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity with SEQ ID NO. 254. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 22 and a light chain variable domain consisting of SEQ ID NO. 254, i.e.the antibody is 40H222L.

In one embodiment, the first antibody may be 40 from table 1 and the second antibody may be 269 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs 25, 26 and 27, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs 278, 279 and 280, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 22, and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 274. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 22 and a light chain variable domain consisting of SEQ ID NO. 274, i.e.the antibody is 40H269L.

In one embodiment, the first antibody may be 40 from table 1 and the second antibody may be 398 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs 25, 26 and 27, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs 398, 399 and 400, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity with SEQ ID NO. 22, and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity with SEQ ID NO. 394. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 22 and a light chain variable domain consisting of SEQ ID NO. 394, i.e., the antibody is 40H398L.

In one embodiment, the first antibody may be 398 from table 1 and the second antibody may be 150 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOS 395, 396 and 397, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOS 158, 159 and 160, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 392 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 154. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 392 and a light chain variable domain consisting of SEQ ID NO. 154, i.e.the antibody is 398H150L.

In one embodiment, the first antibody may be 398 from table 1 and the second antibody may be 158 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOS 395, 396 and 397, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOS 168, 169 and 170, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 392 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 164. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 392 and a light chain variable domain consisting of SEQ ID NO. 164, i.e.the antibody is 398H158L.

In one embodiment, the first antibody may be 398 from table 1 and the second antibody may be 175 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOS 395, 396 and 397, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOS 208, 209 and 210, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 392 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 204. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 392 and a light chain variable domain consisting of SEQ ID NO. 204, i.e.the antibody is 398H175L.

In one embodiment, the first antibody may be 398 from table 1 and the second antibody may be 222 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOS 395, 396 and 397, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOS 258, 259 and 260, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 392 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 254. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 392 and a light chain variable domain consisting of SEQ ID NO. 254, i.e.the antibody is 398H222L.

In one embodiment, the first antibody may be 398 from table 1 and the second antibody may be 269 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOS 395, 396 and 397, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOS 278, 279 and 280, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 392 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 274. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO:392 and a light chain variable domain consisting of SEQ ID NO:274, i.e.the antibody is 398H269L.

In one embodiment, the first antibody may be 398 from table 1 and the second antibody may be 40 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOS 395, 396 and 397, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOS 28, 29 and 30, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 392 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 24. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 392 and a light chain variable domain consisting of SEQ ID NO. 24, i.e.the antibody is 398H40L.

The antibody can comprise a heavy chain variable domain comprising CDRH1, CDRH2 and CDRH3 from the first antibody in table 1 and a light chain variable domain comprising CDRL1, CDRL2 and CDRL3 from the second antibody in table 1, wherein the first antibody and the second antibody are derived from the same germline light chain IG kappa V3-20. Thus, the first or second table 1 antibody may be selected from 55, 159, 165, 222, 253, and 318.

In one embodiment, the first antibody may be 55 from table 1 and the second antibody may be 159 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOS 65, 66 and 67, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOS 178, 179 and 180, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 62, and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 174. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 62 and a light chain variable domain consisting of SEQ ID NO. 174, i.e.the antibody is 55H159L.

In one embodiment, the first antibody may be 55 from table 1 and the second antibody may be 222 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 65, 66 and 67, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 258, 259 and 260, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 62, and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 254. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 62 and a light chain variable domain consisting of SEQ ID NO. 254, i.e.the antibody is 55H222L.

In one embodiment, the first antibody may be 159 from table 1 and the second antibody may be 55 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 175, 176 and 177, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 68, 69 and 70, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 172 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 64. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 172 and a light chain variable domain consisting of SEQ ID NO. 64, i.e.the antibody is 159H55L.

In one embodiment, the first antibody may be 159 from table 1 and the second antibody may be 165 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 175, 176 and 177, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 188, 189 and 190, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 172 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 184. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 172 and a light chain variable domain consisting of SEQ ID NO. 184, i.e.the antibody is 159H165L.

In one embodiment, the first antibody may be 159 from table 1 and the second antibody may be 222 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 175, 176 and 177, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 258, 259 and 260, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 172 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 254. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 172 and a light chain variable domain consisting of SEQ ID NO. 254, i.e.the antibody is 159H222L.

In one embodiment, the first antibody may be 159 from table 1 and the second antibody may be 253 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 175, 176 and 177, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 268, 269 and 270, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 172 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 264. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 172 and a light chain variable domain consisting of SEQ ID NO. 264, i.e.the antibody is 159H253L.

In one embodiment, the first antibody may be 159 from table 1 and the second antibody may be 318 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 175, 176 and 177, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 338, 339 and 340, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 172, and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 334. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 172 and a light chain variable domain consisting of SEQ ID NO. 334, i.e.the antibody is 159H318L.

In one embodiment, the first antibody may be 165 from table 1 and the second antibody may be 159 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOS 185, 186 and 187, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOS 178, 179 and 180, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 182 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 174. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 182 and a light chain variable domain consisting of SEQ ID NO. 174, i.e.the antibody is 165H159L.

In one embodiment, the first antibody may be 165 from table 1 and the second antibody may be 222 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 185, 186 and 187, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 258, 259 and 260, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 182 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 254. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 182 and a light chain variable domain consisting of SEQ ID NO. 254, i.e.the antibody is 165H222L.

In one embodiment, the first antibody may be 222 from table 1 and the second antibody may be 55 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 255, 256 and 257, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 68, 69 and 70, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 252 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 64. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 252 and a light chain variable domain consisting of SEQ ID NO. 64, i.e.the antibody is 222H55L.

In one embodiment, the first antibody may be 222 from table 1 and the second antibody may be 159 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOS 255, 256 and 257, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOS 178, 179 and 180, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 252 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 174. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 252 and a light chain variable domain consisting of SEQ ID NO. 174, i.e.the antibody is 222H159L.

In one embodiment, the first antibody may be 222 from table 1 and the second antibody may be 165 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NO. 255, 256 and 257, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NO. 188, 189 and 190, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 252 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 184. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 252 and a light chain variable domain consisting of SEQ ID NO. 184, i.e.the antibody is 222H165L.

In one embodiment, the first antibody may be 222 from table 1 and the second antibody may be 253 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 255, 256 and 257, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 268, 269 and 270, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 252 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 264. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 252 and a light chain variable domain consisting of SEQ ID NO. 264, i.e.the antibody is 222H253L.

In one embodiment, the first antibody may be 222 from table 1 and the second antibody may be 318 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 255, 256 and 257, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 338, 339 and 340, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 252 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 334. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 252 and a light chain variable domain consisting of SEQ ID NO. 334, i.e.the antibody is 222H318L.

In one embodiment, the first antibody may be 253 from table 1 and the second antibody may be 159 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOS 265, 266 and 267, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOS 178, 179 and 180, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO:262, and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO: 174. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO:262 and a light chain variable domain consisting of SEQ ID NO:174, i.e., the antibody is 253H159L.

In one embodiment, the first antibody may be 253 from table 1 and the second antibody may be 222 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOS 265, 266 and 267, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOS 258, 259 and 260, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO:262, and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO: 254. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO:262 and a light chain variable domain consisting of SEQ ID NO:254, i.e., the antibody is 253H222L.

In one embodiment, the first antibody may be 318 from table 1 and the second antibody may be 159 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOS 335, 336 and 337, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOS 178, 179 and 180, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 332, and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 174. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 332 and a light chain variable domain consisting of SEQ ID NO. 174, i.e.the antibody is 318H159L.

In one embodiment, the first antibody may be 318 from table 1 and the second antibody may be 222 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs 335, 336 and 337, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs 258, 259 and 260, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity with SEQ ID NO. 332, and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity with SEQ ID NO. 254. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 332 and a light chain variable domain consisting of SEQ ID NO. 254, i.e.the antibody is 318H222L.

The antibody can comprise a heavy chain variable domain comprising CDRH1, CDRH2 and CDRH3 from the first antibody in table 1 and a light chain variable domain comprising CDRL1, CDRL2 and CDRL3 from the second antibody in table 1, wherein the first antibody and the second antibody are derived from the same germline light chain IG kappa V1-9. Thus, the first or second table 1 antibody may be selected from 150, 158 and 269.

The antibody may comprise a heavy chain variable domain comprising CDRH1, CDRH2 and CDRH3 from the first antibody in table 1 and a light chain variable domain comprising CDRL1, CDRL2 and CDRL3 from the second antibody in table 1, wherein the first and second antibodies are derived from the same germline heavy chain IGHV1-58. Thus, the first or second table 1 antibody may be selected from 55, 165, 253 and 318.

In one embodiment, the first antibody may be 253 from table 1 and the second antibody may be 55 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOS 265, 266 and 267, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOS 68, 69 and 70, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO:262, and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO: 64. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO:262 and a light chain variable domain consisting of SEQ ID NO:64, i.e., the antibody is 253H55L. The antibody has potent cross-lineage neutralization, e.g., it is effective against all of the SARS-CoV-2 strains tested in the examples (as shown in Table 18 and FIG. 35).

In one embodiment, the first antibody may be 253 from table 1 and the second antibody may be 165 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOS 265, 266 and 267, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOS 188, 189 and 190, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO:262, and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO: 186. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO:262 and a light chain variable domain consisting of SEQ ID NO:186, i.e., the antibody is 253H165L. The antibody has potent cross-lineage neutralization, e.g., it is effective against all of the SARS-CoV-2 strains tested in the examples (as shown in Table 18 and FIG. 35).

In one embodiment, the first antibody may be 55 from table 1 and the second antibody may be 165 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NO. 65, 66 and 67, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NO. 188, 189 and 190, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 62, and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 184. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 62 and a light chain variable domain consisting of SEQ ID NO. 184, i.e.the antibody is 55H165L.

In one embodiment, the first antibody may be 55 from table 1 and the second antibody may be 253 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 65, 66 and 67, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 268, 269 and 270, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 62, and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 264. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 62 and a light chain variable domain consisting of SEQ ID NO. 264, i.e.the antibody is 55H253L.

In one embodiment, the first antibody may be 55 from table 1 and the second antibody may be 318 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOS 65, 66 and 67, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOS 338, 339 and 340, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 62, and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 334. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 62 and a light chain variable domain consisting of SEQ ID NO. 334, i.e.the antibody is 55H318L.

In one embodiment, the first antibody may be 165 from table 1 and the second antibody may be 55 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 185, 186 and 187, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 68, 69 and 70, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 182 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 64. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 182 and a light chain variable domain consisting of SEQ ID NO. 62, i.e.the antibody is 165H55L.

In one embodiment, the first antibody may be 165 from table 1 and the second antibody may be 253 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 185, 186 and 187, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 268, 269 and 270, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 182 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 264. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 182 and a light chain variable domain consisting of SEQ ID NO. 264, i.e.the antibody is 165H253L.

In one embodiment, the first antibody may be 165 from table 1 and the second antibody may be 318 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOS 185, 186 and 187, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOS 338, 339 and 340, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 182 and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO 334. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 182 and a light chain variable domain consisting of SEQ ID NO. 334, i.e.the antibody is 165H318L.

In one embodiment, the first antibody may be 253 from table 1 and the second antibody may be 318 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOS 265, 266 and 267, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOS 338, 339 and 340, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO:262, and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO: 334. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO:262 and a light chain variable domain consisting of SEQ ID NO:334, i.e., the antibody is 253H318L.

In one embodiment, the first antibody may be 318 from table 1 and the second antibody may be 55 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID nos. 335, 336 and 337, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID nos. 68, 69 and 70, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity with SEQ ID NO. 332, and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity with SEQ ID NO. 64. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 332 and a light chain variable domain consisting of SEQ ID NO. 64, i.e.the antibody is 318H55L.

In one embodiment, the first antibody may be 318 from table 1 and the second antibody may be 165 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOS 335, 336 and 337, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOS 188, 189 and 190, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity with SEQ ID NO. 332, and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity with SEQ ID NO. 184. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 332 and a light chain variable domain consisting of SEQ ID NO. 184, i.e.the antibody is 318H165L.

In one embodiment, the first antibody may be 318 from table 1 and the second antibody may be 253 from table 1. Thus, an antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOS 335, 336 and 337, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOS 268, 269 and 270, respectively. The antibody may comprise a heavy chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 332, and a light chain variable domain having 80% > or 90% > or 95% > or 96% > or 97% > or 98% > or 99% or 100% sequence identity to SEQ ID NO. 264. The antibody may comprise a heavy chain variable domain consisting of SEQ ID NO. 332 and a light chain variable domain consisting of SEQ ID NO. 264, i.e.the antibody is 318H253L.

Antibody conjugates

The invention also relates to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof) or a radioisotope (i.e., a radioactive conjugate). Conjugates of antibodies and cytotoxic agents may be made using a variety of bifunctional protein coupling agents known in the art.

The antibodies of the invention may be conjugated to molecules that modulate or alter serum half-life. The antibodies of the invention may bind to albumin, for example, to modulate serum half-life. In one embodiment, the antibodies of the invention will also include a binding region specific for albumin. In another embodiment, the antibodies of the invention may include a peptide linker that is an albumin binding peptide. Examples of albumin binding peptides are included in WO 2015/197772 and WO 2007/106120, the entire contents of both of which are incorporated by reference.

Polynucleotides, vectors and host cells

The invention also provides one or more isolated polynucleotides (e.g., DNA) encoding an antibody of the invention. In one embodiment, polynucleotide sequences are co-present on more than one polynucleotide, but together they are capable of encoding an antibody of the invention. For example, a polynucleotide may encode the heavy and/or light chain variable regions of an antibody of the invention. The polynucleotide may encode the complete heavy and/or light chain of an antibody of the invention. Typically, one polynucleotide will encode each of the heavy and light chains.

Polynucleotides encoding antibodies of the invention may be obtained by methods well known to those skilled in the art. For example, DNA sequences encoding part or all of the heavy and light chains of an antibody may be synthesized from the corresponding amino acid sequences as desired.

General methods by which vectors can be constructed, transfection methods and culture methods are well known to those skilled in the art. In this respect, reference is made to "Current Protocols in Molecular Biology [ guidelines of molecular biology laboratory ]",1999, F.M. Ausubel (editions), wiley Interscience, new York [ journal database of Willi Press, new York ] and the Maniatis handbook published by Cold spring harbor Press.

The polynucleotides of the invention may be provided in the form of an expression cassette comprising a control sequence operably linked to an insertion sequence, thereby allowing expression of the antibodies of the invention in vivo. Accordingly, the invention also provides one or more expression cassettes encoding one or more polynucleotides encoding the antibodies of the invention. These expression cassettes are in turn typically provided within a vector (e.g., a plasmid or recombinant viral vector). Thus, in one embodiment, the invention provides a vector encoding an antibody of the invention. In another embodiment, the invention provides vectors that collectively encode the antibodies of the invention. These vectors may be cloning vectors or expression vectors. Suitable vectors may be any vector capable of carrying a sufficient amount of genetic information and allowing expression of the polypeptides of the invention.

The polynucleotides, expression cassettes or vectors of the invention are introduced into host cells, for example by transfection. Accordingly, the invention also provides a host cell comprising one or more polynucleotides, expression cassettes or vectors of the invention. The polynucleotides, expression cassettes or vectors of the invention may be introduced transiently or permanently into a host cell, allowing for expression of antibodies from one or more of the polynucleotides, expression cassettes or vectors. Such host cells include transient or preferably stable higher eukaryotic cell lines (such as mammalian cells or insect cells), lower eukaryotic cells (such as yeast) or prokaryotic cells (such as bacterial cells). Specific examples of cells include mammalian HEK293 (such as HEK293F, HEK293T, HEK293S or HEK Expi 293F), CHO, heLa, NS0 and COS cells or any other cell line used herein (such as the cell lines used in the examples). Preferably, the cell line selected will be one that is not only stable but also allows for mature glycosylation.

The invention also provides a process for producing an antibody of the invention comprising culturing a host cell containing one or more vectors of the invention under conditions suitable for expression of the antibody from one or more polynucleotides of the invention, and isolating the antibody from said culturing.

Antibody combinations

The inventors have found that certain table 1 antibodies are particularly effective when used in combination, e.g., to maximize therapeutic effect and/or improve diagnostic ability. Useful combinations include antibodies that do not cross-compete with each other and/or bind to non-overlapping epitopes, as exemplified in tables 4 and 5.

Thus, the present invention provides a combination of antibodies of the invention, wherein each antibody is capable of binding to the spike protein of coronavirus SARS-CoV-2, wherein each antibody: (a) At least three CDRs comprising any one of the 42 antibodies in table 1; or (b) binds to the same epitope as antibodies 159, 45 or 384 or competes with these antibodies. In certain embodiments, the table 1 antibodies may be:

-a pair of antibodies listed in the row of table 4;

-a pair of antibodies listed in the row of table 5;

three antibodies listed in the row of table 5;

a pair of antibodies listed in the row of table 4 and antibody 159;

a pair of antibodies listed in the row of table 5 and antibody 159;

three antibodies listed in the row of table 5 and antibody 159;

-any two or more antibodies selected from the group consisting of: 384. 159, 253H55L, 253H165L, 253, 88, 40, and 316;

-any two or more antibodies selected from the group consisting of: 253. 253H55L and 253H165L, 222, 318, 55 and 165;

-any two or more antibodies selected from the group consisting of: 158H222L, 222, 150H222L, 384, 159, 253H55L, 253H165L, 253, 88, 40 and 316; or alternatively

-any two or more antibodies selected from the group consisting of: 158H222L, 222, 150H222L, 253H55L and 253H165L, 222, 318, 55 and 165.

In one embodiment, the invention provides a combination of any of the antibodies described in table 1.

In one embodiment, the invention provides a combination of any of the antibodies disclosed herein (such as any of the antibodies listed in tables 1, 21, 22, 23, 24, and/or 25).

The antibody combinations of the invention may be used as therapeutic mixtures. Accordingly, the present invention also provides a pharmaceutical composition comprising the antibody combination of the invention, as further explained below.

The antibody combinations of the invention are useful for diagnosis. Accordingly, the invention also provides a diagnostic kit comprising the antibody combination of the invention. Also provided herein are methods of diagnosing a disease or complication associated with a coronavirus infection in a subject, as further explained below.

Pharmaceutical composition

The invention provides a pharmaceutical composition comprising an antibody of the invention. The composition may comprise a combination (such as two, three or four) of the antibodies of the invention. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier.

The compositions of the present invention may include one or more pharmaceutically acceptable salts. By "pharmaceutically acceptable salt" is meant a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects. Examples of such salts include acid addition salts and base addition salts.

Suitable pharmaceutically acceptable carriers include aqueous carriers or diluents. Examples of suitable aqueous carriers include water, buffered water, and brine.

Other suitable pharmaceutically acceptable carriers include ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). In many cases, it will be desirable to include isotonic agents (e.g., sugars), polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.

Therapeutic compositions must generally be sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentrations.

The pharmaceutical compositions of the invention may comprise additional therapeutic agents, such as antiviral agents. Antiviral agents bind to coronaviruses and inhibit viral activity. Alternatively, the antiviral agent may not bind directly to coronavirus, but still affect viral activity/infectivity. The antiviral agent may be an additional anti-coronavirus antibody that binds somewhere on SARS-CoV-2 other than the spike protein. Examples of antiviral agents useful in the present invention include Remdesivir (Remdesivir), lopinavir (Lopinavir), ritonavir (ritonavir), APN01, and favalavir (Favilavir).

The additional therapeutic agent may be an anti-inflammatory agent, such as a corticosteroid (e.g., dexamethasone) or a non-steroidal anti-inflammatory drug (e.g., tolizumab).

The additional therapeutic agent may be an anti-coronavirus vaccine.

The pharmaceutical composition can be administered subcutaneously, intravenously, intradermally, intramuscularly, intranasally or orally.

Also within the scope of the invention are kits comprising an antibody or other composition of the invention and instructions for use. The kit may further comprise one or more additional agents, such as additional therapeutic or prophylactic agents as discussed herein.

The method and use of the invention

The invention further relates to the use of the antibodies, antibody combinations and pharmaceutical compositions described herein, e.g. in a method of treating the human or animal body by therapy or in a diagnostic method. The method of treatment may be therapeutic or prophylactic.

For example, the invention relates to methods of treating coronavirus (e.g., SARS-CoV-2) infection, diseases or complications associated therewith (e.g., COVID-19). The method may comprise administering a therapeutically effective amount of an antibody, a combination of antibodies, or a pharmaceutical composition of the invention. The method can further comprise identifying the presence of a coronavirus (e.g., SARS-CoV-2) in the sample from the subject. The invention also relates to an antibody, antibody combination or pharmaceutical composition according to the invention for use in a method of treating a coronavirus (e.g., SARS-CoV-2) infection, a disease or complication associated therewith (e.g., covd-19).

The invention also relates to a method of formulating a composition for use in treating a coronavirus (e.g., SARS-CoV-2) infection, a disease or complication associated therewith (e.g., covd-19), wherein the method comprises admixing an antibody, antibody combination or pharmaceutical composition according to the invention with an acceptable carrier to prepare the composition.

The invention also relates to the use of an antibody, antibody combination or pharmaceutical composition according to the invention for the manufacture of a medicament for the treatment of coronavirus (e.g. SARS-CoV-2) infection or a disease or complication associated therewith (e.g. covd-19).

The invention also relates to the prevention, treatment or diagnosis of coronavirus infection caused by any SARS-CoV-2 strain as described herein. Coronavirus infection may be caused by any SARS-CoV-2 strain, including members of lineages a, a.1, a.2, a.3, a.5, B, b.1, b.1.1, b.2, b.3, b.4, b.1.1.7, b.1.351, p.1, b.1.617.2 or b.1.1.529. In particular, the invention relates to the prevention, treatment or diagnosis of coronavirus infection caused by SARS-CoV-2 strain from lineage b.1.1.7, b.1.351, p.1, b.1.617.2 or b.1.1.529. The invention also relates to the prevention, treatment or diagnosis of coronavirus infection caused by a SARS-CoV-2 strain from lineage b.1.1.7, b.1.351, p.1, b.1.617.2, b.1.1.529, b.1.526.2, b.1.617.1, b.1.258, c.37 or c.36.3.

The invention also provides an antibody, antibody combination or pharmaceutical composition of the invention for use in treating a coronavirus infection or disease or complication associated therewith caused by a SARS-CoV-2 strain comprising one or more mutations, for example in a spike protein, relative to hCoV-19/Wuhan/WIV04/2019 (WIV 04) (GISAID accession number epi_isl_ 402124). In other words, the SARS-CoV-2 strain can be, for example, a modified hCoV-19/Wuhan/WIV04/2019 (WIV) strain that includes one or more modifications in the spike protein.

These mutations may be N501Y in the spike protein relative to the spike protein of hCoV-19/Wuhan/WIV04/2019 (WIV); residues 69 to 70 are deleted, residue 144 is deleted, A570D, D614G, P681H, T716I, S982A and/or D1118H. In particular, the SARS-CoV-2 strain comprises an N501Y mutation in the spike protein. The SARS-CoV-2 strain can comprise all of the mutations in the spike proteins listed above. The SARS-CoV-2 strain can be a member of the B.1.1.7 lineage.

For example, the SARS-CoV-2 strain can comprise deletions of residues 69-70 and N501Y in the spike protein relative to the spike protein in hCoV-19/Wuhan/WIV 04/2019. Alternatively, the SARS-CoV-2 strain can comprise a deletion of residues 69-70 in the spike protein; deletion of residue 144; E484K, A570D, D614G, P681H, T716I, S982A and D1118H.

These mutations may be the deletion of K417N, E484K, N501Y, L18F, D80G, D215G, 242-244, R246I, D614G and/or A701V in the spike protein relative to the spike protein of hCoV-19/Wuhan/WIV04/2019 (WIV). In particular, the SARS-CoV-2 strain comprises an E484K mutation in the spike protein. The SARS-CoV-2 strain can comprise all of the mutations in the spike proteins listed above. The SARS-CoV-2 strain can be a member of the B.1.351 lineage.

For example, the SARS-CoV-2 strain can comprise K417N, E484K, N501Y, D80G, D215G, deletion of residues 242-244, D614G and/or A701V in the spike protein relative to the spike protein of hCoV-19/Wuhan/WIV04/2019 (WIV 04). Alternatively, the SARS-CoV-2 strain can comprise a deletion of residues 242-244 and N501Y in the spike protein. Alternatively, the SARS-CoV-2 strain can comprise a deletion of residues 242-244 and E484K in the spike protein.

These mutations may be K417T, E484K, N501Y, L, 5262K, N501, F, T20N, P26S, D138Y, R190S, H Y and/or T1027I in the spike protein relative to the spike protein of hCoV-19/Wuhan/WIV04/2019 (WIV). In particular, the SARS-CoV-2 strain comprises an E484K mutation in the spike protein. The SARS-CoV-2 strain can comprise all of the mutations in the spike proteins listed above. The SARS-CoV-2 strain can be a member of the Y501.V2 lineage.

These mutations may be L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D G, H655Y, T1027I and/or V1176F in the spike protein relative to the spike protein of hCoV-19/Wuhan/WIV04/2019 (WIV). The SARS-CoV-2 strain can comprise all of the mutations in the spike proteins listed above. The SARS-CoV-2 strain can be a member of the P.1 lineage.

The mutation may be a mutation (e.g., a substitution) at position 417 in the spike protein, relative to the spike protein of hCoV-19/Wuhan/WIV04/2019 (WIV) (GISAID accession number: EPI_ISL_ 402124), wherein the substitution is from a lysine residue to another amino acid residue, such as asparagine (N) or threonine (T).

The mutation may be a mutation (e.g., a substitution) at position 501 in the spike protein, relative to the spike protein of hCoV-19/Wuhan/WIV04/2019 (WIV) (GISAID accession number: EPI_ISL_ 402124), wherein the substitution is from an asparagine residue to another amino acid residue, such as tyrosine.

The mutation may be a mutation (e.g., a substitution) at position 484 in the spike protein, relative to the spike protein of hCoV-19/Wuhan/WIV04/2019 (WIV) (GISAID accession number: EPI_ISL_ 402124), wherein the substitution is from a glutamic acid residue to another amino acid residue, such as lysine.

The mutations may be mutations (e.g., substitutions) at positions 417, 484 and 501 in the spike protein relative to the spike protein of hCoV-19/Wuhan/WIV04/2019 (WIV) (GISAID accession number: EPI_ISL_ 402124) as shown above.

For example, the SARS-CoV-2 strain can comprise a mutation at position 19, 142, 156, 157, 158, 452, 478, 614, 618 and/or 950 in the spike protein relative to the spike protein of hCoV-19/Wuhan/WIV04/2019 (WIV) (GISAID accession number: EPI_ISL_ 402124). The SARS-Cov-2 strain can comprise a member of the lineage that replaces the T19R, G142D, R158G, L452R, T478K, D614G, P681R, D950N, e.g., the B1.617.2 (delta) strain or derived therefrom.

The mutation may be a mutation at position 452 in the spike protein relative to the spike protein of hCoV-19/Wuhan/WIV04/2019 (WIV) (GISAID accession number: EPI_ISL_ 402124). For example, the mutation may be a substitution from leucine (L) to another amino acid residue, such as arginine (R) or glutamine (Q). The SARS-Cov-2 strain can comprise a mutation L452R, such as the B.1.617.2 (delta) strain or a member of the lineage derived therefrom, the B.1.617.1 (kappa) strain or a member of the lineage derived therefrom, or the C.36.3 strain or a member of the lineage derived therefrom. The SARS-Cov-2 strain can comprise a mutation L452Q, such as the C.37 (lambda) strain or a member of the lineage derived therefrom.

The mutation may be a mutation at position 478 in the spike protein relative to the spike protein of hCoV-19/Wuhan/WIV04/2019 (WIV) (GISAID accession number: EPI_ISL_ 402124). For example, the mutation may be a substitution from threonine (T) to another amino acid residue, such as lysine (K). The SARS-Cov-2 strain can comprise a mutation T478K, such as the B.1.617.2 (delta) strain or a member of the lineage derived therefrom.

The mutation may be a mutation at position 339, 371, 373, 375, 417, 440, 446, 477, 478, 484, 493, 496, 498, 501 and/or 505 in the spike protein relative to the spike protein of hCoV-19/Wuhan/WIV04/2019 (WIV) (GISAID accession number: EPI_ISL_ 402124). For example, the mutation may be a substitution from threonine (T) to another amino acid residue, such as lysine (K). The SARS-Cov-2 strain can comprise a member of the B.1.1.529 (omicron) strain or lineage derived therefrom in place of the G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y H.

Antibodies 58, 222, 253 and 253H/55L are particularly effective at neutralizing SARS-Cov-2 strain comprising a mutation at position 339, 371, 373, 375, 417, 440, 446, 477, 478, 484, 493, 496, 498, 501, 505 in the spike protein relative to the position 339, 371, 373, 375, 417, 440, 446 in the spike protein of hCoV-19/Wuhan/WIV04/2019 (WIV 04). Thus, the invention may relate to these antibodies for use in the treatment, prevention or diagnosis of coronavirus infection caused by a SARS-Cov-2 strain comprising a mutation at positions 339, 371, 373, 375, 417, 440, 446, 477, 478, 484, 493, 496, 498, 501, 505 in the spike protein relative to the position 339, 371, 373, 417, 440, 446, 477, 478, 484, 493, 496, 498 in the spike protein of hCoV-19/Wuhan/WIV04/2019 (WIV). Similarly, the invention relates to methods of using these antibodies and the use of these antibodies in the treatment, prevention or diagnosis of coronavirus infection caused by SARS-Cov-2 strains comprising mutations at positions 339, 371, 373, 375, 417, 440, 446, 477, 478, 484, 493, 496, 498, 501, 505 in the spike protein relative to the spike protein of hCoV-19/Wuhan/WIV04/2019 (WIV 04).

The SARS-CoV-2 strain can comprise all of the mutations described herein.

Methods and uses of the invention may include inhibiting a disease state (such as covd-19), e.g., arresting its development; and/or to alleviate a disease state (such as covd-19), for example, causing regression of the disease state until a desired endpoint is reached.

Methods and uses of the invention may include improving or lessening the severity of symptoms of a disease state (such as covd-19), improving or reducing its duration or frequency (e.g., lessening pain or discomfort), and such improvement may or may not directly affect the disease. The symptoms or complications may be fever, headache, fatigue, loss of appetite, myalgia, diarrhea, vomiting, abdominal pain, dehydration, respiratory tract infections, cytokine storms, acute Respiratory Distress Syndrome (ARDS) sepsis and/or organ failure (e.g., heart, kidney, liver, GI, lung).

The methods and uses of the invention can result in a reduction in viral load of coronavirus (e.g., SARS-CoV-2), for example, by ≡10%,. Gtoreq.20%,. Gtoreq.30%,. Gtoreq.40%,. Gtoreq.50%,. Gtoreq.60%,. Gtoreq.70%,. Gtoreq.80%,. Gtoreq.90% or 100% as compared to before treatment. Methods for determining viral load are well known in the art, such as infection assays.

Methods and uses of the invention may include preventing a coronavirus infection from occurring in a subject (e.g., a human), particularly when the subject is susceptible to complications associated with the coronavirus infection.

The invention also relates to identifying subjects infected with a coronavirus, such as SARS-CoV-2. For example, the methods and uses of the invention may involve identifying the presence of a coronavirus (e.g., SARS-CoV-2) or a protein or protein fragment thereof in a sample. The detection may be performed in vitro or in vivo. In certain embodiments, the invention relates to population screening.

The present invention relates to the identification of any SARS-CoV-2 strain, including members of lineages A, A.1, A.2, A.3, A.5, B, B.1, B.1.1, B.2, B.3, B.4, B.1.1.7, B.1.351, P.1, B.1.617.2 or B.1.1.529. In particular, the invention relates to the identification of SARS-CoV-2 strain from pedigree B.1.1.7, B.1.351 or P.1. The invention also relates to the identification of SARS-CoV-2 strain from pedigree B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.1.529, B.1.526.2, B.1.617.1, B.1.258, C.37 or C.36.3. Various strains of SARS-CoV-2 are discussed in more detail above.

It has also been identified that many of the antibodies herein cross-react with SARS-CoV-1. Thus, in one embodiment, the invention relates to the use of an antibody, antibody combination or pharmaceutical composition according to the invention to identify the presence of SARS-CoV-1, e.g. for use in diagnosing a SARS-CoV-1 infection or a disease or complication associated therewith.

The invention may also relate to a method of identifying escape mutants of SARS-CoV-2, comprising contacting a sample with a combination of antibodies of the invention and identifying whether each antibody binds to a virus. The term "escape mutant" refers to variants of SARS-CoV-2 that contain non-silent mutations that may affect the efficacy of existing treatments for SARS-CoV-2 infection. Typically, the non-silent mutation is on an epitope recognized by a prior art antibody and/or an antibody described herein that specifically binds to an epitope of SARS-CoV-2, e.g., on the spike protein of SARS-CoV-2. If the antibody does not bind to the target, it may be shown that the target comprises a mutation that may alter the efficacy of the existing SARS-CoV-2 treatment.

Methods and uses of the invention can include contacting a sample with an antibody or antibody combination of the invention, and detecting the presence or absence of an antibody-antigen complex, wherein the presence of the antibody-antigen complex is indicative of a subject being infected with SARS-CoV-2.

Methods for determining the presence of antibody-antigen complexes are known in the art. For example, in vitro detection techniques include enzyme-linked immunosorbent assays (ELISA), western blots, immunoprecipitation, and immunofluorescence. In vivo techniques include introducing a labeled anti-analyte protein antibody into a subject. For example, the antibody may be labeled with a radioactive label, and its presence and location in the subject may be detected by standard imaging techniques. Detection techniques may provide qualitative or quantitative readings, depending on the assay employed.

In general, the present invention relates to methods and uses for a human subject in need thereof. However, non-human animals such as rats, rabbits, sheep, pigs, cows, cats or dogs are also contemplated.

The subject may be at risk of exposure to a coronavirus infection, such as a healthcare worker or a person who has been in contact with an infected individual. The subject may have visited or plan to visit a country where coronaviruses are known or suspected to have exploded. Subjects may also be at greater risk, such as immunocompromised individuals, for example individuals receiving immunosuppressive therapy or individuals suffering from human immunodeficiency syndrome (HIV) or acquired immunodeficiency syndrome (AIDS).

The subject may be asymptomatic or before symptoms appear.

The subject may be in the early, mid or late stages of the disease.

The subject may be in a hospital or community at the time of first onset and/or later in the hospital.

The subject may be male or female. In certain embodiments, the subject is generally male.

The subject may not be infected with a coronavirus, such as SARS-CoV-2.

The subject may be susceptible to more severe symptoms or complications associated with the coronavirus infection. The methods or uses of the invention may include the step of identifying whether the patient is at risk of developing a more severe symptom or complication associated with the coronavirus.

In embodiments of the invention involving prophylaxis or treatment, the subject may or may not have been diagnosed with an infectious coronavirus, such as SARS-CoV-2.

The present invention relates to analyzing a sample from a subject. The sample may be tissue, cells, and biological fluids isolated from the subject, as well as tissues, cells, and fluids present in the subject. The sample may be blood and a portion or component of blood, including serum, plasma, or lymph. Typically, the sample is from a pharyngeal swab, a nasal swab, or saliva.

The antibody-antigen complex detection assay may be performed in situ, in which case the sample is a tissue section (fixed and/or frozen) of tissue obtained from a biopsy or resection of the subject.

In embodiments of the invention where antibody pharmaceutical compositions and combinations are administered, they may be administered subcutaneously, intravenously, intradermally, orally, intranasally, intramuscularly or intracranially. Typically, antibody pharmaceutical compositions and combinations are administered intravenously or subcutaneously.

The dosage of the antibody may vary depending on the age and size of the subject, and the disease, condition, and route of administration. The antibody may be administered at a dose of about 0.1mg/kg body weight to a dose of about 100mg/kg body weight, such as at a dose of about 5mg/kg to about 10 mg/kg. The antibody may also be administered at a dose of about 50mg/kg, 10mg/kg, or about 5mg/kg body weight.

The combination of the invention may be administered, for example, at a dose of about 5mg/kg to about 10mg/kg of each antibody or at a dose of about 10mg/kg or about 5mg/kg of each antibody. Alternatively, the combination may be administered at a dose of about 5mg/kg total (e.g., a dose of 1.67mg/kg for each antibody in the three antibody combinations).

The antibodies or antibody combinations of the invention may be administered in a multi-dose regimen. For example, the initial dose may be followed by a second or more subsequent doses. The second and subsequent doses may be spaced apart at appropriate times.

As discussed above, the antibodies of the invention are typically used in single pharmaceutical compositions/combinations (co-formulated). However, the invention also generally includes the combined use of the antibodies of the invention in separate formulations/compositions. The invention also includes the use of an antibody as described above in combination with an additional therapeutic agent.

The combined administration of two or more agents and/or antibodies can be accomplished in a number of different ways. In one embodiment, all components may be administered together in a single composition. In another embodiment, each component may be administered separately as part of a combination therapy.

For example, an antibody of the invention may be administered before, after, or simultaneously with another antibody of the invention or binding fragment thereof. Particularly useful combinations are shown, for example, in tables 4 and 5.

For example, the antibodies of the invention may be administered before, after, or simultaneously with an antiviral or anti-inflammatory agent.

In embodiments of the invention directed to detecting the presence of a coronavirus (e.g., SARS-CoV-2) or a protein or protein fragment thereof in a sample, the antibody contains a detectable label. Methods of attaching a tag to an antibody are known in the art, for example, by directly labeling the antibody by coupling (i.e., physically linking) a detectable substance to the antibody. Alternatively, the antibody may be labeled indirectly, for example by reaction with another reagent that is labeled directly. Examples of indirect labeling include detection of primary antibodies using a fluorescent-labeled secondary antibody and end-labeling of DNA probes with biotin so that they can be detected with fluorescent-labeled streptavidin.

The detecting may further comprise: (i) Agents are known that can be used to detect coronaviruses (e.g., SARS-CoV-2) or proteins or protein fragments thereof, such as antibodies to other epitopes of spike proteins or other proteins of coronaviruses, such as anti-nucleocapsid antibodies; and/or (ii) agents known to be unable to detect the presence of a coronavirus (e.g., SARS-CoV-2) or a protein or protein fragment thereof, i.e., to provide a negative control.

In certain embodiments, the antibodies are modified to have increased stability. Suitable modifications are explained above.

The invention also includes kits for detecting the presence of coronavirus (e.g., SARS-CoV-2) in a sample. For example, the kit may comprise: a labeled antibody or a combination of labeled antibodies of the invention; means for determining the amount of coronavirus (e.g., SARS-CoV-2) in the sample; and means for comparing the amount of coronavirus (e.g., SARS-CoV-2) in the sample to a standard. The labeled antibody or combination of labeled antibodies may be packaged in a suitable container. The kit may further comprise instructions for using the kit to detect coronavirus (e.g., SARS-CoV-2) in a sample. The kit may further include other agents known to be useful for detecting the presence of coronavirus, as discussed above.

For example, the antibodies or antibody combinations of the invention are used in lateral flow assays. Typically, a lateral flow test kit is a handheld device with an absorbent pad, which is based on a series of capillary beds, such as porous paper sheets, microstructured polymers, or sintered polymers. The test runs the liquid sample along the pad surface with reactive molecules that show a visually positive or negative result. The test may further include the use of other agents known to be useful in detecting the presence of coronavirus (e.g., SARS-CoV-2) or a protein or protein fragment thereof, as discussed above, such as anti-nucleocapsid antibodies.

Others

It will be appreciated that the different applications of the disclosed antibody combinations or the pharmaceutical compositions of the invention may be adapted to the specific needs of the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting.

In addition, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes two or more "antibodies".

Furthermore, when reference is made herein to ". Gtoreq.x", this means equal to or greater than x. When ". Ltoreq.x" is referred to herein, this means less than or equal to x.

When referring to sequence identity between two sequences, their sequences are compared. Sequences having identity share identical nucleotides at defined positions within the nucleic acid molecule. Thus, a first nucleic acid sequence that shares at least 70% nucleic acid sequence identity with a second sequence requires that at least 70% of the nucleotides in the first nucleic acid sequence are identical to the corresponding nucleotides in the second sequence after the first nucleic acid sequence is aligned with the second sequence.

The sequences are typically aligned for identity calculations using mathematical algorithms such as those developed in Karlin and Altschul (Proc. Natl. Acad. Sci. USA [ Proc. Natl. Acad. Sci. USA (1990): 2264 2268), such as those developed in Karlin and Altschul (Proc. Natl. Acad. Sci. USA [ Proc. Natl. Acad. Sci. USA ]90 (1993): 5873 5877). This algorithm was incorporated into the XBLAST program of Altschul et al (J. MoI. Biol. [ J. Mol. Biol. ]215 (1990): 403410). To obtain a gap alignment, gap BLAST as described in Altschul et al (Nucleic Acids Res. [ nucleic acids Ind. 25 (1997): 3389 3402) can be used. When utilizing BLAST and gapped BLAST programs, default parameters for the respective programs can be used.

The amino acid position numbering provided herein uses the IMGT numbering system @http://www.imgt.orgThe method comprises the steps of carrying out a first treatment on the surface of the Lefranc MP,1997,J,Immunol journal of immunology]Today,18,509), but in some cases the KABAT numbering system or absolute numbering of amino acids based on the sequence listing may be used.

All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

The following examples illustrate the invention.

Examples

Antibodies are critical for immune protection against SARS-CoV-2, some of which are used as therapeutic agents in emergency situations. As shown in the examples below, the inventors identified 377 human monoclonal antibodies (mabs) that recognized viral spikes and focused on 80 binding Receptor Binding Domains (RBDs). By mapping the antigenic sites using unique computational methods and comparing with inhibitory activity, the inventors showed that the binding sites were widely dispersed, but the neutralizing epitopes were highly concentrated. Almost all highly potent neutralizing mabs (IC 50<0.1 μg/ml) block receptor interactions, but have a unique epitope that binds the N-terminal domain. Many mabs use a common V gene and approach the germline, which is a good megahead for vaccine responses. The 19 Fab-antigen structures (some RBDs complexed with two fabs) revealed two novel patterns of engagement of potent inhibitory mabs. Several Fab's are glycosylated, enhancing neutralization of three of them, for two of which the sugar is in contact with the antigen. The most potent mabs have prophylactic or therapeutic protective effects in animal models.

Example 1 characterization of mAb

A group of 42 patients diagnosed by qRT-PCR (Table 2) that have been confirmed to have SARS-CoV-2 infection was studied. ELISA was performed against full-length stable S protein (Wuhan-Hu-1 strain, MN 908947), in which residues 986 and 987 in the linker between the two helices in S2 were mutated to Pro-Pro sequences to prevent conversion to post-fusion helix conformation (Walls et al 2020; wrapp et al 2020), RBD (aa 330-532) or N protein (FIG. 9A). Antibody titers varied from patient to patient and there was a strong correlation between neutralization titers or levels of memory B cells expressing anti-S and disease severity (Chen et al 2020B) (fig. 9B-C).

To generate mabs, two strategies were used. First, B cells expressing IgG were sorted, 4 cells per well, cultured with IL-2, IL-21 and 3T3-msCD40L cells for 13-14 days, and supernatants were tested for reactivity to S protein; positive clones were identified by RT-PCR (FIG. 10A). In the second method, B cells were stained with labeled S or RBD, and single positive cells were sorted and RT-PCR was performed (FIG. 10B). Cell recovery was higher in severe covd-19 cases (fig. 10C), and mabs from 16 patients (9 mild, 7 severe) were isolated in total.

377 antibodies were generated, which were found to react with full-length S by ELISA. mAb were further screened for reactivity to S1 (34%), S2 (53%), RBD (21%) and NTD (11%), the remaining 13% being reactive only to full length trimer spikes (fig. 11A). Analysis of the antibody sequences revealed low levels of somatic mutation in germline sequences of both heavy (average 4.11±2.75 amino acids) and light (average 4.10±2.84 amino acids) chains (fig. 11B). Generally, in-and inter-individual responses are highly polyclonal, with different V gene usage (fig. 11C). The cross-reactivity of 377 anti-S antibodies raised from SARS-CoV-2 patients with full-length S proteins from all human alpha and beta coronaviruses was tested (FIG. 1A). Cross-reactivity was observed in SARS-CoV-1 (52%), MERS (7%), OC43 (6%), HKU1 (7%), 229E (1%) and NL63 (1%). However, cross-reactivity was limited to SARS-CoV-1 for antibodies recognizing RBD, which shared 74% sequence identity with SARS-CoV-2, much more than with other human CoVs (19-21%). Antibodies cross-reactive between the RBDs of SARS-CoV-2 and SARS-CoV-1 showed similar low levels of germline mutations for the S-reactive antibodies of the whole pool. However, for antibodies cross-reactive between SARS-CoV-2 and the four seasonal coronaviruses, there are more germ line mutations in the heavy chain in particular (FIG. 11D). A reasonable explanation for the increase in germ line mutations in cross-reactive clones is that they are selected from pools of seasonal coronavirus-specific B cells, rather than being de novo produced by SARS-CoV-2.

EXAMPLE 2 neutralization Activity of SARS-CoV-2mAb

The Focal Reduction Neutralization Test (FRNT) was used to study the neutralization activity of all 377 mabs. Only 5% of non-RBD mabs showed neutralizing activity (IC ₅₀ <10 μg/ml), whereas 60% of RBD-specific mabs showed neutralizing activity (fig. 1B).

A total of 19 out of 80 anti-RBD antibodies were generated<IC 0.1 μg/ml ₅₀ Level (fig. 1C), defined herein as a strong neutralizing agent. FRNT50 values for selected antibodies are shown in table 3. Many antibodies outside RBD have weak neutralizing activity (IC ₅₀ The value is 0.29-7.38 mug/ml). Antibody mAb 159 binding to NTD (see below) was an IC at 5ng/ml ₅₀ One of the most potent inhibitory antibodies obtained.

The ability of the anti-RBD mAb to block interaction with ACE2 was measured using a competitive ELISA. For antibodies that showed neutralization, there was a broad correlation between inhibition potency and ACE2 blocking, while NTD binding mAb 159 did not block ACE2 binding (fig. 1C).

To investigate the contribution of RBD-binding antibodies to neutralization in polyclonal serum, serum from 8 convalescent donors was immunodepleted with recombinant RBD; depletion of anti-RBD activity was confirmed by ELISA. Neutralization assays performed in RBD depleted and mock depleted samples showed the major contribution made by anti-RBD antibodies (55-87% reduction), but also demonstrated significant role of non-RBD antibodies in the polyclonal neutralization reaction to SARS CoV-2 (fig. 1D).

EXAMPLE 3 mapping of RBD antigen surfaces

Paired competition between antibodies was measured in a 96-well plate format using Biological Layer Interferometry (BLI). 79 antibodies were used and filled a total of 4404 out of 6340 off-diagonal elements of the square competition matrix (see example 13).

To facilitate interpretation of the results, the naming convention is used for RBDs by comparison to the torso (FIG. 2A). The predicted locations covering most of the RBD surfaces were divided into 5 groups using a clustering algorithm (method and cluster4x (Ginn, 2020)) (fig. 2B, C). The left flank cluster differs from the other 4 clusters, which show significant competition at their boundaries and interact sequentially from left shoulder, neck, right shoulder to right flank. The competition between the left shoulder and neck was the strongest, but the neck and right shoulder groups also strongly cross-competed (fig. 2C).

ACE2 binding sites are shown in figure 2D and the positions of 76 individual antibodies (plus the outside) are depicted in figure 2E. Neck clustering is the attachment site of many antibodies with a common IGVH 3-53V region (Yuan et al 2020 b) and strongly overlaps with ACE2 binding sites (fig. 2D-E). Left flank clusters include the previously defined structures EY6A, CR3022 and H014, all of which are reported to exhibit neutralizing activity, but do not compete with ACE2 binding (Yuan et al, 2020a; huo et al, 2020; zhou et al, 2020; lv et al, 2020; wrobel et al, 2020). Although the left flank was largely separated from the neck and shoulder, the two mabs (38, 178) still competed well and were closer to the left shoulder than the more isolated antibodies (1, 22, 177) (fig. 2E). Some regions of RBD are notable for lack of antibody binding. Both right and left flank clusters interact with neck and shoulder clusters, but this does not create a complete antibody "band" around the waist of the RBD. Antibodies against the N and C termini were not seen due to incomplete presentation on RBD or occlusion by other portions of the spike.

In fig. 2F, the antibody neutralization sites are plotted on RBD. There is generally a good correlation between overlap and neutralization with ACE2 footprint. However, there are notable examples of non-neutralizing antibodies as good ACE2 blockers. Based on competition data, non-competitive strong neutralizing mAb pairs can be identified and triplets can be identified if the efficacy threshold is relaxed (tables 4 and 5). Such combinations may prove useful for therapeutic mixtures.

There is certainly a neutralization mechanism other than ACE2 blocking, such as 159 binding to NTD remote from the ACE2 binding site (see below). Interestingly, antibodies co-localized in the left and right flank clusters, respectively, with known neutralizing/protective antibodies EY6A/H014 and S309 (Huo et al 2020; zhou et al 2020; lv et al 2020) did not show significant neutralization in the assay.

Example 4 biophysical characterization of selected antibodies

The kinetics of RBD attachment of the 20 potent RBD binding agents are shown in table 3. K of Fab fragment _D Values are in the range of 0.7 to 7.6nM, and the dissociation rate that may be correlated with therapeutic efficacy is about 1,000-10,000s (Ylera et al, 2013). Expression levels, thermostability, monodispersity and freeze-thaw robustness of the 34 mabs are shown in table 6. All are stable at elevated temperatures, with Tm of 65-80 ℃ being observed for the first time (Walter et al 2012), more than 99% of the mass being in a single species. Almost all can accommodate 20 freeze-thaw cycles.

EXAMPLE 5 potentStructural analysis of monoclonal antibodies-focusing on limited epitopes

Based on the neutralization data (table 3), the antibodies were sent for structural analysis. The structure of 19 complexes (typically one or two fabs (8, by crystallography) bound to RBD alone or a single Fab or mAb (11, by cryo-EM) bound to trimeric spikes) was determined and these are presented in fig. 3 (see also methods, tables 7 and 8 and fig. 12, 13). The organization of the spikes is shown in fig. 4A. Antibody 159 bound to NTD (fig. 4B), while all other antibodies studied bound to RBD. Most RBD binders (40, 150, 158 and 269) bind to well-defined sites in the neck cluster, 253, 316 and 384 bind more toward the front of the left shoulder, and 88 binds toward the rear of the left shoulder (although the footprints overlap). Antibody 75 binds at the right shoulder. The footprints of all these antibodies overlap with the footprint of ACE2 (figure 3).

By selecting antibodies as potent neutralizing agents in the FRNT assay, a large number of high affinity antibodies are omitted. This can be seen for example in antibody 45, which has a K of 0.018 μg/ml _D . The mAb showed weak neutralization (IC ₅₀ 2 μg/ml) and predicted to map to the right flank (fig. 4C). Structural measurements of 45 in the ternary complex with the potent neutralizing agent 88 and RBD revealed binding in the predicted position (previously unreported site adjacent to the potent neutralizing agent S309) (Pinto et al 2020; piccoli et al 2020) (FIGS. 3, 4C, 4D) demonstrating the value of predictive mapping in identifying neoepitopes.

Example 6 binding of potent antibody 384 in a previously unreported pattern

Antibody 384 is the most potent neutralizing mAb (IC ₅₀ 2 ng/ml). The binding pattern is different from any other SARS-CoV-2 antibody reported so far. It approaches the binding site on top of the neck and left shoulder from the front, and the footprint is relatively small, as(heavy chain contribution->While light chain contribution->). Although the orientation of the bound 384Fab is similar to a set of previously reported Fab, including CV07-270, p2b-2f6 and bd629 (Krey et al 2020; ju et al 2020; du et al 2020), it moves toward the left shoulder +.>So that it does not contact the right chest (fig. 3, 4E). Only CDRs H2 and H3 of Fab 384HC interacted with antigen (fig. 5A). Remarkably, the 18 residue long H3 of Fab 384 binds across the top of the neck to reach the H3 binding site of a group of Fab with very short H3, including B38, CB6 and CC12.3 (discussed in example 7) (Wu et al, 2020B; shi et al, 2020; yuan et al, 2020B; hurlbart et al, 2020; wu et al, 2020a; du et al, 2020; clark et al, 2020), such that hydrophobic interactions occur from F104 and L105 at the tip to L455 and F456 of the RBD (fig. 5A). However, the primary interaction contributing to binding affinity and orientation is with RBD residues 482-486 on top of the shoulder. W107 of H3 has a strong pi-interaction with G485, Y59 of H2 contacts V483 and forms a bifurcated H bond with the carbonyl oxygen of G482 and the amino nitrogen of E484, which in turn forms a salt bridge with R52 and H bonds with the side chains of T57 and Y59 (FIG. 5A). E484-F486 also forms a double-stranded antiparallel beta sheet with residues A92-A94 of L3 and creates a stacking interaction from F486 to Y32 of L1. The advantage of backbone RBD interactions may confer resistance to escape of mutations.

EXAMPLE 7 repeated use of the heavy chain V region demonstrates a potent public response

The identified potent neutralizing agents often use the public HC V region (shared by most people compared to the private patient-specific response). Five potent mabs used IGVH3-53 (with 3-10 non-silent mutations) (fig. 5B). Competition data shows that these all bind at similar sites. The structure of the three members 150, 158 and 269 (others 175 and 222) of the group was determined, and the engagement patterns of all three members were found to be nearly identical (fig. 3, 14B). These areHas a shorter H3 (11 residues) and binds at the rear of the neck, with a similar footprint of aboutThe flat binding sites of RBD and the proximity angle of Fab limit their H3 length and the number of contacts to RBD (fig. 5C), however, this is compensated by interactions involving all CDRs from H1, H2 and light chain. In the case of 158, the four residues from H3 have a direct contact +.>And form two hydrogen bonds with RBD, contributing +.>In contrast, 6 residues of H1 and 5 residues of H2 participate in interactions with RBD and together form 6 hydrogen bonds, while the three CDRs of the light chain contribute 6 residues and 5 hydrogen bonds for binding (fig. 5C). The H3 length matches the optimal length of the V region previously reported (Yuan et al, 2020 b), and there is indeed a strong similarity between the H3 sequence of mAb 150 (SEQ ID NO: 433) and mAb CC1.12 previously reported (Yuan et al, 2020 b) (FIG. 14A). Thus, H1 and H2 determine the pattern of conjugation, which is common to many previous studies of antibodies with this V region (FIG. 14C) (Yuan et al, 2020 b).

Repeated conferring potent (IC) ₅₀ <0.1 μg/ml) is IGVH1-58 (mAb: 55. 165, 253, and 318), all of which are powerful). These have even fewer non-silent mutations (2 to 5) and longer HC CDR3 (12-16 residues). The three antibodies (55, 165, 253) have disulfide bonds in their CDR3 that strongly compete with each other for binding and mapping to the neck epitope, but do not compete with mAb 318. In mAb 253, disulfide bonds flank the glycosylation sequence (see below). The crystal structure of the complex comprising Fab 253 demonstrates its binding within the dominant neck epitope (fig. 3). In contrast, competition mapping indicated that Fab 318 binds at the right shoulder epitope (fig. 2E). For this V region, CDR3 appears to be more critical for recognition and can switch binding to different epitopes on the same antigen.Notably, this does not preclude strong binding to near germline V-region sequences.

The final V region with at least 2 potent neutralizers was IGHV3-66, which was found to have a total of 5-fold of 2 potent neutralizers (282 and 40). These two (with relatively few mutations from germline and CDR3 lengths 12 and 13, respectively) compete strongly. Again, the complex structure of one (Fab 40) was determined and demonstrated that the antibody bound directly in the dominant neck epitope as expected from competition data, with little distinction from those using IGHV3-53 (fig. 5D). IGHV3-66 mAb (398) had a much longer H3, 21 residues, and was predicted to bind on the edges of the neck epitope (FIG. 2E).

IGHV3.11 is found in the most potent neutralizing agent 384, but is also used by CV07-270 (Krey et al 2020). CV07-270 swings forward and sideways (FIG. 4E compared to 384) so that it does not compete with ACE2 binding, indicating that 384's potency derives from extended H3 interactions across the ACE2 binding site.

While IGHV3-30 was found in 11 RBD binders, none was a potent neutralizing agent. The structures of two representatives, 75 (ternary complex with 253) and 45 (ternary complex with 88) were determined (table 7). 75 bind on the right shoulder and overlap with the ACE2 binding site (fig. 3), whereas the only HC-RBD contact is via an extended 20 residue H3 (fig. 5E). The IGHV3-30 RBD binders varied in H3 length from 12 to 20 residues, indicating that they bound at different sites as demonstrated by a radically different binding of 45 with an H3 length of 14 residues to the left flank (fig. 3).

In summary, the primary public V regions used for potent antibodies are typically targeted to cervical epitopes, often utilizing the common binding patterns determined by the V regions (although they may occasionally switch epitopes), but this is not the case for weaker neutralizing agents. This may explain the overwhelming manifestation of the common binding pattern at the cervical epitope in the structure determined so far (fig. 14C).

Example 8 light chain mixing can increase neutralization titres

For three clusters of potent anti-RBD antibodies in which >2 members shared the same IGVH (IGHV 3-53, IGHV1-58, and IGHV 3-66), a mixing experiment was performed in which each IGVH matched all IGVL within the cluster (FIG. 6A). Chimeric antibodies were expressed and neutralized and compared to the original mAb clone. Unexpectedly, when the heavy chain of mAb 253 (IGVH 1-58, IGVK 3-20) was combined with the light chain of mAbs 55 and 165 (also IGVH1-58, IGVK3-20, but containing the IGKJ1 region in contrast to IGKJ2 in mAb 253), a 10-fold increase in neutralization titer was found (FIG. 6B). Notably, in mabs 55 and 165, the only difference in contact residues was the Tyr substitution Trp (fig. 6C). Structural analysis of Fab complexes with RBD revealed that the larger hydrophobic tryptophan side chains stabilized the hydrophobic region of the antibody and were in close proximity to the critical hydrophobic region of RBD (E484-F486) used by many potent neutralizers, while the smaller tyrosine side chains produced less contact.

In summary, the mapping method defines five binding clusters or epitopes. By analogy with the human torso, four of these clusters form a continuous band extending from the left shoulder to the neck, right shoulder and down the right flank of the torso, while the fifth forms a more discrete location toward the left flank. These sites are widely distributed on the surface, however 21 of the most potent (IC ₅₀ <0.1 μg/ml) and all but one of the mabs blocked receptor attachment to the neck. The only exception mAb 159 binds NTD and the mechanism of neutralization is not clear, fab 159 lack of neutralization suggests that aggregation may play a role, however this domain is often associated with receptor binding in other coronaviruses and 159 may interfere with co-receptor binding (Li, 2015).

There is now a basic database of antibody/antigen complexes of SARS-CoV-2 spike (84 PDB deposits by 12 months 2020, including nanobody structures). The number of unique structures is much smaller than this, and the concern for strong neutralization of the public V region means that many of these structures have nearly identical binding patterns therein (fig. 14). Systematic analysis was performed using neutralization and mapping to directly determine the structure of 19 complexes by crystallography and cryoEM in order to profile the high resolution details of the binding of the major class of potent neutralizing agents. Highly potent ACE2 blocking mabs map to two sites in the neck and left shoulder regions, residues E484-F486 bridge the epitope and can approach Fab binding from a variety of different angles of attack. Notably, the mutation F486L, which may affect the binding of some of these antibodies, has been identified in minks as a recurrent mutation associated with host adaptation (van Dorp et al 2020).

EXAMPLE 9 role of N-linked glycans in antibody interactions

15-25% of IgG are known to carry N-linked glycans in their variable region, sometimes having an effect on antigen binding. Of the 80 RBD-binding antibodies described herein, 14 (17.5%) contained glycosylated sequences resulting from somatic mutations in their variable regions. For 8 mabs (1, 88, 132, 253, 263, 316, 337, 382), these sequences were in HC, and for 5 mabs, they were in CDRs. Several of the HC mutations but none of the LC mutations were found in potent inhibitory antibodies (neutralizing IC ₅₀ <0.1 μg/ml). Two of these (88 and 316) can be deglycosylated without denaturation, and BLI analysis showed that this has negligible effect on RBD/Fab affinity (K _D =0.8/1.2 nM and 1.0/2.0nM,88 and 316 are deglycosylated/glycosylated, respectively), although the association rate is slightly faster in the absence of sugar (e.g., 1.4x10 with mAb 88) ⁵ 1/Ms ratio of 3.8X10 ⁵ 1/Ms). However, mutations that abrogate glycosylation had a detrimental effect on neutralization of both antibodies and 253H165L chimeras (fig. 15). The structure of mabs 88, 316 and 253 that were complexed with RBD and spikes was thus determined (fig. 3, 6D, 15, tables 7, 8).

Antibodies 88 and 316 contain glycosylation sites in H1 (N35) and H2 (N59), respectively. The crystal structure of RBD-316Fab complex is shown inA well defined density of 3 glycans (including α1,6 linked fucose) is shown at resolution (fig. 6D and 15E). The structure of Fab 88 was determined in ternary complexes with 45 and RBD to resolution +.>(88 ChCl domains are disorderedBut VhVl domains have a well-defined density). Antibody 88 binds to the rear of the neck and 316 binds to the top of the neck, with the orientation being radically different, whereas the H3 of the two fabs overlap well (fig. 6D and S7). The glycan of Fab 88 wraps around the back of the left shoulder like a necklace and the glycan of Fab 316 sits on top of the same shoulder. Fab 88 has->Footprint of (HC, LC and glycan are +.>And->) While Fab 316 hasFootprint of (HC, LC and glycan are +.>And->). Residues E484-F486 of RBD interact extensively in these antibodies with residues L1 and L3 from the 3 CDRs and LC as described above for mAb 384, so for 316, the side chain of E484H-bonds with N52 and S55 of H2 and Y33 of H1, G485 contacts W50 of H2, and F486 interacts strongly with Y93 and W99 of L3 and Y34 of L1. This suggests that E484-F486 constitutes a hotspot for the epitope. These residues are accessible from a variety of different angles of attack, so Fab 384, 316 and 88 all interact with this region, although their attitudes on RBD are significantly different. In contrast, H3 of 253 overlapped with glycans of mAb 88, whereas glycans of mAb 253 did not interact directly with RBD (fig. 6D).

In all cases, the sugar appears near the top of the left shoulder, and in 3 cases there are 2 interactions directly with the antigen, but rather weak. Although somatic mutations are quite rare, the frequency of continuum generation is high, which is interesting, indicating the presence of positive selection.

Although the most potent neutralizing mAb approaches the germline, somatic mutation introduces an N-linked glycosylation site into the variable region of 17.5% of the potent neutralizing agent. These may contribute to interactions with RBD and although they appear to have a relatively small impact on affinity, they significantly enhance neutralization.

Example 10 binding in the case of trimeric spike

On isolated stable spikes, the RBD was found to have two orientations: "up" and "down" (Roy et al 2020). These two orientations form a family of conformations, with up to 20 degrees of conformational change (Zhou et al, 2020), and the downward conformation may include a more closely packed "locked" conformation (Ke et al, 2020; toelzer et al, 2020; carrique et al, 2020; xiong et al, 2020). The structure seen by cryo-EM has RBD in a typical up or down conformation (see fig. 7A), although antibody binding sometimes introduces minor changes in RBD orientation. The most common configurations observed for the spike constructs used are 1 RBD-up and 2-down. ACE2 can only attach to an upward conformation that is considered less stable, facilitating conversion to a post-fusion state. In this configuration, fab 40, 150, 158 and chimeras 253H55L and 253H165L are seen to bind to the spike in this one up configuration. 253H55L also bound to the full downward configuration (1 Fab/trimer), as do Fab 316 (3 Fab/trimer) and Fab 384 (1 Fab/trimer). In contrast, fab88 binds in the all-up configuration (3 Fab/trimers) (table 9 and fig. 7A).

Fab 384 bound primarily one RBD per trimer, although analysis of the different particle categories revealed that some of the weak density decorated other RBDs, also in the downward position, with slight shifts seen between the different categories of RBDs (fig. 16). This can be attributed to the more favorable RBD conformation that can only be maintained by one RBD at a time.

In order to visualize binding of highly potent mAb 159, it was necessary to incubate the spike with 159IgG (Fab alone showed no binding). This revealsAll three NTDs with the spike modified by 159 with RBDs are in one up or all down configuration (fig. 16). 159 binding site is about a distance from the previously reported binding site for NTD binding agent 4A8 (Chi et al, 2020)Wherein CDR-H3 binds on the NTD side between loops 144-153 and 246-258 (FIG. 7B). CDR-H3 of 159 is 11 residues shorter than 4A8 (Chi et al 2020), and binds at the top center of the NTD and N-terminus of the NTD, which interact with residues 144-147, 155-158, 250-253. All 3 CDRs of the heavy chain contribute +.>While the light chain has little contact with NTD +.> Similar to 4A8 (Chi et al 2020) (fig. 7B, C).

EXAMPLE 11 potency of interaction

Binding of full length and Fab fragments to whole SARS-CoV-2 was measured by ELISA and these were compared to neutralization curves of selected antibodies for which structural information was available (fig. 7D and table 9). For anti-NTD mAb-159, the binding of full length and Fab to virions was almost identical, which was too far apart in line with NTD on trimer to be bivalent conjugated Consistent (FIG. 7C), and indicates that mAb-159 cannot reach between adjacent spike trimers on the surface of virions. Interestingly, while IgG-159 is a potent neutralizing agent, fab-159 does not have neutralizing activity, suggesting that the Fc portion is critical for activity, although its mechanism is not immediately apparent andand is not involved in blocking ACE2 interactions.

The binding and neutralization losses of the attached mAb-88 to Fab were quite slight in all upward conformations compared to IgG (fig. 7D and 9), but binding to all downward forms of the spike mAb (253, 316, 384) was much more pronounced. Thus, mAb-384 showed 79-fold reduction in virus binding and 486-fold loss of neutralizing activity when reduced to Fab, suggesting the use of two Fab arms when the antibody interacts with the virion, and also highlights the excellent K of Fab-159 _D 2.5 to 81 times better than the other Fab depicted in fig. 7D and table 9. Finally, the relationship between antibody binding and neutralization was estimated using the following formula: occupancy percentage = BMax [ Ab ]]/(Kd+[Ab]) Wherein BMax is the percent maximum binding, [ Ab ]]Is the Ab concentration required to reach 50% frnt, and Kd is the Ab concentration required to reach half maximum binding. mAb-384 can reach NT50, with an estimated average occupancy of 12% of the maximum available antibody binding site on each virion, probably due in part to the avidity conferred by bivalent attachments (Table 9). Divalent attachment to the downward conformation may also lock all three RBDs, thereby excluding attachment to ACE2. Some of the variations in the effects seen in fig. 7D and table 9 may be due to the interplay between the angle and position of the antibody arm's attack on the RBD and constraints on system flexibility.

The correlation between Fab and IgG binding/neutralization and the pattern of attachment to pre-fusion spikes was determined as seen in cryoEM. When Fab and full length IgG1 (e.g., 316 and 384) are compared, those antibodies that bind to spikes in the downward conformation appear to show a clear affinity enhancement for binding and neutralization, indicating that there is a relationship between attachment pattern and neutralization, as can also be seen from the potent neutralization against antibodies that bind at the left and right hypochondriac (S309 and EY6A/H014 (Pinto et al 2020; zhou et al 2020; lv et al 2020)) epitopes that have not been reported to be strongly neutralized in the assays used herein.

EXAMPLE 12 in vivo efficacy

The most promising efficacy of neutralizing human mabs was determined in vivo. K18-hACE2 transgenic mouse model using SARS-CoV-2 pathogenesis, wherein human ACE2 expressionDriven by an epithelial cell-specific cytokeratin-18 gene promoter (McCray et al, 2007; winkler et al, 2020). In this model, SARS-CoV-2 infected animals develop severe lung disease and high levels of lung viral infection with concomitant immune cell infiltration and tissue damage (Winkler et al 2020). Initially, by using 10 ³ SARS-CoV-2 of PFU 1 day (D-1) intraperitoneal injection 1 day prior to intranasal (i.n.) challenge single 250 μg (10 mg/kg) doses of mAb 40 and 88 were administered as prophylaxis. Passively transferring mAb 40 or 88, instead of isotype control mAb (hE 16), prevented SARS-CoV-2-induced weight loss (fig. 17A). In lung homogenates of animals treated with antibodies 40 and 88, no infectious virus was detected 7 days post infection (dpi), whereas there was a considerable amount in animals treated with isotype control mAb (fig. 17B). Consistent with these results, viral RNA levels were reduced by approximately 10,000-100,000 fold compared to isotype control mAb treated animals (fig. 17C). In peripheral organs (including heart, spleen or brain), viral RNA levels were reduced or undetectable in animals treated with mAb 40 or 88 (fig. 17D-G). Furthermore, viral RNA levels at 7dpi were significantly lower in nasal washes of animals treated with mAb 40 and 88 compared to isotype control.

To further evaluate the in vivo efficacy of mabs, 10 was used ³ SARS-CoV-2 of each PFU was evaluated for therapeutic activity at 1dpi (D+1) in a larger group. Although different degrees of protection were observed in each mAb, weight loss was significantly reduced in all animals treated with anti-SARS-CoV-2 mAb at 6 and 7dpi compared to isotype control (fig. 8A). Although the lung infectious virus level of isotype control mAb treated animals was 10 ⁶ PFU/g tissues, but little infectious virus was detected in animals treated with mAb 40, 88, 159, 384 or 253H55L (FIG. 8B). In animals treated with mAbs 40, 159, 384 and 253H55L, the levels of pneumoviral RNA at 7dpi were also reduced. mAb 88 showed an average reduction of-100 fold (fig. 8C). At disseminated sites of infection, particularly heart, spleen and brain, all anti-SARS-CoV-2 mabs showed protective activity, but mabs 384 and 253H55L reduced viral RNA levels the most (fig. 8D, E, G). In nasal washes, mabs 159 and 384 showed the greatest reduction in viral RNA waterFlat capacity (fig. 8F). Taken together, these data indicate that several mabs in this group can reduce infection in the upper, lower and distant sites when administered post-infection.

In summary, the most potent antibodies identified were demonstrated to have protective effects in animal models upon prophylactic and therapeutic administration. Designed competition mapping methods propose a series of antibody combinations with non-overlapping epitopes. Thus, these results may be helpful for immunotherapy.

Example 13 materials and methods

Materials and methods of examples 1 to 12.

Trimeric spike of SARS-CoV-2

To construct an expression plasmid for SARS-CoV-2 spike protein, the genes encoding residues 1-1208 having the mutation of RRAR (SEQ ID NO: 409) to GSAS (SEQ ID NO: 410) at the furin cleavage site (residues 682-685), proline substitution at residues 986 and 987 followed by the T4 fibrin trimerization domain, the HRV3C protease cleavage site, the twin Strep tag and the 8XHIS tag (SEQ ID NO: 411) were synthesized and optimized for mammalian expression (Wrapp et al 2020). The optimized coding sequence was cloned into the mammalian expression vector pHLsec.

Trimeric spikes of SARS-CoV, MERS-CoV, OC63-CoV, HKU1-CoV, 229E-CoV, NL63-CoV

Synthetic fragments encoding human codon optimized spike glycoprotein sequences from CoV-229E (GenBank accession NC-002645.1; amino acids 1-1113), coV-HKU1 (GenBank accession NC-006577.2; amino acids 1-1300), coV-NL63 (GenBank accession NC-005831.2; amino acids 1-1289), coV-OC43 (GenBank accession NC-006213.1; amino acids 1-1297), coV-MERS (GenBank accession AFS88936.1; amino acids 1-1291) (Zhao et al 2013), coV-SARS1 (GenBank accession AY27874; amino acids 11-1195) (Simmons et al 2004) and CoV-SARS2 (GenBank accession MN908947; amino acids 1-1208) were used to construct expression plasmids. Fragments were cloned as previously reported by Wrapp et al (Wrapp et al 2020).

Mutations encoding stable proline residues and eliminating putative furin cleavage sites were inserted in each sequence as follows: for CoV-229E, TI > PP (aa 871-872); for CoV-HKU1, RRKR (SEQ ID NO: 412) > GSAS (SEQ ID NO: 410) (aa 756-759) and AL > PP (aa 1071-1072); for CoV-NL63, RRSR (SEQ ID NO: 413) > GSAS (SEQ ID NO: 410) (aa 754-757) and SI > PP (aa 1052-1053); for CoV-OC43, AL > PP (aa 1070-1071); for CoV-MERS, RSVG (SEQ ID NO: 414) > ASVG (SEQ ID NO: 415) (aa 748), RSAR (SEQ ID NO: 416) > GSAS (SEQ ID NO: 410) (aa 884-887) and VL > PP 1060-1061; for CoV-SARS1, KV > PP (aa 968-969); for CoV-SARS2, RRAR (SEQ ID NO: 409) > GSAS (SEQ ID NO: 410) (aa 682-685) and KV > PP (aa 986-987). All sequences were verified by DNA sequencing.

DNA plasmids encoding Strep-Tag-tagged spike proteins were transfected into HEK293T cells and incubated for 7 days at 37 ℃. Affinity purification of CoV spike protein trimer. In the case of CoV-229E and CoV-NL63, the spike protein is further purified by SEC.

anti-RBD antibodies were depleted from plasma samples.

Nickel-bearing agarose beads were incubated overnight with His-tagged RBDs. Beads incubated in the absence of RBD antigen were used as a simulated control containing only beads. The beads were pre-cleared with pooled SARS-CoV-2 negative plasma. The beads are incubated with a human plasma sample of interest. The remaining depleted samples were collected, filter sterilized, and completely depleted by RBD direct ELISA test. ACE2 and RBD. Constructs were as described in Huo et al 2020 (Huo et al 2020) and produced as described in Zhou et al 2020 (Zhou et al 2020).

Isolation of human monoclonal antibodies from peripheral B cells by memory B cell stimulationTo produce human monoclonal antibodies from peripheral blood B cells, CD22+ B cells were isolated from PBMC using CD22 microbeads (130-046-401; miltenyi Biotec, meinai Biotech). Pre-enriched B cells were stained with anti-IgM-APC, igA-FITC and IgD-FITC. Double negative memory B cells (IgM-, igA-/D-cells) were sorted by FACS and plated at 384 wells at a density of 4B cells per wellAnd on the plate. Cell proliferation was stimulated and IgG was produced by culturing with irradiated 3T3-msCD40L feeder cells (12535; NID AIDS kit program), 100U/ml IL-2 (200-02; peprotech, peplectane Co.) and 50ng/ml IL-21 (200-21; peplectane Co.) for 13-14 days. Supernatants were harvested from each well and screened for SARS-CoV-2 binding specificity by ELISA. Lysis buffer was added to positive wells containing SARS-CoV-2 specific B cells and stored immediately at-80℃for future use in Ig gene amplification and cloning.

Isolation of spike and RBD specific single B cells by FACS

To isolate spike and RBD specific B cells, PBMC were stained sequentially with LIVE/DEAD Fixable Aqua dye (Invitrogen) followed by recombinant trimeric spike-twin-Strep or RBD-biotin. Cells were then stained with an antibody mixture consisting of CD3-FITC, CD14-FITC, CD56-FITC, CD16-FITC, igM-FITC, igA-FITC, igD-FITC, igG-BV786, CD19-BUV395 and Strep-MAB-DY549 (iba) or streptavidin-APC (Biolegend)) to detect the Strep tag of spike or biotin of RBD. Spike or RBD specific single B cells were gated as cd19+, igg+, CD3-, CD14-, CD56-, CD16-, igM-, igA-, igD-, spike+ or rbd+ and sorted into each well of a 96-well PCR plate containing rnase inhibitor (N2611; promega). Plates were briefly centrifuged and frozen on dry ice and then stored at-80 ℃ for future use in Ig gene amplification and cloning.

Cloning and expression of SARSCOV 2-specific human mAbs.

Genes encoding Ig VH, ig V.kappa.and V.lambda.were recovered from the positive wells using RT-PCR (210210; QIAGEN). Nested PCR (203205; kaiji) was then performed to amplify the genes encoding gamma, lambda and kappa chains with a "mixture" of primers specific for human IgG. PCR products of the genes encoding the heavy and light chains were combined with expression vectors of human IgG1 or immunoglobulin kappa or lambda chains by Gibson (Gibson) assembly. To express the antibodies, plasmids encoding heavy and light chains were co-transfected into 293T cell lines by the polyethylenimine method (408727; sigma) and supernatants containing the antibodies were harvested for further characterization.

Construction of Fab expression plasmid

The heavy chain expression plasmid of the specific antibody was used as a template to amplify the first fragment, the heavy chain vector comprising the variable region and CH1, up to Kabat amino acid number 233. Amplifying the second fragment of the thrombin cleavage site and a twin-Strep-tag having an overlapping end with the first fragment. The two fragments were ligated by gibbon assembly to prepare Fab heavy chain expression plasmids.

Construction of scFv antibody plasmids

Heavy and light chain expression plasmids of specific antibodies were used as templates to amplify the variable region genes of the heavy and light chains, respectively. First, the heavy chain gene product with an AgeI-SalII restriction site was cloned into an scFv vector, which is a modified human IgG expression vector with a linker between the H chain and L chain genes, followed by a thrombin cleavage site and a twin-Strep-tag. The light chain gene product with the NheI-NotI restriction site was cloned into an scFv vector containing a heavy chain gene insert to generate an scFv expression plasmid.

Fab and scFv production and purification

Protein production was performed in HEK293T cells by transient transfection with polyethylenimine in FreeStyle 293 medium. For Fab antibody production, fab heavy chain expression plasmids were co-transfected with the corresponding light chains. For scFv antibody production, scFv expression plasmids of specific antibodies were used for transfection. After 5 days of incubation at 37℃and 5% CO2, the culture supernatant was harvested and filtered using a 0.22mm Polyethersulfone (PES) filter. Fab and scFv antibodies were purified by Strep-Tactin affinity chromatography (IBA life sciences) according to Strep-Tactin XT handbook.

Determination of plasma and antibody binding to recombinant proteins by ELISA

MAXISORP immunoplates (442404; nelker (NUNC)) coated with 0.125 μg of Strep MAB-Classic were incubated with double strep-tag recombinant spikes of SARS-CoV-2, SARS-CoV, MERS-CoV, OC43-CoV, HKU1-CoV, 229E-CoV and NL 43-CoV. Serial dilutions of plasma or mAb were added followed by ALP conjugated anti-human IgG (a 9544; sigma). The reaction was developed by adding PNPP substrate and quenched with NaOH. To determine binding to SARS-CoV-2RBD, SARS-CoV-2NP, SARS-CoV-2 spike S1 (40591-V08H; yinqiao Shenzhou Biol (Sino Biological Inc)) and SARS-CoV-2 spike S2 (40590-V08B; yinqiao Shenzhou Biol) the immune plates were coated with Tetra-His antibody (34670; kaiji) followed by recombination of SARS-CoV-2RBD, SARS-CoV-2NP, SARS-CoV-2 spike S1 and SARS-CoV-2 spike S2 with a 5. Mu.g/mL His tag. Plasma endpoint titer (EPT) was defined as the reciprocal plasma dilution corresponding to twice the mean OD value obtained in the simulation. The EC50 of mabs was evaluated using nonlinear regression (curve fitting).

Whole virus ELISA

To determine the binding affinity of the antibodies to SARS-CoV-2 virus, the virus was captured on a plate coated with mouse anti-SARS-CoV-2 spike (mAb 31 with mouse Fc) and then incubated with serial dilutions of SARS-CoV-2 specific human mAb (full length IgG or Fab) followed by incubation with ALP conjugated anti-human IgG (A8542, sigma). The reaction was developed with PNPP substrate and quenched with NaOH. Results are expressed as a percentage of total binding.

Focal reduction neutralization assay (FRNT)

The neutralization potential of abs was measured using a Focus Reduction Neutralization Test (FRNT), in which the reduction in the number of infected foci was compared to a no antibody negative control well. Briefly, serial dilutions of Ab were mixed with real SARS-CoV-2/human/AUS/VIC01/2020 (Caly et al 2020) and incubated for 1 hour at 37 ℃. The mixture was then transferred to Vero cell monolayers and incubated for 2 hours, then 1.5% semi-solid carboxymethylcellulose (CMC) cover medium was added to each well to limit virus spread. Foci formation assay was then performed by staining Vero cells with human anti-NP mAb (mAb 206), followed by peroxidase conjugated goat anti-human IgG (a 0170; sigma). Finally, by adding truebu e peroxidase substrate visualizes lesions (infected cells). Percent lesion reduction was calculated and IC was determined ₅₀ 。

NTD binding assay

mAbs were screened for binding to MDCK-SIAT1 cells expressing the N-terminal domain (NTD) of SARS-CoV-2 spike glycoprotein (MDCK-NTD, from Alain Townsend professor). Briefly, MDCK-NTD cells were incubated overnight. mAb supernatant from transfected 293T cells was added and incubated. A secondary antibody goat anti-human IgG Fc specific FITC (F9512, sigma-Aldrich) was then added (50 μl per well) and incubated. After washing twice, wells were fixed with 1% formaldehyde in PBS. Bound antibody was detected by fluorescence intensity.

ELISA-based ACE2 binding inhibition assay

For the ACE2 competition ELISA, 250ng of ACE2 protein was immobilized onto MAXIXORP immunization plates and the plates were blocked with 2% bsa in PBS. Simultaneously, serial dilutions of Ab were mixed with recombinant RBD-mFc (40592-V05H; yinqiao China Biotechnology Co.) and incubated at 37℃for 1 hour. The mixture was then transferred to ACE2 coated plates and incubated for 1 hour, followed by the addition of goat anti-mouse IgG Fc-AP (Inje, # A16093) at a 1:2000 dilution. The reaction was developed by adding PNPP substrate and quenched with NaOH. Absorbance was measured at 405 nm. ACE2/RBD binding inhibition was calculated by comparison to control wells without antibody. Determination of IC using probit program from SPSS software package ₅₀ 。

Spike protein production for structural analysis

The stable cell line generating vector pNeoSec was used to clone the SARS-Cov2 spike ectodomain that contained amino acids 27-1208 that had mutations at the furin cleavage site (RRAR (SEQ ID NO: 409) > GSAS (SEQ ID NO: 410) at residues 682-685) and PP (KV > PP at residues 986-987). There is a twin strep ii tag at the N-terminus and fusion at the C-terminus with the T4 fibrin trimerization domain, HRV 3C cleavage site and His-8 tag. Human Embryonic Kidney (HEK) Expi293F cells (zemoeimer technology (Thermo Fisher Scientific)) were transfected with the construct as described previously along with the phiC31 integrase expression plasmid (Zhao et al, 2014). Polyclonal G418 resistant (1 mg/ml) cell populations were used for protein production. The Expi293F cells were grown adherently at 30 ℃ in roller bottles with high glucose DMEM (sigma) with 2% fbs for 6 days. Soluble spike proteins were captured from the dialyzed conditioned medium using a pre-filled 5ml HisTrap excel column (general electric medical life sciences (GE Healthcare Life Sciences)). After the 20mM imidazole PBS wash step, the proteins were eluted in 300mM Phosphate Buffered Saline (PBS) containing imidazole. Proteins were further purified using 16/600Superdex 200 size exclusion chromatography and either acidic buffer (20 mM acetate, 150mM NaCl, pH 4.6) (for low pH spike incubation) or neutral buffer (2 mM Tris, 150mM NaCl, pH 7.5).

RBD generation for structural analysis

The stable HEK293S cell line expressing His-tagged RBD was cultured in DMEM (high glucose, sigma) supplemented with 10% fbs (invitrogen), 1mM glutamine and 1x non-essential amino acids at 37 ℃. Cells were transferred to roller bottles (Greiner) and cultured at 30 ℃ in DMEM supplemented with 2% fbs, 1mM glutamine and 1x non-essential amino acids for 10 days for protein expression. To purify the proteins, the dialyzed medium was passed through a 5mL HisTrap Nickel column (GE Healthcare). The column was washed with buffer 20mM Tris pH7.4, 200mM NaCl, 30mM imidazole, and RBD eluted using buffer 20mM Tris pH7.4, 200mM NaCl, 300mM imidazole. Endoglycosidase H1 (. About.1 mgml) was added in a volume of 30. Mu.l ^-1 ) Added to 30mg RBD and incubated for 2 hours at room temperature. The samples were then further purified using a Superdex 75HiLoad 16/600 gel filtration column (general electric medical Co.) using 10mM HEPES pH7.4, 150mM NaCl. The purified RBD was concentrated to 10.6mgml using a 10-kDa ultracentrifuge filter (Amicon), inc ^-1 And stored at-80 ℃.

Preparation of Fab from IgG

Fab fragments were digested with papain from purified IgG using Pierce Fab preparation kit (Semerfeier Co., thermo Fisher) according to the manufacturer's protocol.

Physical measurement

Thermal stability was assessed using Thermofluor (DSF). Briefly, 3 μg of Ab formulation was used in 50 μl of reaction containing 10mM HEPES pH 7.5, 100mM NaCl, 3 XSYPROorange (Sieimer's Feisher). Samples were heated from 25 ℃ to 97 ℃ in an RT-PCR machine (Agilent), MX3005 p) and fluorescence was monitored at 25 ℃ after each 1 ℃ heating. The melting temperature (Tm) was calculated by fitting a 5-parameter sigmoid curve using JTSA software (P.bond, https:// Paulsbond. Co. Uk/JTSA). Polydispersity was assessed by DLS using 10 μg Ab formulation in une instrument (non chain Labs). The freeze-thawing experiments were performed on 4 mabs with 1mg/ml material by the following procedure: aliquots (centrifuged at 20000g for 10 min) were flash frozen using LN2, thawed and centrifuged, and the absorbance of the soluble fraction was measured at 280 nm.

Crystallization

Purified RBDs were combined with Strep-labeled Fab150, fab58, scFv269 and Fab316, respectively, in a 1:1 molar ratio, at final concentrations of 13.2, 9.4, 12.7 and 13.0mg ml, respectively ^-1 . RBD was combined with Fab45 and Strep-tagged Fab88, fab75 and Fab253, and Fab75 and Strep-tagged chimeric Fab 253H55L at a molar ratio of 1:1:1, with final concentrations of 7mgml, respectively ^-1 . Glycosylated RBD was combined with Fab S309 (Pinto et al 2020) and Fab384 in a molar ratio of 1:1:1, final concentration of 8mgml ^-1 . Crystals of RBD-150 complex were formed in Molecular Dimensions Morpheus condition C2 containing 0.09M NPS (nitrate, phosphate and sulfate), 0.1M MES/imidazole pH 6.5, 10% (w/v) PEG 8000 and 20% (v/v) ethylene glycol, and crystals were also formed in Hampton Research PEGRx condition D11 containing 0.1M imidazole pH 7.0 and 12% (w/v) PEG 20000. The crystals of RBD-158 were obtained from Index condition C01 containing 3.5M NaCOOH pH 7.0, while some crystals contained 0.15M (NH 4) ₂ SO ₄ Proplex condition C1, 0.1M Tris pH 8.0 and 15% (w/v) PEG 4000, and at 0.15M (NH 4) ₂ SO ₄ Further in 0.1M Tris pH 7.6 and 14.6% (w/v) PEG 4000And (5) optimizing. Crystals of complexed RBD-scFv269 were obtained from Index condition F01 containing 0.2M proline, 0.1M HEPES pH 7.5 and 10% (w/v) PEG 3350. Crystals of RBD-316 complex were obtained from Index condition G10 containing 0.2M MgCl2, 0.1M bis-Tris pH 5.5 and 25% (w/v) PEG 3350. Crystals of RBD-45-88 complex were obtained from PEGRx condition G12 containing 10% (v/v) 2-propanol, 0.1M sodium acetate trihydrate pH 4.0, 22% (w/v) PEG 6000. Crystals of RBD-75-253 complex were obtained from PEGRx condition D8 containing 0.1M BIS-TRIS pH 6.5, 16% (w/v) PEG 10000. Crystals of RBD-75-253H55L were obtained from Index condition F5 containing 0.1M ammonium acetate, 0.1M bis-Tris pH 5.5 and 17% (w/v) PEG 10000. For RBD-S309-384 ternary complex, good crystals were obtained from Morpheus condition H1 containing 0.1M amino acid (Glu, ala, gly, lys, ser), 0.1M MES/imidazole/pH 6.5, 10% (w/v) PEG 20000 and 20% (w/v) PEG MME 550.

X-ray data collection, structure determination and refinement

Diffraction data were collected at 100K at beam line I03 of british diamond light source company (Diamond Light Source). The structure was determined by molecular replacement with PHASER (Liebschner et al, 2019) using a search model of the RBD, vhvl and ChCl domains of closely related Fab in the sequence of each complex. The ChCl domain of Fab 88 in the RBD-88-45 complex is disordered. Data collection and structure refinement statistics are given in table 7.

Cryo-EM grid preparation

For all Fab or IgG-spike complexes, aliquots of 3 μ L S to 0.6 μm (as measured by OD) to Fab (1:6 molar ratio) were prepared, aspirated and applied almost immediately to a freshly glow discharge Cu carrier C flat 2/1-200 mesh porous carbon coated grid (high intensity, 20s, plasma cleaner PDC-002-CE, ha Like Plasma company (Harrick Plasma)). Excess liquid was removed by blotting with-1 force at 4.5 ℃ and 100% reported humidity for 5-5.5s using a vitro filter paper (grade 595, tedpe la inc.) and then flash frozen into liquid ethane using a vitro Mark IV (zemer femto).

Cryo-EM data collection and processing

40. 253H55L and 253H165L spike complex:

films were collected in compressed tiff format on a Titan Krios G2 (Siemeco) with K3 detector (Gatan) operating at 300kV in super resolution counting mode using a custom version of EPU 2.5 (Siemeco). Using a defocus range of 0.8-2.6 μm, a nominal magnification of x 105,000, corresponding toCalibration pixel size of the pixels and total dose is +.>

The twice-binned movies were then motion corrected and aligned using a repeat (3.1) scheduler (Zivanov et al, 2018) using alignment based on a 5 x 5 patch. CTF estimation was performed on full-frame unweighted micrographs using the GCTF (1.06) (Zhang, 2016) module in crysparc (v2.14.1-real time) (Punjani et al, 2017).

88. 150, 158, 159IgG, 316, and 384 spike complexes:

data were collected at 88, 150, 158 on a Titan Krios G2 (Simer Feisher) with K2 camera and a GIF quantum energy filter (Gatan) with 30eV slit, running at 300 kV. For 159 (IgG), 384 and 316, data were collected as for 88, 150 and 158, except that 20keV slots were used. Custom scripts with SerialEM (version 3.8.0 beta) (Mastronard e, 2005) were used at a nominal magnification of 165kX (corresponding to each pixel) A calibrated pixel size) of the image sensor set up fast multi-shot data acquisition. Applying +.f over 40 frames using defocus range of-0.8 μm to-2.6 μm> Is effective in preventing or treating a disease. The original film was motion and CTF corrected at speed using crysparc real-time patch-motion and patch-CTF correction (Punjani et al, 2017).

40. 253H55L, 253H165L, 88, 150, 158, 159IgG, 316, and 384 complexes:

after manual inspection of CTF and motion estimation, images of poor quality are discarded. The particles were then spot picked in crySPARC (Punjani et al, 2017) and initially extracted with four bins. After examining the 2D class, the class of interest is selected to produce a template for complete particle selection. The binned particles are then subjected to one to three rounds of non-reference 2D classification followed by 3D classification with de novo derived models, followed by further refinement and binning.

For both 150 and 158, two separate sets of data are placed on the same grid and the sets of refinement particles from each set are separated by exposure group prior to combining. For 150, a total of 77,265 exposed group splitting particles (51,554 out of 4726 movies and 25,711 out of 2079 movies) were initially combined, reclassified into five categories, and the two best categories (42,655 particles) were subjected to further non-uniform refinement, evident in the density of Fab bound to one RBD in the "up" conformation. Notably, the discarded class included a high proportion of uncolored S (28,463 particles, reported at gsfsc=0.143 as resolution Factor B is->)。

Classification using heterogeneous refinement in crysparc was found to be generally poor, in contrast to 3D variability analysis that was used to attempt to better resolve the complete spike-Fab structure. Localized refinement using a mask centered around the Fab/RBD region (not reported here) is also used, but the map is still insufficient to build the model clearly at the RBD/Fab interface, andfar inferior to crystallographic pictures. The 3D variability analysis was found to be essential for isolation of RBD up and RBD down conformations of 159-IgG. This result is presented for 159-IgG and 384. Briefly, using 3D variability analysis module utilizationThe resolution filter and mask around the RBD/Fab region divided the data into eight clusters. The mask is first generated by fitting model rigid bodies of spikes and Fab into a detailed map in chirea, then selecting the model region that includes RBD and Fab and clipping the desired portion of the map using a "color region" module. The resulting map was smoothed with a Gaussian filter (Pettersen Ef Fau-Goddard et al 2004), converted to a Mask format using a Relion3.1'Mask Create', and then imported into a crySPARC. The resolution estimate is taken from the gold standard-FSC (fsc=0.143) reported in the local resolution module in crysparc (Punjani et al, 2017).

Competitive assay for antibodies

Competition assays for anti-RBD antibodies were performed on a Fortebio Octet RED e machine using a Fortebio anti-HIS (HIS 2) biosensor. Mu.g ml was added to 2. Mu.g ml ^-1 His-tagged Beta RBD dissolved in running buffer (10mM HEPES pH 7.4 and 150mM NaCl) was used as ligand and first immobilized on the biosensor. The biosensor was then washed in running buffer to remove unbound RBD. Each biosensor was immersed in a different saturated antibody (Ab 1) to saturate the bound RBD, but one biosensor was immersed in the running buffer to serve as a reference during this step. The concentration of the saturated antibody used was 15. Mu.g ml ^-1 . If 15. Mu.g ml ^-1 Insufficient to achieve saturation, a higher concentration is applied. All biosensors were then washed again with running buffer and immersed in wells containing the same competing antibody (Ab 2). The concentration of competing antibody used was 5. Mu.g ml ^-1 . The Y-axis value of the signal of the different saturated antibodies in this step is divided by the value of the reference channel to obtain the ratio result of the different Ab1-Ab2 pairs. A ratio result approaching 0 indicates complete contention, while a 1 indicates no contention. Together, a total of50 IgG and 4 Fab (Fab 40, EY6A (Zhou et al 2020), FD5D (unpublished) and S309 (Pinto et al 2020)) were used as saturated antibodies and 80 IgG were used as competing antibodies.

Competitive mapping of antibodies

Total assembly bin of antibody: the competition values were prepared for cluster analysis and binning by limiting all competition values to between 0 and 1. The competition value between antibodies i and j is averaged with the competition value of j and i (when both are available). Antibodies were clustered into three different groups by single-valued decomposition of a competition value matrix using Cluster4x (Ginn, 2020).

Preparation of RBD surfaces and grids: the surface of the receptor binding domain is found on PyMOL (The PyMOL Molecular Graphics System [ PyMOL molecular graphics System)]1.2r3pre version, (-) Schrodinger companyLLC)) is generated from chain E of PDB code 6 YLA. The mesh is generated and iteratively shrunk and constrained to the surface of the RBD to provide a smoother surface to guide antibody refinement, reducing complex surface features that may lead to impractical exploration of local minima.

Immobilization of the position of antibodies of known structure: to provide objective positions of those antibodies of known structure (FD 5D (not published), EY6A (Zhou et al 2020), S309 (Pinto et al 2020) and mAb 40) to reflect the enclosed region, all non-hydrogen antibody atoms were found to be at any RBD atomIn, and as such all RBD atoms are at the antibody atom And (3) inner part. From each group, the atom with the lowest sum of squares length among all other members is identified and the midpoint of the two atoms is locked to the nearest vertex on the mesh. The solvent molecules are omitted, but in the case of S309, the sugar-oligomer cofactor is included in the antibody atomic group.

Objective function: in evaluating the objective function, either all unique antibody pairs (all pairs) or only unique pairs where one antibody is immobilized (immobilized pairs) are considered, depending on the stage of the minimization protocol. Estimating the level of competition for each antibody pair as described by f (x) in equation 1

Wherein r is the working radius of the antibody, set toRepresenting the approximate antibody radius. The distance between antibody pairs is given by d (in angstroms) for a given functional evaluation. The objective function is the sum of the squared differences between the competition estimates and the competition values from the SPR data.

Obtaining a set of self-consistent refined antibody positions: using LBFGS refinement, minimization is done globally by 1000 large cycles of Monte Carlo-sequence samples. The random starting position for each antibody was generated by randomly assigning starting vertices on the RBD grid and taking into account the data points of the pairs with at least one immobilized antibody, the objective function was minimized for 20 cycles, followed by 40 cycles for all data points. Between each cycle, the antibody positions are locked on the nearest mesh vertices. Depending on the starting position of the antibody, the result is a mixture of well-refined and poorly refined solutions. The results are arranged in ascending order according to the objective function score. The antibody position of each result was transferred as a pseudo-C-alpha position into cluster4x (Ginn, 2020). Self-consistent solutions are rich in lower objective function scores and are separated for further analysis using cluster4 x. Thus, the average position of each antibody was locked to the nearest vertex on the grid, and RMSD was calculated from all contributed antibody positions.

Cell and virus (mouse experiment)

Vero CCL81 (American type culture Collection (ATCC)) and Vero-furin cells (Mukherjee et al 2016) were cultured at 37℃in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 10mM HEPES and 100U/ml penicillin-streptomycin. The 2019n-CoV/USA_WA1/2019 isolate of SARS-CoV-2 WAs obtained from the United states disease control center (CDC). Propagating the virus stock by seeding Vero CCL81 cells and collecting the supernatant after cytopathic effect is observed; the chips were removed by centrifugation at 500 Xg for 5 minutes. The supernatant was aliquoted and stored at-80 ℃. All work on infectious SARS-CoV-2 was done in the institutional biosafety committee of the university of Washington medical institute approving BSL3 and A-BSL3 facilities using appropriate positive pressure air respirators and protective equipment.

Mouse experiment

Animal studies were performed according to recommendations in Guide for the Care and Use of Laboratory Animals of the National Institutes of Health [ national institutes of health laboratory animal care and use guidelines ]. The protocol was approved by the laboratory animal management and use committee of the university of washington, medical college (support No. a 3381-01). Virus inoculation was performed under anesthesia induced and maintained by ketamine hydrochloride and xylazine, and all efforts were made to minimize pain in the animals.

Heterozygous K18-hACE C57BL/6J mice (strain: 2B6.Cg-Tg (K18-ACE 2) 2 Prlmn/J) were obtained from Jackson laboratories (The Jackson Laboratory). 10 for intranasal administration ³ SARS-CoV-2 of each PFU was inoculated into seven to eight weeks old male and female animals.

Measurement of viral load

Tissues were weighed and homogenized with zirconia beads in 1000 μl DMEM medium supplemented with 2% heat inactivated FBS in a MagNA Lyser instrument (rogowski life sciences (Roche Life Science)). The tissue homogenate was clarified by centrifugation at 10,000rpm for 5 minutes and stored at-80 ℃. RNA was extracted on a Kingfusher Flex extraction robot (Semer Firex technology (Thermo Scientific)) using MagMax mirVana Total RNA isolation kit (Semer Firex technology). RNA was reverse transcribed and amplified using TaqMan RNA-to-CT 1-step kit (Sieimerfeier). Reverse transcription was performed at 48℃for 15 minutes and then at 95℃for 2 minutes. Amplification was completed in 50 cycles as follows: 95℃for 15 seconds and 60℃for 1 minute. The copy of SARS-CoV-2N gene RNA in the sample was determined using the previously disclosed assay (PubMed ID 32553273). Briefly, taqMan assays were designed to target highly conserved regions of the N gene (forward primer: ATGCTGCAATCGTGCTACAA (SEQ ID NO: 417)); reverse primer: GACTGCCGCCTCTGCTC (SEQ ID NO: 418); and (3) probe: 56-FAM/TCAAGGAAC/ZEN/AACATTGCCAA/3 IABkFQ/(SEQ ID NO: 419)). This region is included in the RNA standard to allow for copy number determination. The reaction mixture contained primers and probes at final concentrations of 500 and 100nM, respectively.

Plaque assay. Vero-furin cells (Mukherjee et al 2016) at 2.5X10 ⁵ The density of individual cells/wells was seeded in flat bottom 12-well tissue culture plates. The next day, the medium was removed and replaced with 200 μl of 10-fold serial dilutions of the material to be titrated diluted in dmem+2% fbs. After incubation at 37℃for 1 hour, 1mL of methylcellulose coating was added. Plates were incubated for 72 hours and then fixed with 4% paraformaldehyde in phosphate buffered saline (final concentration) for 20 minutes. Plates were stained with 0.05% (w/v) crystal violet stain in 20% methanol and washed twice with distilled deionized water before plaque counting.

Affinity assay using biological layer interferometry

The binding affinity of antibodies to RBD or spike was determined using Octet RED 96e (ForteBio). anti-RBD IgG was immobilized to an AR2G biosensor (foterbuo corporation), while RBD was used as serial diluted analyte. For IgG159, the spike was immobilized to the AR2G biosensor, with IgG159 acting as a serial dilution of the analyte. Kd values were calculated using a 1:1 global fit model using Data Analysis HT 11.1 (foterbuo).

Example 14 characterization of N501Y mutation in RBD

RBD can be compared to classical trunk, in which the shoulders and neck are involved in interactions with ACE2 receptors (fig. 19A, B). In this case, residue 501 is located within the footprint of the receptor on the right shoulder and is involved in hydrophobic interactions, especially with the side chains of residues Y41 and K353 of ACE2, where mutation of 501 from N to Y provides the opportunity to enhance interactions (fig. 19B, C).

Influence on ACE2 affinity

Mutations at 501 have been reported to increase the spike affinity of ACE2 (Starr et al, 2020; gu et al, 2020), although these data are not directed to mutations to Y. In contrast, zahradini k et al (zahradini k et al 2021) report on the direct selection of N501Y when evolving RBD to enhance affinity. The effect of this mutation on RBD binding to ACE2 was thus investigated using Biological Layer Interferometry (BLI) (fig. 19D). The results show a significant increase in binding affinity (7-fold) due to the slower dissociation rate: WT RBD (501N) -ACE2: k (K) _D 75.1nM(K _on 3.88E4/Ms，K _off 2.92E-3/s)，RBD(501Y)-ACE2：K _D 10.7nM(K _on 6.38E4/Ms，K _off 6.85E-4/s). This is consistent with enhanced interactions of the tyrosine side chains with the side chains of residues Y41 and K353 of ACE2 (fig. 19C). In the case of multivalent interactions at the cell surface, this effect will be amplified. Only this point can explain the increase in selection and propagation of the N501Y mutation.

Influence on affinity of monoclonal antibodies

To investigate the effect of the N501Y mutation on antibody binding, the set of 377 monoclonal antibodies (80 of which mapped to RBD) were used, which were generated from SARS-CoV-2 cases infected during the first wave pandemic in the united kingdom using samples collected 6 months prior to 2020. Potent neutralizing agents tend to have relatively few somatic mutations (5.33 and 4.33 amino acids on average in the heavy and light chains (HC, LC), respectively), and many common antibody reactions (i.e., those using common V region genes), including IGHV3-53 (5 potent mabs), IGHV1-58 (4 potent mabs), and IGHV3-66 (2 potent mabs), are present in the collection of RBD-specific mabs.

Analysis of the change in the position of N501Y relative to the binding of all structurally characterized potent monoclonal antibodies indicated that more than half of the binding of the antibodies would be unaffected by this change (fig. 20A). However, a class of public antibodies has attracted particular attention, namely those using IGHV3-53 (Yuan et al 2020; wu et al 2020). These antibodies and IGHV3-66 antibodies bind to N501Y below the Light Chain (LC) CDR1 region and they are expected to be affected by mutations, as for them, unlike ACE2, interactions with asparagine are very beneficial (fig. 20B).

To examine the effect on antibody binding, BLI experiments were performed to more strongly neutralise binding of mAb to RBD containing 501Y and 501N (example 17, figure 20C). The result is mapped to the RBD in fig. 20D. There is little effect on many potent antibodies, such as IGVH1-58 antibodies: 55. 165, 253, and 318. There is a significant-3 fold effect on mAb 40 (IGHV 3-66) and on most important IGHV3-53 antibodies (150, 158, and 175). However, there is a correlation between LC of IGHV3-53 antibody and the size of the effect, so the common IGLV1-9 antibodies (mabs 150 and 158) showed a sustained decrease in affinity by about 3-fold (fig. 21A). In contrast, mAb 222 showed no decrease with IGLV 3-20. When modeling using the most similar light chain from PDB, it did not contact residue 501, which accounts for this effect (fig. 21B). However, mAb 269 appears to be highly sensitive to this mutation (30-fold effect). The structure of the single chain Fv version of this antibody complexed with WT RBD (fig. 21C) showed similar interactions as observed in mabs 150 and 158. To further understand this, inThe crystal structure of Fab 269 complexed with RBD with 501Y was determined at resolution (example 17, table 10). The results are shown in fig. 21C. Essentially, it appears that this mutation introduced a rather small substitution of the L1 loop (fig. 21D), but there was a concomitant effect of the adjacent L3 loop (fig. 21E), with a significant switch at the position of Y94, thereby eliminating the contact with residues R403 and E406 of RBD. Finally, the effect on the regenerative antibodies REGN10933 and REGN10987 in the current clinical trial was very small (fig. 20C).

Example 15 influence of B.1.1.7 mutations on neutralization by potent mAbs

Next, neutralization assays were performed with a powerful mAb targeting the ACE2 interaction surface of RBD. Neutralization was performed using the Focus Reduction Neutralization Test (FRNT) and using virus strains Victoria and b.1.1.7 obtained from the british public health department (Public Health England) (fig. 22A, table 11). For some antibodies (40, 88, 222, 316, 384, 398), the FRNT 50 values between b.1.1.7 and Victoria strains were minimally affected (< 2-fold difference). However, for other antibodies, there was a decrease in neutralization titer of b.1.1.7, particularly mAb 269 (where neutralization was almost completely lost) and mAb 278 (which failed to reach 100% neutralization, showing a maximum of only 78%). Comparing all these results, an average reduction in FRNT titres of 4.3-fold (p < 0.0001) was found between Victoria and b.1.1.7 strains. Finally, 2 groups of monoclonal antibodies were observed, which have reached an advanced clinical trial for SARS-CoV-2: the pair REGN10933 and REGN10987, and the aslicon mabs AZD1061, AZD8895, and AZD7442 (combination of AZD1061 and AZD 8895) (fig. 22B, tables 11 and 12). Neutralization of REGN10987 was not affected by b.1.1.7, while REGN10933 showed a slight decrease but still remained potent in activity (fig. 22B, tables 11 and 12). Neutralization of AZ antibodies was also hardly affected.

EXAMPLE 16 neutralizing Activity of convalescent plasma and vaccine serum

During the first wave infection, a large number of samples were collected from cases in recovery (4-9 weeks post infection) for monoclonal antibody production before the b.1.1.7 strain appeared. Stored plasma from these cases was used for neutralization assays comparing Victoria and b.1.1.7 (fig. 23A). Analysis of 34 convalescent samples (including WHONC 20/130 reference serum) showed almost identical FRNT50 values for a few sera, but the average FRNT50 dilution of B.1.1.7 strain was 3-fold lower (p < 0.0001) than that of Victoria strain.

Neutralization of b.1.1.7 and Victoria strains was also determined using serum obtained from recipients of oxford-alsikan and a parchment vaccine. For the AZD1222 vaccine, serum was obtained at baseline and after 14 and 28 days of the second dose. For the psilosis, serum was obtained 7-17 days after the second dose of vaccine, which was administered 3 weeks after the first dose (participants were negative for serum response at the time of group entry). Neutralization assays for b.1.1.7 and Victoria strains showed 1.7-fold (n=10p=0.002) and 2.6-fold (n=15p < 0.0001) reduction in neutralization titers between b.1.1.7 and Victoria strains, respectively, for the aslicon vaccine 14 days and 28 days after the second dose (fig. 23B). For the psilosis vaccine BNT162b2, also 2.6-fold (n=25p < 0.0001) (fig. 23C).

Finally, plasma from 13 patients infected with b.1.1.7 (all with spike gene deletions in the viral PCR test and 11 verified by sequencing) was obtained at different time points after infection and neutralization between b.1.1.7 and Victorian strains was compared (fig. 24). At early time points, the neutralization titers were low or absent, except for 1 disease collected on day 1 of disease, which case showed the same neutralization of both viruses, and was the highest of all samples we measured at 1:136884 in this study, which we speculated to be likely to represent reinfection b.1.1.7. There was no significant difference between the neutralization titers of the two viruses for the samples as a whole.

In summary, neutralization assays of convalescence and vaccine serum revealed that higher concentrations of serum were required for b.1.1.7 virus to achieve neutralization, although there was no evidence that b.1.1.7 virus could evade neutralization of serum produced by early SARS-CoV-2 strains or vaccines.

Neutralization reactions against Victoria strains were less effective on b.1.1.7 and part of this effect was due to the N501Y mutation, as demonstrated by the weaker binding of many antibodies in which N501Y is the only difference to RBD. Reduced binding and neutralization is particularly apparent for some but not all members of the public VH3-53 class of mabs, where the light chain is very close to Y501. However, B.1.1.7 contains other mutations that may be involved in neutralization, in particular deletions at 69-70 and 144 in NTD. NTD-binding antibodies that do not block interaction with ACE2 have been described by many groups as being capable of neutralizing SARS-CoV-2 (Chi et al 2020; liu et al 2020; cerutti et al 2021), some of which exhibit IC50 values below 10ng/ml. In this study, b.1.1.7 showed only a 5.7-fold decrease in FRNT50 of mAb 159 (FRNT 50 Victoria 11ng/ml b.1.1.7 ng/ml), indicating that the binding site was not completely destroyed despite deletion of residue 144 at the edge of the footprint of the antibody.

The expression level of ACE2 has been shown to correlate with the likelihood of infection by SARS-CoV-1 (Jia et al 2005), and a higher affinity for ACE2 of SARS-CoV-2 has been considered to be the basis for its greater spread. It is reasonable to hypothesize that further increases in affinity will increase the likelihood of random events of viral attachment, leading to localization for sufficient time to trigger internalization of the virus, possibly by recruiting additional receptors. As indicated by zahradnik, j. Et al (zahradnik et al 2021), in the case of public health measures reducing R0 below 1, there will be a selection pressure that increases the affinity of the receptor.

This increase in transmission is exacerbated by a decrease in neutralizing efficacy of antibodies produced by previous infections. It is predicted that modification of the ACE2 binding surface of RBD will directly disrupt binding of affinity-lost antibodies to mutated residues. However, antibodies that compete for neutralization by ACE2, even if not directly affected by the mutation, will have to compete with ACE2 for binding to RBD, and RBD mutations that increase affinity of ACE2 will shift the equilibrium from mAb/RBD interactions to RBD/ACE2, making virus neutralization more difficult.

Mutations at position 484 of the spike may have similar dual effects, and zahradni k, j. While most efforts have been directed to the generation of antibodies that neutralize by blocking ACE2 binding, other mechanisms are possible (Huo et al 2020; zhou et al 2020), and in fact partially or non-neutralizing antibodies may confer protection (Dunand et al 2016). Such antibodies may not be affected by mutations in the ACE2 binding site, and they are worth more thorough research because they will form excellent components in therapeutic mixtures. In addition, natural exposure and vaccination may confer protective immunity against symptomatic and severe covd-19 via memory T cell responses (Sariol and Perlman,2020; altmann and Boyton, 2020).

Recent descriptions of many viral variants that appear to develop independently have attracted attention as it may mark the emergence of strains capable of evading vaccine-induced antibody responses. There is now an urgent need to closely investigate the emergence of the novel SARS-CoV2 strain worldwide and to quickly understand the consequences of immune escape. It is necessary to determine the relevant factors that protect against SARS-CoV-2 and understand how T cells contribute to protection in addition to the antibody response. It must also be understood whether the emerging strains (including B.1.1.7, 501Y.V2 and P.1) lead to more severe disease and whether they can evade natural or vaccine-induced immune responses (Zhu et al, 2021).

EXAMPLE 17 materials and methods

For examples 14-16.

COG-UK sequence analysis

All COG-UK sequences were downloaded 24 days 1 month 2020 and the translated protein sequence was approximately identical to the wild type reference sequence from the start codon and stop codon between nucleotides 21000-25000 and the mutation 501Y was filtered. Sequence alignment was performed and the identified mutations were plotted as red spheres (single point mutations) or black spheres (deletions) at the simulated C-alpha position of the spike structure, the size being proportional to the logarithm of the number of mutations. Residues mutated at a rate of greater than 0.3% compared to wild type are clearly labeled.

Cloning of native RBD, RBD N501Y and ACE2

The native RBD and ACE2 constructs are identical to those of Zhou et al (Zhou et al 2020). For cloning RBD N501Y, a construct of the native RBD was used as a template, and PCR was performed using two primers of the RBD (forward primer 5'-CTACGGCTTTCAGCCCACATACGGTGTGGGCTACCAGCCTT-3' (SEQ ID NO: 420) and reverse primer 5'-AAGGCTGGTAGCCCACACCGTATGTGGGCTGAAAGCCGTAG-3' (SEQ ID NO: 421)) and two primers of the pNEO vector (forward primer 5'-CAGCTCCTGGGCAACGTGCT-3' (SEQ ID NO: 422) and reverse primer 5'-CGTAAAAGGAGCAACATAG-3' (SEQ ID NO: 423)). The amplified DNA fragment was digested with restriction enzymes AgeI and KpnI, and then ligated with the digested pNEO vector. Except for the N501Y mutation, this construct encodes a protein identical to the native RBD.

Protein production

Protein expression and purification were performed as described in Zhou et al (Zhou et al 2020).

Preparation of 269Fab

Fab fragments of 269 antibodies were digested and purified using Pierce Fab preparation kit according to the manufacturer's protocol.

Crystallization

269Fab and RBD N501Y were mixed in a 1:1 molar ratio to a final concentration of 9.9mg ml ^-1 . After incubation for 30 minutes at room temperature, the samples were used for initial crystal screening in a crystal quick 96 well X plate (grignard first biosystems (Greiner Bio-One)) using a cartesian robot using a nanoliter sitting-drop vapor diffusion method as previously described (Walter et al, 2003). Crystals for the complex were obtained from Molecular Dimensions Proplex screen, condition B10 containing 0.15M ammonium sulfate, 0.1M MES pH 6.0 and 15% peg 4000.

Biological layer interferometry

The BLI experiments were run on an Octet Red 96e machine (Buddha). To measure the binding affinity of monoclonal antibodies to native RBD and RBD N501Y, RBD and RBD N501Y were immobilized to an AR2G biosensor (foterbuo corporation), respectively. Monoclonal antibodies are used as analytes. To measure the binding affinity of native RBD and RBD N501Y to ACE2, native RBD and RBD N501Y were immobilized to an AR2G biosensor, respectively. Serial dilutions of ACE2 were used as analyte. Data were recorded using software Data Acquisition 11.1 (Buddha Corp.) and analyzed using software Data Analysis HT 11.1 (Buddha Corp.) using a 1:1 fitting model.

X-ray data collection, structure determination and refinement

The crystals were mounted in a ring and immersed for one second in a solution containing 25% glycerol and 75% mother liquor, then frozen in liquid nitrogen prior to data collection. Diffraction data were collected at 100K at beam line I03 of british diamond light source company. Diffraction images were recorded on an Eiger2 XE 16M detector rotated by 0.1 ° (each image was exposed for 0.007s with a beam of lightA size of 80×20 μm, a beam transmission rate of 100%, and a wavelength of ). The data is indexed, integrated and scaled using an automated data processing program, xia2-dials (Winter, 2010; winter et al, 2018). A720℃dataset was collected from 2 frozen crystals with a resolution of +.>

The crystal has a unit cell size of a= 195.1,And β=100.6° of space group C2. The structure was determined by molecular replacement with PHASER (McCoy et al, 2007) using a search model of the ChCl domains of the SARS-CoV-2RBD/COVOX-scFv269 complex (PDB ID, 7 BEM) and the SARS-CoV-2RBD/COVOX-158 complex (PDB ID, 7 BEK). The crystal asymmetry unit has an N501Y RBD/COVOX-269Fab complex, and the solvent content is-51%. Reconstruction of the cyclic model with COOT (Emsley and Cowtan, 2004) and refinement with PHENIX (Liebschner et al 2019) yields a resolution of all data of +.>R is as follows _work =0.197 and R _free ＝0.222。

The electron density of the side chain of Y501 is weak. However, when the structure is refined with asparagine at 501, there is a strong but diffuse positive density around the side chain, indicating the presence of flexible tyrosine residues (fig. 26). Mass spectrometry and bio-layer interferometry data confirm that tyrosine is indeed at 501.

Data collection and structure refinement statistics are given in table 10. Structural comparison residues forming the RBD/Fab interface were identified using SHP (Stuart et al, 1979), using PISA (Krissinel and Henrick, 2007), and mapped using PyMOL (PyMOL molecular graphics System, version 1.2r3pre, schrodinger).

Virus (virus)Stock solution

SARS-CoV-2/human/AUS/VIC01/2020 (Caly et al 2020) and SAR-CoV-2/B.1.1.7 supplied by UK public health department (Public Health England) were grown in Vero (ATCC CCL-81) cells. Cells were infected with SARS-CoV-2 virus at a multiplicity of infection of 0.0001. When 80% cpe was observed, the virus-containing supernatant was harvested and spun at 2000rpm at 4 ℃ and then stored at-80 ℃. Viral titers were determined by performing a lesion formation assay on Vero cells. Sequence verification was performed on both the 5 th generation Victoria and 2 nd generation b.1.1.7 stock, containing the expected spike protein sequence with no change in furin cleavage site.

Focal reduction neutralization assay (FRNT)

The neutralization potential of abs was measured using a Focus Reduction Neutralization Test (FRNT), in which the reduction in the number of infected foci was compared to a no antibody negative control well. Briefly, serial dilutions of Ab or plasma were mixed with SARS-CoV-2 strain Victoria or B.1.1.7 and incubated for 1 hour at 37 ℃. The mixture was then transferred in duplicate to 96-well cell culture treated flat bottom microplates containing confluent Vero cell monolayers and incubated for an additional 2 hours before 1.5% semi-solid carboxymethylcellulose (CMC) cover medium was added to each well to limit virus spread. Foci formation assay was then performed by staining Vero cells with human anti-NP mAb (mAb 206), followed by peroxidase conjugated goat anti-human IgG (a 0170; sigma). Finally, approximately 100 lesions (infected cells) per well were visualized in the absence of antibodies by adding truebue peroxidase substrate. Virus infected cell foci were counted using AID ELISpot software on a classical AID ELISpot reader. The percent lesion reduction was calculated and IC50 was determined using the probit program from the SPSS software package.

Psilosis vaccine

The serum of the psilosis was obtained 7-17 days after the second dose of vaccine, which was administered 3 weeks after the first dose (to the knowledge of the participants, they were negative for serum response at the time of group entry).

The study was approved by the oxford transformed gastroenterology unit (Oxford Translational Gastrointestinal Unit) GI biological library study 16/YH/0247[ ethical committee of study (REC) of yokken and henb-sheffield ]. The study was conducted in accordance with principles of the guidelines of the quality control Specification (Good Clinical Practice) (GCP) for clinical trials of drugs in accordance with the declaration of Helsinki (Declaration of Helsinki) (2008) and International conference of coordination (International Conference on Harmonization) (ICH). Written informed consent was obtained for all patients enrolled in the study. The vaccinators were medical staff of the NHS foundation of the university of Oxford hospital, who had not previously been infected with SARS-C0V-2. Each patient received two doses of the covd-19 mRNA vaccine BNT162b2 (30 micrograms) and was administered intramuscularly as a series of two doses (0.3 mL each) at 18-28 days intervals after dilution. The average age of the vaccinators was 43 years (ranging from 25-63 years), 11 men and 14 women.

Alaslicon-oxford vaccine research procedure and sample processing

All details of the random control trial of the ChAdOx1nCoV-19 (AZD 1222) have been previously published (PMID: 33220855/PMID: 32702298). These studies were registered with ISRCTN (15281137 and 89951424) and clinical trimals. Written informed consent was obtained for all participants and tested according to the principles of the helsinki statement (Declaration of Helsinki) and the pharmaceutical clinical trial quality management code (Good Clinical Practice). These studies were sponsored by the university of Oxford (UK) and approved by the national ethics committee (the south-middle burkest's research ethics committee, ref. 20/SC/0145 and 20/SC/0179) and the united kingdom regulatory agency (pharmaceutical and health care product regulatory agency). The independent DSMB reviews all mid-term security reports. Copies of the protocol are included in the previous publications (PMID: 33220855/PMID: 32702298).

Data from vaccinated volunteers receiving two vaccinations are included herein. Vaccine dose is 5×10 ¹⁰ The virus particles (standard dose; SD/SD cohort n=21) or half dose as its first dose (low dose) standard as its second doseDose (LD/SD cohort n=4). The interval between the first and second doses is in the range of 8-14 weeks. Blood samples were collected on the day of vaccination and on pre-specified days after vaccination (e.g., 14 days and 28 days after boosting) and serum was isolated.

Example 18.B.1.351 mutant changes

Many isolates of b.1.351 have been described, all of which have the key mutations K417N, E484K and N501Y in RBD. Tegally et al (Tegaly et al 2021) reported an isolate containing 10 changes (L18F, D80A, D215G, L242-244 deletion, R246I, K417N, E484K, N501Y, D614G, A V) relative to the Wuhan sequence. Sequencing of the strain used in this report (cases from the uk) showed only 8 changes and lacked L18F and R246I compared to Tegaly et al isolates. Coronavirus genomic sequences were analyzed in British strains obtained from the COG-UK database (Tatusov et al, 2000) and in south Africa strains obtained from GISAID (https:// www.gisaid.org). It appears that b.1.1.7 and b.1.351 rapidly take up overwhelming advantages in the uk and south africa, respectively. In the evolution of the b.1.1.7 variant in the uk and the b.1.351 variant in south africa, a large number of NTD-only deletion mutants (Δ69-70 in b.1.1.7 and Δ242-244 in b.1.153) and 501Y-only mutants were observed in both countries, and then strains with both deletions and 501Y were progressively dominant (fig. 27A, B). Thereafter, the count of the two "single mutant" variants was decreased. Characteristic mutations of b.1.351 as found in south africa are shown (figure 27C, D, E). In addition, by 2 months of 2021, 21 of the b.1.1.7 sequences were observed in the COG-UK database to have independently obtained the 484K (rather than 417N) mutation found in the b.1.351 variant, and 90 sequences showed these mutations in the background of b.1.351 (as defined by the deletion with the characteristic Δ242-244 NTD).

EXAMPLE 19 neutralization of convalescent plasma to B.1.351

Plasma was collected from a group of infected patients during the uk first wave SARS-CoV-2 infection. Samples were collected from recovery cases 4-9 weeks after infection at month 6 of 2020 before the emergence of b.1.1.7. Also included are the plasma recently collected from patients infected with b.1.1.7.

Neutralization titers against Victoria (early Whan-related strain of SARS-CoV-2) (Seemann et al 2020) were compared to B.1.351 using the Focus Reduction Neutralization Test (FRNT). For early recovery samples (n=34), the neutralization titer for b.1.351 was reduced by an average of 13.3-fold (p= < 0.0001) compared to Victoria (fig. 28A, table 14). A few convalescence samples (e.g., 4, 6, 15) remained well neutralized to b.1.351, but for the majority, the titer was greatly and significantly reduced, with 18/34 samples failing to reach 50% neutralization at a plasma dilution of 1:20, with some samples showing almost complete reduction in neutralization activity. Overall, neutralization titers between Victoria and b.1.351 were reduced 13.3-fold (geometric mean) (p < 0.0001) in 34 convalescence plasma samples (fig. 28C).

Neutralization was also performed using plasma recently collected from patients with b.1.1.7 (n=13) at different time points, all of these cases had S gene knockouts on diagnostic PCR (TaqPath, b.1.1.7 specific to sameire' S company), and 11 cases had virus sequencing confirming b.1.1.7 (fig. 28B, table 14). Both Victoria and b.1.351 had lower neutralization titers at early time points, but in one case (b.1.1.7p4), the samples taken 1 day after admission showed very high titers against Victoria (1:136,884) and b.1.351 (1:81,493), and this could represent reinfection b.1.1.7. Overall, titers between Victoria and b.1.351 were reduced 3.1-fold (geometric mean) in serum from patients infected with b.1.1.7 (fig. 28D).

EXAMPLE 20 neutralization of B.1.351 by vaccine serum

Neutralization of Victoria and b.1.351 was measured using vaccine serum obtained from individuals vaccinated with the psilosis-bayer vaccine BNT162b2 and oxford-aslicon AZD 1222. For the psilon-byntaceae, vaccinated serum (n=25) was obtained from the healthcare worker 4-17 days after the second dose, which was administered 3 weeks after the first dose (fig. 29A and table 15). For the aliskiren vaccine, samples were obtained 14 days or 28 days after the second vaccine dose (n=25), with dosing intervals of 8-14 weeks (fig. 29B, table 15). For the psilon-bayer vaccinee serum, the geometric mean titer of b.1.351 was 7.6 times lower (p= < 0.0001) than Victoria (fig. 29C), and for the oxford-alslicon vaccinee serum, the geometric mean b.1.351 titer was 9 times lower (p < 0.0001) than Victoria (fig. 29D and table 15).

The neutralizing titer against Victoria strains induced by the serum of the psittaceae vaccine was 3.6 times higher than that of the oxford-alslicon vaccine (p= < 0.0001). Although the overall decrease in titres was very similar, 7.6-fold and 9-fold, respectively, more samples failed to reach a 50% frnt titre (9/25) for b.1.351 than for the gabion vaccine (2/25) because the as Li Kangdi degrees began at a lower base.

EXAMPLE 21 neutralization of B.1.351 by monoclonal antibody

A library of 377 human monoclonal antibodies to spike proteins was generated from convalescence samples obtained from patients infected during the first wave of SARS-CoV-2 in the united kingdom. Neutralization assays were performed for 20 of the most potent mabs (FRNT 50 titers <50 μg/ml) (19 anti-RBD and 1 anti-NTD) against UK b.1.1.7 strain, victoria strain and b.1.351 strain (fig. 22A, tables 12 and 13). Data for Victoria and b.1.351 strains are also shown in fig. 30 and table 16.

The impact on mAb neutralization was severe, with a > 10-fold decrease in neutralizing titer of the 14/20 antibodies, most of which showed full knockout activity. This is consistent with the key roles of K417, E484, and N501 (particularly E484) in antibody recognition below and the ACE2 interaction surface of RBD described in fig. 31A-G.

Interestingly, the single potent NTD-binding antibody mAb 159 included in these assays also showed a complete knockout of activity against b.1.351 (which contained a deletion of amino acids 242-244 in the NTD portion of the epitope of mAb 159). As can be seen from fig. 31H, I, RBD loops 246-253 interact with the heavy chain of mAb 159 and the heavy chain of 4A8 (the only other strong neutralizing NTD binding agent with the reported structure (Chi et al, 2020)). The 242-244 deletion will undoubtedly alter the presentation of the loop, compromising binding to these mabs. Binding at this so-called "super site" has been reported to have potential therapeutic relevance (mccall et al 2021). The b.1.1.7, b.1.351 and p.1 lineages all converged due to deletions or systematic alterations in NTD. Although p.1 does not have an NTD deletion, it is expected that altering L18F, T20N and P26S (Faria et al, 2021) would significantly affect binding at the NTD epitope. Since these converging characteristics may not be caused by the selection pressure from the antibody response, there still appears to be potential biological drivers to be discovered, such as increased receptor binding and potentially increased transmissibility conferred by RBD mutations, which may lead to the epitope being extremely sensitive to and escaping from antibody binding.

EXAMPLE 22 neutralization of B.1.351 by monoclonal antibody in late clinical trials

Many monoclonal antibodies are in post clinical trials as therapies or prophylactics against SARS-CoV 2. The regenerator and the aslicon used a mixture of 2 monoclonal antibodies to resist mutational escape of the virus. Neutralizing assays were performed with the regenerator pairs REGN10933 and REGN10987 and the aslicon pairs mAb AZD106 and AZD8895 (fig. 22B, 30B). Neutralization of REGN10987 was not affected by b.1.351, whereas REGN10933 was severely damaged (factor 317) (fig. 30B). Compared to Victoria, AZ has little effect on b.1.351 on neutralization of antibodies.

Table 12 shows that many of the most potent mabs against Victoria strains maintained high potency against b.1.1.7 strain. In particular, mabs 40, 55, 58, 222, 281, 316, 384, 394 and 398 maintain strong potency against b.1.1.7. Many of the most potent mabs also maintained high potency against south african strains (b.1.351). In particular, mabs 55, 58, 150, 165, 222, 253, 278 and 318 maintained strong potency against b.1.351.

Example 23 understanding neutralization was abolished: binding of ACE2 to B.1.351RBD

The triple mutations K417N, E484R and N501Y are characteristic of B.1.351 RBD. These residues lie within the ACE2 footprint (FIG. 27E), and in vitro evolution of optimizing affinity for ACE2 suggests that they confer higher affinity for the receptor (Starr et al, 2020; zahradnI, et al, 2021). To investigate this effect, the kinetics of soluble ACE2 binding to recombinant RBD was measured by bio-layer interferometry (BLI) (fig. 32, A, B). As expected, the affinity for b.1.351RBD was very high, in fact 19-fold higher than that for Victoria RBD and 2.7-fold higher than that for b.1.1.7. KD of 4.0nm, kon of 4.78E4/Ms and Koff of 1.93E-4/s, thus the off-rate is about 1.5 hours, which will further exacerbate the efficacy decline observed in neutralization assays, as lower affinity antibodies will have difficulty competing with ACE2 unless they have a very slow off-rate or show affinity effects blocking attachment. Thus, while the potent RBD binders of this group all have higher affinities (KD of 75.1 and 10.7nM, respectively) than between ACE2 and Victoria or b.1.1.7RBD, five of the 19 have affinities lower than or equal to ACE2 and b.1.351 RBD. Further small increases in affinity (e.g., 2-fold) will defeat almost all antibodies.

Example 24 profiling of the effects of RBD mutations on RBD binding

To see the order of more than two-thirds of neutralization abrogation in the 19 potent mabs binding RBD, KD binding to recombinant RBD was measured by BLI (fig. 32, C, D, table 16). While 17 fabs with KD lower than 4nM (affinity of ACE2 to b.1.351) for Victoria test, this was reduced to 4 for b.1.351 (or 2 if the engineered light chain version of 253 was removed, with 7 fabs failing to achieve an affinity close to μm). These results follow approximately the neutralization results (compare panels C and D of fig. 32, see table 16), indicating that the observed pattern of effect on neutralization is primarily due to amino acid substitutions in RBD, K417N, E484K, and N501Y.

The basis of these effects can be understood in the context of anatomical descriptions of RBD, we have defined four almost continuous structural epitopes (left shoulder, neck, right shoulder and right flank) and a separate left flank epitope for the human torso (fig. 32E). Herein, the ACE2 binding site extends through the neck and both shoulders. N501Y is on the right shoulder, K417N is at the back of the neck, and E484R is on the left shoulder. Although these three mutations are nominally in different epitopes, the overlapping nature of these epitopes means that the residues are close enough that more than one may directly affect the binding of any one antibody. In addition, allosteric effects (structural equivalents that are genetically upper) may exist, thereby affecting the extendability over a certain distance. This, combined with the observation that only a relatively small fraction of footprint residues are critical for binding, explains the difference between structural epitopes (footprints) and functional epitopes (Cunningham and Wells, 1993). Despite these warnings, most of the effects observed can be directly explained by reference to previous structural knowledge.

Many of the reported Fab/SARS-CoV-2RBD complexes are directed against antibodies using the public HC V region IGHV3-53 (Yuan et al 2020), and these antibodies are represented in this group by five antibodies that are potent against Victoria virus. The neutralizing and binding capacity of four of these antibodies, 150, 158, 175 and 269, was severely compromised or abolished, with 222 being the exception, since its binding was not affected by the b.1.351 variant (fig. 32F, G). The IGHV3-53 antibody family binds at the same epitope in the posterior neck of RBD in a very similar proximity orientation as IGHV3-66 Fab also shares. Most of them are in direct contact with K417 and N501, but none with E484. The rather short HC CDR3 of these Fab is typically located directly above K417, forming hydrogen bonds or salt bridges and hydrophobic interactions, while N501 interacts with the LC CDR-1 loop (fig. 31). However, mAb 150 is slightly different, forming a salt bridge between K417 and LC CDR3D92 and an H bond between N501 and S30 in LC CDR1 (fig. 31B), while 158 more typically forms a hydrogen bond from the carbonyl oxygen of G100 and K417 of HC CDR3 and a hydrophobic contact from S30 to N501 of LC CDR 1. Thus, the combined effect of the K417N and N501Y mutations is expected to severely impair the binding of most IGHV3-53 and IGHV3-66 mAbs. However, one member 222 of this class is not affected by the b.1.1.7 or b.1.351 variants.

Fab 88 binds RBD at the rear of the left shoulder, residues G104 and K108 of HC CDR3 contact E484, while LC CDR2 produces extensive hydrophobic interactions as well as backbone hydrogen bonding from Y51 and a salt bridge from D53 to K417 (fig. 31A). It is expected that the charge at E484 changes from negative to positive and shortening of the side chain of residue 417 from K to N abrogates all of these interactions, accounting for the hundreds of times loss of KD. 384 is one of the most potent neutralizing mabs we found against Victoria virus. The mAb approaches the binding site from the front of the left shoulder, burying the 82% solvent accessible region of E484 by hydrogen bonding with Y50, T57 and Y59 and salt bridging with R52 of HC CDR2 (fig. 31D), explaining the catastrophic effect of the E484K mutation on binding (table 16).

mAb 222 is not the only antibody that exhibits resistance to b.1.351. The FRNT50 titers of mabs 55, 165, 253 and 318 between Victoria and b.1.351 were also relatively equal, indicating that their epitopes were not interfered with by the K417N, E484K and N501Y mutations. Antibodies 55, 165 and 253 are related to each other and it was shown that combining the light chain of 55 or 165 with the heavy chain of 253 resulted in an increase in neutralization titer>1log. Chimeras 253H/55L and 253H/165L both neutralize B.1.351, FRNT ₅₀ Titers were 9 and 13ng/ml, respectively. 253 and the structures of these chimeric Fab with RBD or spike showed that they bind almost identically to the same epitope and do not contact any of the three mutation site residues, correlating well with the neutralization and BLI binding data (fig. 31C).

Example 25 methods of examples 18 to 24

Virus stock solution

SARS-CoV-2/human/AUS/VIC01/2020 (Caly et al 2020) provided by UK public health department (Public Health England) and SARS-CoV-2/B.1.1.7 were both grown in Vero (ATCC CCL-81) cells. Cells were infected with SARS-CoV-2 virus using MOI of 0.0001. The virus-containing supernatant was harvested at 80% CPE and spun at 2000rpm at 4℃and then stored at-80 ℃. Viral titers were determined by performing a lesion formation assay on Vero cells. Both the 5 th generation Victoria and 5 th generation b.1.157 stock were sequenced to verify that they contained the expected spike protein sequence and no change in furin cleavage sites. The B1.351 virus used in these studies contained the following mutations: D80A, D G, L-244 deletion, K417N, E484K, N501Y, D614G, A V.

Bacterial strains and cell culture

Vero (ATCC CCL-81) cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) high glucose (Sigma Aldrich) supplemented with 10% Fetal Bovine Serum (FBS), 2mM Glutamax (Ji Bike company (Gibco), 35050061) and 100U/ml penicillin-streptomycin at 37 ℃. Human mAbs were expressed in HEK293T cells cultured in UltraDOMA PF protein-free medium (catalog No. 12-727F, longsha Corp (LONZA)) at 37℃and 5% CO 2.

Transformation of plasmid pNEO-RBD K417N, E484K, N Y was performed using E.coli DH 5. Alpha. Bacteria. Single colonies were picked and grown with 50. Mu.g mL ^-1 Kanamycin LB broth at 37℃at 200rpm in a shaker overnight. At 37℃and 5% CO ₂ HEK293T (ATCC CRL-11268) cells were cultured in DMEM high glucose (Sigma Aldrich Co.) supplemented with 10% FBS, 1%100X Mem Neaa (Ji Bike Co.) and 1%100X L-glutamine (Ji Bike Co.). To express RBD, RBD K417N, E484K, N Y and ACE2 HEK293T cells were cultured at 37 ℃ in DMEM high glucose (sigma) supplemented with 2% fbs, 1%100x Mem Neaa and 1%100x L-glutamine for transfection.

Participants (participants)

Participants were recruited through three studies: sepsis immunology [ oxford REC C, reference: 19/SC/0296], ISARIC/WHO severe reinfection clinical characterization protocol [ oxford REC C, reference: 13/SC/0149] and oxford gastrointestinal disease: covd sub-study [ sheffield REC, reference: 16/YH/0247]. Diagnosis was confirmed by reporting symptoms consistent with covd-19 and detecting SARS-CoV-2 positives from approved laboratory-detected upper respiratory (nasal/laryngeal) swabs using reverse transcriptase polymerase chain reaction (RT-PCR). Blood samples were collected at least 14 days after symptoms had occurred after informed consent. Clinical information is captured for all individuals at the time of sampling, including the severity of the disease (classified as mild, severe or severe infection according to recommendations of the world health organization) and the time between symptoms and sampling and the age of the participants.

Serum from a psilosis vaccinator

Alaslicon-oxford vaccine research procedure and sample processing

All details of the random control trial of the ChAdOx1nCoV-19 (AZD 1222) have been previously published (PMID: 33220855/PMID: 32702298). These studies were registered with ISRCTN (15281137 and 89951424) and clinical trimals. Copies of the protocol are included in the previous publications (PMID: 33220855/PMID: 32702298).

Data from vaccinated volunteers receiving two vaccinations are included herein. Vaccine dose is 5×10 ¹⁰ The number of virus particles (standard dose; SD/SD cohort n=21) or the half dose as its first dose (low dose) or the standard dose as its second dose (LD/SD cohort n=4). The interval between the first and second doses is in the range of 8-14 weeks. Blood samples were collected on the day of vaccination and on pre-specified days after vaccination (e.g., 14 days and 28 days after boosting) and serum was isolated.

COG-UK sequence analysis

Downloading the COG-UK at 2.2.2021 (Tatusov et al, 2000) and the GISAID sequence at 1.30.2021 from south Africa (https:// www.gisaid.org /) and obtaining the spike protein sequence after nucleotide 21000, followed by sequence alignment and mutation identification. The b.1.351 variant was filtered using selection criteria 501Y and Δ242. The b.1.1.7 variants were filtered using selection criteria 501Y and Δ69. The structural positions of mutations were modeled as red (single point mutation), black (deletion) or blue (addition) on spike structure, the size was proportional to the logarithm of the incidence, and those mutations with an incidence of more than 5% in the population were clearly labeled.

Focal reduction neutralization assay (FRNT)

The neutralization potential of abs was measured using a Focus Reduction Neutralization Test (FRNT), in which the reduction in the number of infected foci was compared to a no antibody negative control well. Briefly, serial dilutions of Ab or plasma were mixed with SARS-CoV-2 strain Victoria or B.1.351 and incubated for 1 hour at 37 ℃. The mixture was then transferred in duplicate to 96-well cell culture treated flat bottom microplates containing confluent Vero cell monolayers and incubated for an additional 2 hours before 1.5% semi-solid carboxymethylcellulose (CMC) cover medium was added to each well to limit virus spread. Foci formation assay was then performed by staining Vero cells with human anti-NP mAb (mAb 206), followed by peroxidase conjugated goat anti-human IgG (a 0170; sigma). Finally, approximately 100 lesions (infected cells) per well were visualized in the absence of antibodies by adding truebue peroxidase substrate. Virus infected cell foci were counted using AID ELISpot software on a classical AID ELISpot reader. Using the probit program from the SPSS software package to calculate the percent lesion reduction and determine IC ₅₀ 。

Cloning of native RBD, ACE2 and RBD K417N, E484K, N501Y

The native RBD and ACE2 constructs are identical to those of Zhou et al (Zhou et al 2020). Another construct comprising K417N, E484K, N501Y was generated using PCR, which construct was identical to the native RBD except for the K417N, E484K and N501Y mutations, as confirmed by sequencing.

Protein production

Protein production is as described by Zhou et al (Zhou et al 2020).

Biological layer interferometry

The BLI experiments were run on an Octet Red 96e machine (Buddha). To measure the binding affinity of monoclonal antibodies and ACE2 to native RBD and RBD K417N, E484K, N Y, each RBD was immobilized onto an AR2G biosensor (foterbio corporation). Monoclonal antibodies were used as analytes or serial dilutions of ACE2 were used as analytes. All experiments were run at 30 ℃. Data were recorded using the software Data Acquisition 11.1 (Buddha Corp.) and Data Analysis HT 11.1 (Buddha Corp.) with a 1:1 fitting model for Analysis.

EXAMPLE 26 mutation of P.1

P.1 was first reported by agates in amazon, north brazil at month 12 (Faria et al 2021). Agates developed the first large wave infection in 3 to 6 months 2020, and by 10 months it was estimated that about 75% of individuals in this area were infected, representing a very high rate of attack. Month 12 in 2020, a second large wave infection started, resulting in further hospitalization. This second wave corresponds to the rapid onset of p.1, which was not seen until 12 months ago (when found in 52% of cases), rising to 85% by 1 month 2021 (fig. 40).

P.1 contains a number of changes compared to the previous B.1.1.28 and P.2 cycles in Brazil (Faria et al, 2021). Compared with the Wuhan sequence P.1, the sequence contains the following mutations: L18F, T20N, P S, D35138Y, R S in NTD, K417T, E484K, N501Y in RBD, D614G and H655Y at the C-terminus of S1, and T1027I, V1176F in S2. The altered positions seen in p.1 and their representation of the positions of occurrence on full spike protein and RBD compared to those found in b.1.1.7 and b.1.351 are shown in fig. 33. The mutations K417T, E484K, N501Y in ACE2 interacting surfaces are of most interest as they might promote evasion of neutralizing antibody reactions mainly targeting this region (fig. 33D). British COVID-19 genomics (COG-UK) (Tatusov et al, 2000) and global shared avian influenza data initiative (GISAID) (https:// www.gisaid.org) databases were retrieved. A small number of sequences including the K417T mutation (including the p.1 lineage) were observed in the sequencing of japan, france, belgium, italy, the netherlands and columbia (fig. 40).

Notably, P.1, B.1.1.7 and B.1.351 produced multiple mutations in NTD, two deletions Δ69-70 and Δ144 in B.1.1.7, four amino acid changes and Δ242-244 in B.1.351, and 6 amino acid changes in NTD in P.1, but no deletion. Notably, two of the NTD changes in p.1 introduced N-linked glycosylation sequence subunits T20N (TRT to NRT) and R190S (NLR to NLS, fig. 33E). In the absence of these changes, NTDs fill quite well with glycosylation sites, and in fact it has been shown that a single bare patch surrounded by N-linked glycans attached at N17, N74, N122, and N149 defines a "super-site" restriction in which neutralizing antibodies can attach to NTDs (ceritti et al 2021). Residue 188 is somewhat blocked, while residue 20 is highly exposed, near the attachment site of neutralizing antibody 159, and impinges on the proposed NTD super site.

Example 27 influence of RBD mutations on ACE2 affinity

The affinities of RBD/ACE2 interactions for Wuhan, b.1.1.7 (N501Y) and b.1.351 (K417N, E484K, N Y) RBD were measured in the previous examples. N501Y increases affinity 7-fold, and the combination of mutations 417, 484 and 501 further increases affinity (19-fold increase compared to Wuhan). Here, p.1 RBD (K417T, E484K, N501Y) is shown. The KD of the p.1/ACE2 interaction was 4.8nM, with kon= 1.08E5/Ms and koff=5.18E-4/s (fig. 41, methods), indicating that binding to p.1 is substantially indistinguishable from b.1.351 (4.0 nM).

To better understand RBD-ACE2 interactions, inThe crystal structure of the RBD-ACE2 complex was determined at resolution (example 35, table 17). The RBD-ACE2 engagement pattern of p.1 and the original Wuhan RBD sequence is essentially identical (fig. 34A). RMS deviation between 791 Ca positions +.>Similar to experimental errors in coordinates, and the local structure around each of the three mutations is conserved. However, calculation of the electrostatic potential of the contact surface revealed a significant change, with the p.1 RBD having much greater complementarity consistent with higher affinity. (FIG. 34B, C, D).

Residue 417 is located at the rear of the RBD neck and is a lysine residue forming a salt bridge with D30 of ACE2 in the original SARS-CoV-2 (fig. 34E). Threonine of the p.1 RBD no longer forms this interaction and the resulting gap is open to solvent, so there is no obvious reason that mutations will increase affinity for ACE2, and this is consistent with directed evolution studies in which such mutations are seldom selected in RBDs with increased affinity for ACE2 (zahradni k et al 2021).

Residue 484 was located on the left shoulder of RBD and neither the original Glu nor Lys of P1 was significantly contacted with ACE2, whereas the significant change in charge significantly improved electrostatic complementarity (fig. 34F, G), consistent with the increased affinity.

Residue 501 is located on the right shoulder of RBD and the change from a relatively short Asn side chain to a large aromatic Tyr allows for favorable loop stacking interactions, consistent with increased affinity (fig. 34H).

EXAMPLE 28 binding and neutralization of P.1 RBD by potent human monoclonal antibodies

Of the 20 potent antibody groups with a focal reduction neutralization 50% (FRNT 50) value <100ng/ml, 19 of these mabs have epitopes on RBD and all of these block ACE2/RBD interactions, while mAb 159 binds NTD. Biological Layer Interferometry (BLI) was used to measure the affinity of RBD binding antibodies, and Victoria (SARS-CoV-2/human/AUS/VIC 01/2020) (early isolate of SARS-CoV-2) was found to have a single change S247R in S compared to the Wuhan strain (Seemann et al 2020; caly et al 2020). Monoclonal antibody binding was significantly affected, some showed complete knockdown of activity (fig. 34I). The results for P.1 showed a greater effect than B.1.1.7, but were similar to B.1.351 (Zhou et al, 2021), which was expected because both contained the same 3 residue mutation in RBD, differing only at position 417, K417N in B.1.351, and K417T in P.1. The localization of the binding effect is shown in fig. 34J and reflects the direct interaction with the mutated residue. Notably, mAb 222, although adjacent to the mutated residue, maintained binding potency in all variants, as discussed in the examples below.

EXAMPLE 29 neutralization of P.1 by potent human monoclonal antibodies

Neutralization was measured by a Focus Reduced Neutralization Test (FRNT) using the same set of 20 potent antibodies and compared to neutralization by Victoria and variants b.1.1.7 and b.1.351. Neutralization of monoclonal antibodies was significantly affected by p.1 compared to Victoria, 12/20 showed > 10-fold reduction in FRNT50 titer, and some showed complete knockdown of activity (fig. 35; table 18). The results of P.1 showed a greater effect than B.1.1.7, but were similar to those of B.1.351 (Zhou et al, 2021). There was a good correlation between negative effects on neutralization and on RBD affinity (fig. 34J).

Example 30. Neutralization of P.1 by monoclonal antibodies being developed for clinical use was reduced.

Many potent neutralizing antibodies are being developed for therapeutic or prophylactic clinical use (Ku et al, 2021; baum et al, 2020; kemp et al, 2021). The neutralization assay was performed for P.1 using antibodies S309 Vir (Pinto et al 2020), AZD8895 and AZD1061 from Abies, REGN10987 and REGN10933 from Rens, LY-CoV555 and LY-CoV16 from Gift, and ADG10, ADG20 and ADG30 from Aldajia (FIG. 35B). The affinity of the regenerator and the aslicon antibodies for binding to p.1 RBD was also studied by BLI, and the results (fig. 34I) were very similar to the neutralization results. Neutralization of both gift antibodies was severely impaired, LY-CoV16 and LY-CoV555 showed almost complete loss of neutralization of P.1 and B.1.351, while LY-CoV16 also showed a significant decrease in neutralization of B.1.1.7. There was also a moderate decrease in neutralization of p.1 by AZD8895 from the escape of neutralization of p.1 by REGN 10933. All variants were neutralized by the three adagil antibodies, reaching a plateau at 100% neutralization, and ADG30 showed a slight increase in neutralization of p.1. S309 Vir is largely unaffected, although for several viruses (including P.1) the antibody is not fully neutralised, possibly reflecting incomplete glycosylation at N343, as sugar interactions are critical for binding of the antibody N343 (Pinto et al 2020). Escape from REGN10933 and LY-CoV555 reflects escape of other potent antibodies (including antibodies 316 and 384) that interact strongly with residues 484-486 and are severely impaired by significant changes in E484K, while LY-CoV016 (an IGHV3-53 mAb) is affected by changes at 417 and 501. The depletion of the Ly-CoV-16 and LyCoV-555 antibodies reflects the observation of Starr et al (Starr et al, 2021) (Greaney et al, 2021), that LY-CoV555 is sensitive to the mutation at residue 384 and LY-CoV16 is sensitive to the change at 417.

Example 31 neutralization and reduction of NTD binding antibodies

Compared to Victoria (only 64% neutralization at 10 μg/ml), the neutralization titer of p.1 by NTD-binding mAb159 was reduced 133-fold (fig. 35A). Although P.1 did not have a deletion in NTD like B.1.1.7 (Δ69-70, Δ144) or B.1.351 (Δ242-244), it is clear that 6 NTD mutations in P.1 (L18F, T20N, P26S, D138Y, R190S) disrupted the epitope of mAb159 (FIG. 36A). The failure of the antibody to achieve complete neutralization may be due to the distance bound Fab 159 aboutWhile the L18F mutation was even closer and likely reduced affinity (fig. 36A). Since potent NTD binding antibodies have been proposed with a single super site, binding of many of these is expected to be affected (Cerutti et al 2021).

EXAMPLE 32 neutralization and reduction of VH3-53 public antibodies

Five of the potent monoclonal antibodies used herein (150, 158, 175, 222 and 269) belong to the VH3-53 family, and the other 2 (of 5 of this family) belong to nearly identical VH3-66, and the discussion below applies to these antibodies as well. Binding sites for these antibodies have been described in the previous examples. Most of these antibodies attach to RBD in a very similar manner. These motifs are widely reproduced, VH3-53 being the most common deposited sequence and structure for SARS-CoV-2 neutralizing antibodies. Their engagement with RBD is determined by CDR-H1 (SEQ ID NOS: 449, 452, 455, 458 and 461) and CDR-H2 (SEQ ID NOS: 450, 453, 456, 459 and 462), while CDR-H3 (SEQ ID NOS: 451, 454, 457, 460 and 463) is characterized by a short and relatively few interactions (Yuan et al 2020; barnes et al 2020). The structure of mabs 150, 158 and 269 has been resolved (fig. 36B), indicating that although not in contact with residue 484, CDR-H3 interacts with K417 and CDR-L1 interacts with N501, meaning that binding and neutralization of VH3-53 antibodies is predicted to be impaired by the N501Y changes in variant viruses b.1.1.7, b.1.351 and p.1, whereas additional changes at 417 in p.1 (K417T) and b.1.351 (K417N) might be expected to produce additive effects.

Neutralization of p.1 by 175 and 158 was severely impaired, and neutralization of p.1 by 269 was almost completely lost. However, for 150p.1, neutralization was less damaging than b.1.351 (Zhou et al, 2021), whereas for 222, neutralization was completely unaffected by changes in p.1 and virtually all variants (fig. 35A).

Measurement of 222 affinity for p.1 (kd=1.92±0.01 nM) and Wuhan RBD (kd=1.36±0.08 nM) showed no decrease in interaction strength, despite the change in the putative binding site of p.1 (table 18).

To see how 222 can still neutralize p.1, the crystal structure of the six ternary complexes formed by the recombination of 222 with RBD was resolved for the following: (i) the original virus and carrying the mutation (ii) K417N; (iii) K417T; (iv) N501Y; b.1.351 Changes 417, 484 and 501 specific to (v) and p.1 (vi). All crystals also contain another Fab (EY 6A as crystallization partner) (Zhou et al 2020), are isomorphous and have a resolution of 1.95 to structureWithin (fig. 36C, D, example 35, table 17). As expected, the binding poses of 222 are substantially identical in all structures (paired RMSDs in the ca atoms between pairs of structures are for all residues in the RBD and Fv regions of mAb 222 Fig. 36D).

In the original virus, residue 417 forms a weak salt bridge interaction with heavy chain CDR3 residue E99. Mutation to Asn or Thr abrogates this and has little direct interaction, although weak with heavy chain Y52 and light chain Y92Contact (fig. 36E). However, the buffer molecules/ions rearrange to form bridging interactions, and this may mitigate the loss of salt bridges, in addition, the original salt bridge is weaker and its contribution to binding may be offset by the loss of entropy in the lysine side chains. 222 is slightly longer than the CDR-H3 at 13 residues (SEQ ID NO: 457) found in most potent VH3-53 antibodies, however this seems unlikely to be the reason for 222 resistance, but it appears that there is generally little binding energy from CDR3-H3, since most of the binding energy of the heavy chain contributes to the binding energy from CDR-H1 (SEQ ID NO: 455) and CDR-H2 (SEQ ID NO: 456) that do not interact with RBD residue 417, meaning that many VH3-53 antibodies may be resistant to common N/T mutations (FIG. 36B).

Residue 501 is in contact with CDR-L1 of mAb 222 (SEQ ID NO: 468) (FIG. 36, D, F), however, the interaction with P30 may be slightly enhanced by the N501Y mutation, which provides a stacked interaction with proline, conferring resistance. This is in contrast to the case where direct contact confers sensitivity to the majority of other VH3-53 antibodies (fig. 34I, J and 35A) that escape through mutation to Tyr.

Example 33.222 light chain can rescue neutralization of other VH3-53 mAbs

The reasoning about the relative robustness of mAb 222 to common variants (p.1, b.1.1.7 and b.1.351) compared to other VH3-53 antibodies stems from the selection of light chains, we modeled 222LC with heavy chains of other VH3-53 antibodies to see if they are likely compatible (fig. 36G). Unexpectedly, it seems unlikely that there is a serious spatial conflict. This is in contrast to many of the conflicts that we see when we have the light chain of other VH3-53 antibodies linked to the heavy chain of 222 (figure 36G, H). This suggests that the 222 light chain may be the almost universal light chain of these 3-53 antibodies and may confer resistance to the p.1, b.1.1.7 and b.1.351 variants. This led us to generate chimeric antibodies containing 222LC in combination with HC of other VH3-53 mabs 150, 158, 175 and 269. In all cases, the chimeric antibodies expressed well and neutralization assays were performed against Victoria, b.1.1.7, b.1.351 and p.1 viruses (fig. 37). For B.1.1.7, neutralization of 150HC/222LC, 158HC/222LC and 269HC/222LC was restored to a level close to that seen on Victoria, whereas 175HC/222LC was unable to fully neutralize B.1.1.7. For b.1.351 and p.1, the activity of mabs 150 and 158 was restored in chimeras containing 222LC, with 150HC/222LC showing 50-fold higher potency against b.1.351 (7 ng and 350 ng/ml) and 13-fold higher potency against p.1 (3 ng and 40 ng/ml) than native 150. At 3ng/ml FRNT50, 150HC/222LC was the most potent antibody tested against P.1.

A number of common antibody responses (antibodies derived from the common v gene) have been reported for SARS-CoV-2, mainly VH3-53/VH3-66 and VH1-58 (Yuan et al 2020; barnes et al 2020). Mixing the heavy and light chains from the antibody within VH1-58 can increase the neutralization titer 20-fold relative to the parent antibody (253 HC chimeras with 55LC or 165 LC). Here, it was shown that the use of 222LC to generate chimeras in VH3-53 antibodies was able to confer extensive neutralization of antibodies with reduced neutralizing capacity against viral variants. Furthermore, 150HC and 222LC chimeras achieved 13-fold and 3-fold neutralization titer increases, respectively, compared to the parent 150 and 222 mAb. Due to the similarity between VH3-53 and VH3-66, chimeras between heavy and light chains of such antibodies are also expected to result in increased neutralization titers in a similar manner. The generation of such antibody chimeras in other anti-SARS-CoV 2 antibodies can similarly lead to the discovery of more antibodies with enhanced activity.

EXAMPLE 34 neutralization of P.1 by convalescent plasma and vaccine serum

Convalescent plasma samples were collected from a group of volunteers with SARS-CoV-2 infection as demonstrated by positive diagnostic PCR, as described in the previous examples. Samples were collected during the recovery period, i.e. 4-9 cycles after infection, all samples were taken during the first wave infection in the uk, before month 6 in 2020 and before the b.1.1.7 variants appeared. Plasma was also collected from recently infected b.1.1.7 volunteers as evidenced by viral sequencing or determination of S gene deletion from diagnostic PCR.

Neutralization of P.1 was assessed by FRNT on 34 recovery phase samples (FIG. 38A; table 19A). The p.1 neutralization curve is shown with Victoria along with the neutralization curves of b.1.1.7 and b.1.351. The geometric mean neutralization titer of p.1 was reduced 3.1-fold (p < 0.0001) compared to Victoria. This decrease was similar to b.1.1.7 (2.9 times) and much less than b.1.351 (13.3 times) (fig. 38C). When plasma from individuals who had transfected b.1.1.7 was used, p.1 was compared to Victoria, we only seen a slight decrease in neutralization (1.8-fold, p=0.0039) (fig. 38B and D, table 19B).

Next neutralization assays were performed using serum collected from individuals receiving BNT162b 2-pyro-bayeraceae or ChAdOx1nCoV-19 oxford-alslicon vaccine (fig. 39). For the psilon-bayer takii vaccine, serum was collected 4-14 days after the second dose of vaccine, which was administered three weeks after the first dose (n=25). For the oxford-alsikan vaccine, serum was collected 14 days or 28 days after the second dose, which was administered 8-14 weeks after the first dose (n=25). For the psii-bai-entiac vaccine serum, the geometric mean neutralization titer against p.1 was reduced 2.6-fold relative to Victoria virus (p < 0.0001) (fig. 39A, C), and 2.9-fold for the oxford-alsikan vaccine (p < 0.0001) (fig. 39B, D, table 20).

Neutralization titers against p.1 were similar to those against b.1.1.7, and only a few samples failed to achieve 100% neutralization at a 1:20 dilution of serum, significantly superior to that of b.1.351, with the titers of BNT162b 2-gabox 1 nCoV-19 assiconazole vaccine reduced 7.6-fold and 9-fold, respectively.

The reason for the difference in neutralization of b.1.351 and p.1 by immune serum is not clear, but may reflect the difference in mutations introduced beyond RBD. In addition to mAb 159, a number of potent neutralizing mabs have been reported that map to NTD (Cerutti et al, 2021), and this domain has multiple mutations in all three major variant strains: b.1.1.7 had two deletions, b.1.351 had one deletion and four substitutions, and p.1 had 6 amino acid substitutions, including the creation of two N-linked glycan sequences (fig. 33A-C). Neutralization comparison of a pseudovirus expressing only three RBD mutations of B.1.351 (K417N E484K N501Y) with a pseudovirus expressing a complete set of mutations in B.1.351 spike shows that non-RBD changes greatly increase neutralization escape (Wibmer et al 2021; wang et al 2021). The variation in NTD of the main variants was far less consistent than that found in RBD, and there was no strong trend in electrostatic properties (fig. 33A-C). Thus, it is still unclear what the driving factors for these changes are, although it is reasonable to one or more of immune escape, co-receptor binding, and regulation of RBD dynamics that affect the presentation of the receptor binding site. Nevertheless, these changes appear to be the main cause of non-RBD components of neutralization variation between strains.

Example 35 materials and methods of examples 26 through 34

Virus stock solution

SARS-CoV-2/human/AUS/VIC01/2020 (Caly et al 2020), SARS-CoV-2/B.1.1.7 and SARS-CoV-2/B.1.351 are provided by UK public health department (Public Health England) and P.1 from Brazil pharyngeal swabs is grown in Vero (ATCC CCL-81) cells. Cells were infected with SARS-CoV-2 virus using MOI of 0.0001. The virus-containing supernatant was harvested at 80% CPE and spun at 3000rpm at 4℃and then stored at-80 ℃. Viral titers were determined by performing a lesion formation assay on Vero cells. The 5 th generation Victoria, 2 nd generation b.1.1.7 and 4 th generation b.1.351 stocks were sequenced to verify that they contained the expected spike protein sequences and furin cleavage sites unchanged. The p.1 virus used in these studies contained the following mutations: L18F, T20N, P S, D138Y, R190S, K417T, E464K, N501Y, D614G, H655Y, T1027I, V1176F. The generation 1 P.1 virus was sequence-confirmed, and the furin cleavage site was unchanged.

Bacterial strains and cell culture

Vero (ATCC CCL-81) cells were cultured in Dulbeck's Modified Eagle Medium (DMEM) high glucose (sigma aldrich) supplemented with 10% Fetal Bovine Serum (FBS), 2mM GlutaMAX (Ji Bike company, 35050061) and 100U/ml penicillin-streptomycin at 37 ℃. Human mAbs were expressed in HEK293T cells cultured in UltraDOMA PF protein-free medium (catalog No. 12-727F, longsha Corp (LONZA)) at 37℃and 5% CO 2. Coli DH 5. Alpha. Bacteria were used to transform plasmids encoding wt and mutant RBD proteins. Single colonies were picked and cultured overnight in LB broth with 50. Mu.g of mL-1 kanamycin at 37℃at 200rpm in a shaker. HEK293T (ATCC CRL-11268) cells were cultured in DMEM high glucose (sigma aldrich) supplemented with 10% fbs, 1%100x Mem Neaa (Ji Bike company) and 1%100x L-glutamine (Ji Bike company) at 37 ℃ and 5% co 2. To express RBD, RBD K417T, E484K, N501Y, RBD K417N, RBD K417T, RBD E484K and ACE2, HEK293T cells were cultured in DMEM high glucose (sigma) supplemented with 2% fbs, 1%100x Mem Neaa and 1%100x L-glutamine at 37 ℃ for transfection.

Participants (participants)

P.1 virus from pharyngeal swabs. The international reference laboratory for coronaviruses, FIOCRUZ (WHO), as part of national coronavirus monitoring, was approved by the FIOCRUZ ethics committee (CEP 4.128.241) to continuously receive and analyze samples of suspected cases of covd-19 for virological monitoring. Clinical samples (pharyngeal swabs) containing P.1 were shared with the university of Oxford, england according to MTA IOC FIOCRUZ 21-02.

Serum from a psilosis vaccinator

The serum of the psilosis was obtained 7-17 days after the second dose of BNT162b2 vaccine. The vaccinators were medical personnel of The NHS foundation of The university of oxford hospital, had not previously been infected with SARS-CoV-2, and were included in The group OPTIC study as part of The oxford transformation gastrointestinal GI BioBank study 16/YH/0247 (Jokk county and Henber-Sheffield (Yorkshire & The Humber-Sheffield) Research Ethics Committee (REC)). The study was conducted in accordance with principles of the guidelines of the quality control Specification (Good Clinical Practice) (GCP) for clinical trials of drugs in accordance with the declaration of Helsinki (Declaration of Helsinki) (2008) and International conference of coordination (International Conference on Harmonization) (ICH). Written informed consent was obtained for all patients enrolled in the study. Each patient received two doses of the covd-19 mRNA vaccine BNT162b2 (30 micrograms) and was administered intramuscularly as a series of two doses (0.3 mL each) at 18-28 days intervals after dilution. The average age of the vaccinators was 43 years (ranging from 25-63 years), 11 men and 14 women.

Alaslicon-oxford vaccine research procedure and sample processing

All details of the random control trial of the ChAdOx1 nCoV-19 (AZD 1222) have been previously published (PMID: 33220855/PMID: 32702298). These studies were registered with ISRCTN (15281137 and 89951424) and clinical trimals. Written informed consent was obtained for all participants and tested according to the principles of the helsinki statement (Declaration of Helsinki) and the pharmaceutical clinical trial quality management code (Good Clinical Practice). These studies were sponsored by the university of Oxford (UK) and approved by the national ethics committee (the south-middle burkest's research ethics committee, ref. 20/SC/0145 and 20/SC/0179) and the united kingdom regulatory agency (pharmaceutical and health care product regulatory agency). The independent DSMB reviews all mid-term security reports. Copies of the protocol are included in the previous publications (PMID: 33220855/PMID: 32702298).

Focal reduction neutralization assay (FRNT)

The neutralization potential of abs was measured using a lesion reduction neutralization assay (FRNT), in which the reduction in the number of infected lesions was compared to a negative control well without antibody. Briefly, serial dilutions of Ab or plasma were mixed with SARS-CoV-2 strain Victoria or P.1 and incubated at 37℃for 1 hour. The mixture was then transferred in duplicate to 96-well cell culture treated flat bottom microplates containing confluent Vero cell monolayers and incubated for an additional 2 hours before 1.5% semi-solid carboxymethylcellulose (CMC) cover medium was added to each well to limit virus spread. Foci formation assay was then performed by staining Vero cells with human anti-NP mAb (mAb 206), followed by peroxidase conjugated goat anti-human IgG (a 0170; sigma). Finally, approximately 100 lesions (infected cells) per well were visualized in the absence of antibodies by adding truebue peroxidase substrate. Virus infected cell foci were counted using AID ELISpot software on a classical AID ELISpot reader. The percent lesion reduction was calculated and IC50 was determined using the probit program from the SPSS software package.

Cloning of ACE2 and RBD proteins

The constructs for EY6A Fab, 222Fab, ACE2, WT RBD, b.1.1.7 and b.1.351 mutant RBD were the same as described in the previous examples. For cloning RBD K417T and RBD K417N, PCR was performed using the primers of RBD K417T (forward primer 5'-GGGCAGACCGGCACGATCGCCGACTAC-3' (SEQ ID NO: 424) and reverse primer 5' -GTAGTCGGCGATCGTGCCGGTCTGCCC (SEQ ID NO: 425)) and RBD K417N (forward primer 5'-CAGGGCAGACCGGCAATATCGCCGACTACAATTAC-3' (SEQ ID: 426) and reverse primer 5'-GTAATTGTAGTCGGCGATATTGCCGGTCTGCCCTG-3' (SEQ ID NO: 427)) and the two primers of pNEO vector (forward primer 5'-CAGCTCCTGGGCAACGTGCT-3' (SEQ ID NO: 422) and reverse primer 5'-CGTAA AAGGAGCAACATAG-3' (SEQ ID NO: 423)), respectively, with the plasmid of WT D as template. For cloning of the P.1 RBD, the construct of B.1.351 RBD was used as template and PCR was performed using the primers of the RBD K417T and pNEO vectors described above. The amplified DNA fragment was digested with restriction enzymes AgeI and KpnI, and then ligated with the digested pNEO vector. All constructs were verified by sequencing.

Protein production

Protein production is as described by Zhou et al (Zhou et al 2020). Briefly, the protein-encoding plasmid was transiently expressed in HEK293T (ATCC CRL-11268) cells. The conditioned media was dialyzed and purified using a 5-ml HisTrap Nickel column (general electric Healthcare Co., ltd.) and further purified using a Superdex 75 HiLoad 16/60 gel filtration column (general electric Healthcare Co.).

Biological layer interferometry

The BLI experiments were run on an Octet Red 96e machine (Buddha). To measure the binding affinity of ACE2 to p.1 RBDs, and the affinity of monoclonal antibodies and ACE2 to native RBDs and RBD K417N, RBD K417T, RBD E484K, and RBD K417T E484K N Y (each p.1 RBD), each RBD was immobilized to an AR2G biosensor (foterbuo). Monoclonal antibodies were used as analytes or serial dilutions of ACE2 were used as analytes. All experiments were run at 30 ℃. Data were recorded using the software Data Acquisition 11.1 (Buddha Corp.) and Data Analysis HT 11.1 (Buddha Corp.) with a 1:1 fitting model for Analysis.

Antibody production

The aslicon and the regenerator antibodies were supplied by aslicon corporation, and the sanskril, gill, and adagilo antibodies were supplied by adagilo corporation. For chimeric antibodies, the heavy and light chains of the indicated antibodies were transiently transfected into 293Y cells and the antibodies were purified from the supernatant on protein a.

Crystallization

ACE2 was mixed with p.1 RBD in a 1:1 molar ratio to a final concentration of 12.5mg ml-1. EY6A Fab, 222 Fab and WT or mutant RBD were mixed in a 1:1:1 molar ratio to a final concentration of 7.0mg ml ^-1 . All samples were incubated for 30 minutes at room temperature. Most crystallization experiments were set up using nanoliter sitting-drop vapor diffusion in a crystal quick 96 well X plate (Grignard first biosystems) using a rectangular robot with 100nl of protein per drop plus 100nl of stock solution as described previously (Water et al, 2003). Crystallization of the B.1.1.7 RBD/EY6A/222 complex was established by manual pipetting with 500nl of protein per drop plus 500nl of stock. Good crystals of EY6A Fab and 222Fab complexed with WT, K417T, K417N, B.1.1.7, B.1.351 or P.1 RBD were obtained from Hampton Research PEGRx 2 screening conditions 35 containing 0.15M lithium sulfate, 0.1M citric acid pH 3.5, 18% w/v PEG 6,000. Crystals of the P.1 RBD/ACE2 complex were formed in Hampton Research PEGRx 1 screening conditions 38 containing 0.1M imidazole pH 7.0.

X-ray data collection, structure determination and refinement

Crystals of ternary complexes of WT and mutant RBD/EY6A and 222Fab were mounted in the loop and immersed for one second in a solution containing 25% glycerol and 75% mother liquor, then frozen in liquid nitrogen prior to data collection. rbd/ACE2 crystals do not use cryoprotectants. Diffraction data were collected at 100K at beam line I03 of british diamond light source company. All Data (except some WT RBD-EY6A-222Fab complex images) were collected as part of an automated queuing system that allowed for Unattended automated Data collection (https:// www.diamond.ac.uk/Instruments/Mx/I03/I03-Manual/Unattended-Data-collections. Html). A diffraction image rotated by 0.1℃was recorded on an Eiger2 XE 16M detector (each image had an exposure time of 0.004 or 0.006s, a beam size of 80X 20 μm, a beam transmission rate of 100%, and a wavelength of ). The data is indexed, integrated and scaled using an automated data processing program, xia2-dials (Winter, 2010; winter et al, 2018). A1080℃dataset was collected from 3 positions of the frozen crystals of the WT RBD-EY6A-222Fab complex. 720 ° data for each of the b.1.1.7, p.1 and b.1.351 mutant RBD/EY6A and 222Fab complexes (each from 2 crystals) were collected, and 360 ° data for K417N and K417T RBD with EY6A and 222Fab were collected, and ACE2 complex was collected from a single crystal.

The structure of the WT RBD-EY6A-222 and P.1 RBD-ACE2 complexes was determined by molecular replacement with PHASER (McCoy et al, 2007) using search models of SARS-CoV-2RBD-EY6A-H4 (PDB ID 6 ZCZ) (Zhou et al, 2020) and RBD-158 (PDB ID, 7 BEK) complexes, and RBD and ACE2 complexes (PDB ID, 6LZG (Wang et al, 2020), respectively. For all structures, model reconstruction was performed with COOT (Emsley and Cowtan, 2004) and refinement with PHENIX (Liebschner et al, 2019). The ChCl domain of EY6A is flexible and has poor electron density. Data collection and structure refinement statistics are given in table S1. Structural comparisons residues forming the RBD/Fab interface were identified using SHP (Stuart et al, 1979), using PISA (Krissinel and Henrick, 2007), and mapped using PyMOL (PyMOL molecular graphics System, version 1.2r3pre, schrodinger).

Quantification and statistical analysis

Statistical analysis is reported in the results and legend. Neutralization was measured by FRNT. The percent lesion reduction was calculated and IC50 was determined using the probit program from the SPSS software package. Analysis was performed using the Wilcoxon paired symbol rank test and two-tailed P values were calculated from geometric mean. BLI Data were analyzed using a Data Analysis HT 11.1 (Buddha Corp.) using a 1:1 fitting model.

Example 36 Cross-reactivity of mAb

Live virus neutralization assays were performed using the following viruses, which contained the indicated changes in RBD: victoria (an early Wuhan-related strain), alpha (N501Y), beta (K417N, E484K, N501Y), gamma (K417T, E484K, N Y), delta (L452R, T478K) and Omicron (G339D, S371L, S373P, S F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y H) (FIG. 42, table 26).

Mabs 58, 222, 253H55L from early pandemic samples showed neutralization of Omicron. In particular, mabs 58 and 222 maintain a strong neutralization of omicron. Mab 222 potently neutralized all strains tested. Mab 58 strongly neutralized all but Delta strains.

EXAMPLE 37 further neutralization data of selected antibodies against SARS-CoV-2 antibody

Further neutralization experiments were performed to determine neutralization of SARS-CoV-2 variant by the selected antibodies. As discussed in the detailed description above, antibodies derived from the same heavy chain V gene may exchange light chains to produce antibodies comprising the heavy chain variable region of a first antibody and the light chain variable region of a second antibody, and such new antibodies may have improved neutralization and/or other characteristics when compared to the "parent" antibody.

Tables 21 to 25 provide examples of such antibodies that can be generated by exchanging light chains between antibodies derived from the same heavy chain V gene.

Tables 27 to 28 provide further neutralization data for selected antibodies and antibodies generated by exchanging light chains between antibodies derived from the same heavy chain V gene. Almost all antibodies produced by exchanging light chains between antibodies derived from the same heavy chain V gene exhibit improved neutralization when compared to the "parent" antibody. The data in FIG. 43A depicts mutations in the NTD, RBD and CTD of the spike protein of the SARS-CoV-2 variant when compared to the Wuhan SARS-CoV-2 spike protein sequence. The data in fig. 43B corresponds to the data shown in table 28.

Watch (watch)

TABLE 1 SEQ ID NO for selected antibodies

Table 2. Summary of SARS-CoV-2 infected patients enrolled in the group study.

TABLE 3 neutralization of selected mAbs and drying of the biological layer to affinity (KD) and association (Ka) and dissociation (Kdis) rates Involves measurement of the BLI (although 159BLI measurement is for Fab)

# the neutralizing activity of the selected antibodies against SARS-CoV-2 was determined by FRNT. Data were from 3 independent experiments, with two replicate wells per experiment, and data are shown as mean ± s.e.m.

* FRNT was performed once with two duplicate wells.

Determination of Fab fragments

₅₀ TABLE 4 efficacy threshold from cluster analysis<Non-competing antibody pair for 0.1ug/ml IC

₅₀ TABLE 5 efficacy threshold from cluster analysis<1ug/ml IC non-competing antibody pair

Table 6. Stability test of selected antibodies using thermofluor and Dynamic Light Scattering (DLS).

Table 7.X ray data collects and refines statistical information (molecular substitutions).

^a The presence of transformed NCS. ^b The values in brackets are for the highest resolution shell

Table 8 Cryo-EM data collection, refinement and validation statistics for spike/COVOX-Fab (IgG) complexes.

The numbers in brackets refer to the 30 ° oblique dataset combined with the 0 ° data. Brackets provide values for C1 symmetry. * Only the rigid body is refined.

The lower page is over-bent

TABLE 9 determination of affinity (KD), neutralization potency of full-length IgG and Fab against SARS-CoV-2 for ten selected antibodies ₅₀ (IC) and% occupancy. Data from 2 independent experiments, each with two replicate wells, and data are shown as average ±s.e.m.。

Antibodies to	Binding KD (nM)	Neutralization Ic ₅₀ (nM)	% occupancy rate
				40 IgG	0.33±0.04	0.16±0.03	33.3±1.3
40 Fab	1.23±0.19	45.68±7.15	97.4±0.0
				88 IgG	0.25±0.01	0.07±0.04	21.9±9.5
88 Fab	1.21±0.14	6.95±0.07	85.2±1.6
				150 IgG	0.24±0.01	0.08±0.02	25.1±5.7
150 Fab	0.75±0.10	0.61±0.23	43.2±6.6
				158 IgG	0.50±0.02	0.13±0.02	20.5±2.6
158 Fab	3.73±0.56	4.12±0.04	52.7±3.5
				159 IgG	0.19±0.02	0.08±0.01	30.0±5.2
159 Fab	0.29±0.02	N/A	N/A
				253 IgG	0.17±0.01	0.36±0.02	67.9±2.3
253 Fab	23.38±3.84	N/A	N/A
				253H55L IgG	0.09±0.01	0.02±0.00	19.6±2.1
253H55L Fab	10.19±0.56	5.08±0.47	33.2±0.8
				253H165L IgG	0.09±0.01	0.03±0.00	24.5±0.6
253H165L Fab	5.02±1.40	5.22±1.18	51.3±1.4
				316 IgG	0.13±0.00	0.08±0.02	35.4±6.0
316 Fab	8.76±0.64	27.64±9.47	74.0±8.1
				384IgG	0.10±0.03	0.01±0.00	12.1±2.3
384 Fab	7.86±0.96	4.86±0.16	38.4±3.7

TABLE 10X-ray data collection and refinement statistics

Data collection

Data set RBDN501Y/COVOX-269

Space group C2

Refining

R.m.s. deviation

Key length 0.003

Bond angle (°) 0.6

The numbers in brackets refer to the highest resolution shell of the data.

TABLE 11 neutralization of selected antibodies against SARS-CoV-2 strain Victoria and B.1.1.7

Selected antibodies were assayed for neutralizing activity against SARS-CoV-2 strains Victoria and B.1.1.7 by FRNT. Data were from 2 independent experiments, with two replicate wells per experiment, and data are shown as mean ± s.e.m.

IC50 ratio between strain B.1.1.7 and Victoria

TABLE 12 neutralization and RBD binding of selected antibodies to SARS-CoV-2 strain Victoria and B.1.1.7

IC50 ratio between strain B.1.1.7 and Victoria.

The KD values of Fab associated with N501-RBD and Y501-RBD, as measured by BLI, showed mAb159 to bind to NTD and were not tested.

VH and VL gene usage of monoclonal antibodies is indicated on the right.

TABLE 13 RBD/Fab complexes analyzed for N501 contact

TABLE 14 FRNT50 titres for Victoria and B.1.351 strains (A) 34 convalescence plasma (B) from 13 Plasma from a patient named infected b.1.1.7.

TABLE 14A neutralization of SARS-CoV-2 recovery phase plasma against SARS-CoV-2 strain Victoria and B.1.351

TABLE 14B neutralization of SARS-CoV-2 recovery phase plasma against SARS-CoV-2 strain Victoria and B.1.351

TABLE 15 FRNT50 titres for Victoria and B.1.351 strains (A) from 25-fold-Bay-Entecan epidemic Serum of recipient of seedlings. (B) oxford-African vaccine.

Table 16 FRNT50 titres for Victoria and B.1.351 strains (A) 22 human monoclonal antibodies. (B) Two kinds of materials Regenerator and 2 antibodies to the as Li Kangshan clone. Analysis was performed using the Mann-WhitneyU test and double tail P values were calculated, number Shown as mean ± s.e.m.

Table 16A

Table 16B

TABLE 17 data collection and refinement statistics for RBD complexes

The values in brackets are for the highest resolution shell.

TABLE 18 neutralization of selected antibodies against SARS-CoV-2 strains Victoria, B.1.1.7, B.1.351 and P.1 RBD binding.

Watch 18 (Xuezhi)

In the case where the table is blank, the data of these points have not been obtained yet.

TABLE 19 FRNT50 titres for Victoria and P.1 strain (A) 34 convalescent plasma (B) from 13 sensations Plasma of patients stained with b.1.1.7.

TABLE 19A

TABLE 19B

TABLE 20 FRNT50 titres for Victoria and P.1 strains (A) from 25-part-Bay Entecan vaccine Serum of recipient. (B) Serum from recipients of 26 oxford-alsikan vaccines.

Table 21. Examples of mixed chain antibodies generated from antibodies derived from the same germline heavy chain IGHV 3-53.

Table 22. Examples of mixed chain antibodies generated from antibodies derived from germline heavy chain IGHV3-53 or IGHV 3-66.

Table 23. Examples of mixed chain antibodies generated from antibodies derived from the same germline light chain IG kappa V3-20.

Table 24. Examples of mixed chain antibodies generated from antibodies derived from the same germline light chain IG kappa V1-9.

TABLE 25 Mixed production from antibodies derived from the same germline heavy chain IGHV1-58Examples of chain-fused antibodies.

TABLE 26 IC50 titres of selected antibodies against SARS-CoV-2 variants

TABLE 27 IC50 titres of selected antibodies against SARS-CoV-2 variants

The following table shows 50% focal reduced neutralization titers (FRNT 50) of the indicated monoclonal antibodies against the indicated viruses. Supp stands for tissue culture supernatant, but not other antibodies that use purified antibodies in the assay. Chimeric antibodies from the combination of the Heavy Chain (HC) of one antibody with the Light Chain (LC) of another antibody are represented as follows: 150HC/222LC represents the combination of the heavy chain from antibody 150 with the light chain of antibody 222.

* Blank boxes have no available data

TABLE 28 contains the indicated presence of the spike protein sequences of Wuhan SARS-CoV-2 spike protein sequences when compared to the spike protein sequences of Wuhan SARS-CoV-2 Neutralization IC50% value for a mutant set of pseudoviral constructs

IC50 values of the indicated antibodies are shown. Mixed chain antibodies from the combination of Heavy Chains (HC) of one antibody with Light Chains (LC) of another antibody are represented as follows: 150HC/222LC represents the combination of the heavy chain from antibody 150 with the light chain of antibody 222.

Sequence listing

Amino acid sequences of heavy and light chain variable regions of selected antibodies

Nucleotide sequences of heavy and light chain variable regions of selected antibodies

Amino acid sequence of CDR

Sequence listing

<110> oxford university innovation Co., ltd (Oxford University Innovation Limited)

<120> antibody

<130> N420712WO

<150> GB2101578.9

<151> 2021-02-04

<150> GB2101580.5

<151> 2021-02-04

<150> GB2102401.3

<151> 2021-02-19

<150> GB2103388.1

<151> 2021-03-11

<150> GB2118423.9

<151> 2021-12-17

<150> GB2112297.3

<151> 2021-08-27

<150> GB2115824.1

<151> 2021-11-03

<150> GB2118426.2

<151> 2021-12-17

<160> 473

<170> patent In version 3.5

<210> 1

<211> 379

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 1

gaggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60

tcctgcaagg cttctggagg caccttcagc aactatgcta tcagctgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggaggg atcatcccta tctttggtac agcaaactac 180

gcacagaact tccagggcag agtcacgatt accgcggacg aatccatgag cacagcctac 240

atggagctga gcagcctgag atctgaggac acggccgtat attactgtgc gggaggtggg 300

aggtattgta gtggtggtag gtgccactct gcctactctg cctactgggg ccagggaacc 360

ctggtcaccg tctcctcag 379

<210> 2

<211> 126

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 2

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Asn Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Met Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Gly Gly Gly Arg Tyr Cys Ser Gly Gly Arg Cys His Ser Ala Tyr

100 105 110

Ser Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 3

<211> 322

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 3

gccatccagt tgacccagtc tccaggcacc ctgtctttgc ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120

cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggac 240

cctgaagatt ttgcagtgta ttactgtcag caatatggta gctcactcac tttcggcgga 300

gggaccaaag tggatatcaa ac 322

<210> 4

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 4

Ala Ile Gln Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Pro Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Asp

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys

100 105

<210> 5

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 5

Gly Gly Thr Phe Ser Asn Tyr Ala

1 5

<210> 6

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 6

Ile Ile Pro Ile Phe Gly Thr Ala

1 5

<210> 7

<211> 19

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 7

Ala Gly Gly Gly Arg Tyr Cys Ser Gly Gly Arg Cys His Ser Ala Tyr

1 5 10 15

Ser Ala Tyr

<210> 8

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 8

Gln Ser Val Ser Ser Ser Tyr

1 5

<210> 9

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 9

Gly Ala Ser

1

<210> 10

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 10

Gln Gln Tyr Gly Ser Ser Leu Thr

1 5

<210> 11

<211> 364

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 11

caggtgcagc tggtggagtc tgggggaggc ttggtccacc ctggggggtc cctgagactc 60

tcctgttcag cctctggatt caccttcagt aactatgcta tgcactgggt ccgccaggct 120

ccagggaagg gactggaata tgtttcagct attagtagta gtggggatat cacatactac 180

gcggactccg taaagggcag attcaccatc tccagagaca attccaagaa ctcactgtat 240

cttcaaatga acagtctgag agctgaggac acggctgttt attactgtgt gaaagatgta 300

acgaggacct actacgtagt ctttgactac tggggccagg gaaccctggt caccgtctcc 360

tcag 364

<210> 12

<211> 121

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 12

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val His Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val

35 40 45

Ser Ala Ile Ser Ser Ser Gly Asp Ile Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Lys Asp Val Thr Arg Thr Tyr Tyr Val Val Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 13

<211> 322

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 13

gccatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtgggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agttatttaa attggtatca gcaggaacca 120

gggaaagccc ctaaactcct gatctatgct gcatccagtt tgcaaggtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacacta ccccgtacac ttttggccag 300

gggaccaaag tggatatcaa ac 322

<210> 14

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 14

Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Glu Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Gly Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys

100 105

<210> 15

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 15

Gly Phe Thr Phe Ser Asn Tyr Ala

1 5

<210> 16

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 16

Ile Ser Ser Ser Gly Asp Ile Thr

1 5

<210> 17

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 17

Val Lys Asp Val Thr Arg Thr Tyr Tyr Val Val Phe Asp Tyr

1 5 10

<210> 18

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 18

Gln Ser Ile Ser Ser Tyr

1 5

<210> 19

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 19

Ala Ala Ser

1

<210> 20

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 20

Gln Gln Ser Tyr Thr Thr Pro Tyr Thr

1 5

<210> 21

<211> 358

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 21

caggtgcagc tggtggagtc tgggggaggc ttggtccagc ctggggggtc cctgagactc 60

tcctgtgcag tctctggatt caccgtcagt aggaactaca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcactt atttatagcg gtggtagcac attctacgca 180

gactccgtga agggcagatt caccatctcc agagacaatt ccaagaacac gctgtatctt 240

caaatgaaca gcctgagagc cgaggacacg gctgtgtatt actgtgcgag agatctgttt 300

cataggagtg gttatcacga ctactggggc cagggaaccc tggtcaccgt ctcctcag 358

<210> 22

<211> 119

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 22

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Val Ser Arg Asn

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Leu Ile Tyr Ser Gly Gly Ser Thr Phe Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Leu Phe His Arg Ser Gly Tyr His Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 23

<211> 319

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 23

gtcatctgga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc aggcgagtca ggacattaac aactatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatcttcgat gcctccaatt tggaaacagg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat tttactttca ccatcagcag cctacagcct 240

gaagatattg caacatatta ctgtcaacag tatgataatc tccctgcctt cggcggaggg 300

accaaagtgg atatcaaac 319

<210> 24

<211> 106

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 24

Val Ile Trp Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Asn Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Phe Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro Ala

85 90 95

Phe Gly Gly Gly Thr Lys Val Asp Ile Lys

100 105

<210> 25

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 25

Gly Phe Thr Val Ser Arg Asn Tyr

1 5

<210> 26

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 26

Ile Tyr Ser Gly Gly Ser Thr

1 5

<210> 27

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 27

Ala Arg Asp Leu Phe His Arg Ser Gly Tyr His Asp Tyr

1 5 10

<210> 28

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 28

Gln Asp Ile Asn Asn Tyr

1 5

<210> 29

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 29

Asp Ala Ser

1

<210> 30

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 30

Gln Gln Tyr Asp Asn Leu Pro Ala

1 5

<210> 31

<211> 361

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 31

gaagtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cgtctggatt caccttcagt aactatggca tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcggtt gtatggtatg atggaagcaa gaaatactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa caccctgtat 240

ctgcaaatga acagcctgag agtcgaggac acggctgtgt attactgcgc gagagatttt 300

gcggtggggg aggagatcgc tgactcctgg ggccagggaa ccctggtcac cgtctcctca 360

g 361

<210> 32

<211> 120

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 32

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Val Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Phe Ala Val Gly Glu Glu Ile Ala Asp Ser Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 33

<211> 325

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 33

tcctatgagc tgactcagcc accctcggtg tcagtgtccc caggacaaac ggccaggatc 60

acctgctctg gagatgcatt gccaaaaaaa tatgcttatt ggtaccagca gaagtcaggc 120

caggcccctg tactggtcat ctatgaggac agcaaacgac cctccgggat ccctgagaga 180

ttctctgggt ccagctcagg gacaatggcc accttgacta tcagtggggc ccaggtggag 240

gatgaaggtg actactactg ttactcaaga gacagcagtg gtgatcattg ggtgttcggc 300

gcagggacca agctgaccgt cctag 325

<210> 34

<211> 108

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 34

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Pro Lys Lys Tyr Ala

20 25 30

Tyr Trp Tyr Gln Gln Lys Ser Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Glu Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Thr Met Ala Thr Leu Thr Ile Ser Gly Ala Gln Val Glu

65 70 75 80

Asp Glu Gly Asp Tyr Tyr Cys Tyr Ser Arg Asp Ser Ser Gly Asp His

85 90 95

Trp Val Phe Gly Ala Gly Thr Lys Leu Thr Val Leu

100 105

<210> 35

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 35

Gly Phe Thr Phe Ser Asn Tyr Gly

1 5

<210> 36

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 36

Val Trp Tyr Asp Gly Ser Lys Lys

1 5

<210> 37

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 37

Ala Arg Asp Phe Ala Val Gly Glu Glu Ile Ala Asp Ser

1 5 10

<210> 38

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 38

Ala Leu Pro Lys Lys Tyr

1 5

<210> 39

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 39

Glu Asp Ser

1

<210> 40

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 40

Tyr Ser Arg Asp Ser Ser Gly Asp His Trp Val

1 5 10

<210> 41

<211> 363

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 41

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cctctggatt caccttcagt acctatgcta tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggctgtt ctttcatatg atggaagcaa taaatactac 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgc gaaagggggc 300

tcgtacgcgt actactacta catggacgtc tggggcaaag ggaccacggt caccgtctcc 360

tca 363

<210> 42

<211> 121

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 42

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Leu Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Gly Ser Tyr Ala Tyr Tyr Tyr Tyr Met Asp Val Trp Gly

100 105 110

Lys Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 43

<211> 322

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 43

gacatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc aggcgagtca ggacattagc aactatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctacgat gcatccaatt tggaaacagg ggtcccatca 180

aggttcagtg gaggtggatc tgggacagat tttactttca ccatcaccag cctgcagcct 240

gaagatattg caacatatta ctgtcaacag tatgataatc tcccgctcac tttcggcgga 300

gggaccaaag tggatatcaa ac 322

<210> 44

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 44

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Gly Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Thr Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys

100 105

<210> 45

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 45

Gly Phe Thr Phe Ser Thr Tyr Ala

1 5

<210> 46

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 46

Leu Ser Tyr Asp Gly Ser Asn Lys

1 5

<210> 47

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 47

Ala Lys Gly Gly Ser Tyr Ala Tyr Tyr Tyr Tyr Met Asp Val

1 5 10

<210> 48

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 48

Gln Asp Ile Ser Asn Tyr

1 5

<210> 49

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 49

Asp Ala Ser

1

<210> 50

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 50

Gln Gln Tyr Asp Asn Leu Pro Leu Thr

1 5

<210> 51

<211> 376

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 51

gttcagctgg tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctctggtgg ctccgtcagt agtggtagtt actactggag ctggatccgg 120

cagcccccag ggaagggact ggagtggatt gggtatatgt atttcagtgg gagcaccaac 180

tataatccct ccctcaagag tcgagtcacc atatcattag ccacgtccaa gaaccagttc 240

tccctgaagc tgagctctgt gaccgctgcg gacacggccg tctattactg tgcgagaggg 300

gattacgatt tttggagtgg tccccccggt cgggtggacg tctggggcaa agggaccacg 360

gtcaccgtct cctcag 376

<210> 52

<211> 125

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 52

Val Gln Leu Val Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly

20 25 30

Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Met Tyr Phe Ser Gly Ser Thr Asn Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Leu Ala Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Gly Asp Tyr Asp Phe Trp Ser Gly Pro Pro Gly Arg Val

100 105 110

Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 53

<211> 325

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 53

gaaatagtga tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120

cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240

cctgaagatt ttgcagtgta ttactgtcag cactatggta gttcacccgt aacttttggc 300

caggggacca aagtggatat caaac 325

<210> 54

<211> 108

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 54

Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Gly Ser Ser Pro

85 90 95

Val Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys

100 105

<210> 55

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 55

Gly Gly Ser Val Ser Ser Gly Ser Tyr Tyr

1 5 10

<210> 56

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 56

Met Tyr Phe Ser Gly Ser Thr

1 5

<210> 57

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 57

Ala Arg Gly Asp Tyr Asp Phe Trp Ser Gly Pro Pro Gly Arg Val Asp

1 5 10 15

Val

<210> 58

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 58

Gln Ser Val Ser Ser Ser Tyr

1 5

<210> 59

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 59

Gly Ala Ser

1

<210> 60

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 60

Gln His Tyr Gly Ser Ser Pro Val Thr

1 5

<210> 61

<211> 367

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 61

caggtgcagc tggtgcagtc tgggcctgag gtgaagaagc ctgggacctc agtgaaggtc 60

tcctgcaagg cttctggatt cacctttact agctctgctg tgcagtgggt gcgacaggct 120

cgtggacaac gccttgagtg gataggatgg atcgtcgttg gcagtggtaa cacaaactac 180

gcacagaagt tccaggaaag agtcaccatt accagggaca tgtccacaag cacagcctac 240

atggagatga gcagcctgag atccgaggac acggccgtgt attactgtgc ggcaccggcc 300

tgtggtacca gctgctctga tgcctttgat atctggggcc aagggacaat ggtcaccgtc 360

tcttcag 367

<210> 62

<211> 122

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 62

Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Ser Ser

20 25 30

Ala Val Gln Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Trp Ile Val Val Gly Ser Gly Asn Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Glu Arg Val Thr Ile Thr Arg Asp Met Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Pro Ala Cys Gly Thr Ser Cys Ser Asp Ala Phe Asp Ile Trp

100 105 110

Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 63

<211> 325

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 63

gacatccaga tgacccagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120

cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240

cctgaagatt ttggagtgta ttactgtcag cagtatggta gctcaccgtg gacgttcggc 300

caagggacca aggtggaaat caaac 325

<210> 64

<211> 108

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 64

Asp Ile Gln Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Gly Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 65

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 65

Gly Phe Thr Phe Thr Ser Ser Ala

1 5

<210> 66

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 66

Ile Val Val Gly Ser Gly Asn Thr

1 5

<210> 67

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 67

Ala Ala Pro Ala Cys Gly Thr Ser Cys Ser Asp Ala Phe Asp Ile

1 5 10 15

<210> 68

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 68

Gln Ser Val Ser Ser Ser Tyr

1 5

<210> 69

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 69

Gly Ala Ser

1

<210> 70

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 70

Gln Gln Tyr Gly Ser Ser Pro Trp Thr

1 5

<210> 71

<211> 382

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 71

caggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggcaggtc cctgagactc 60

tcctgtgcag cctctggatt cacctttgat gattatgcca tgcactgggt ccggcaacct 120

ccagggaagg gcctggagtg ggtctcaggt gtcagttgga acagtggtac cataggctat 180

gcggactctg tgaagggccg attcatcatc tccagagaca acgccaagaa ctccctgtat 240

ctgcaaatga acagtctgaa agctgaggac acggccttgt attactgtgc aagagaagtg 300

ggggggactt ttggagtcct tatttcacgc gaggggggac ttgattactg gggccaggga 360

accctggtca ccgtctcctc ag 382

<210> 72

<211> 127

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 72

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Val Ser Trp Asn Ser Gly Thr Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Glu Val Gly Gly Thr Phe Gly Val Leu Ile Ser Arg Glu Gly

100 105 110

Gly Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 73

<211> 325

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 73

tcctatgagc tgacacagcc accctcggtg tcagtggccc caggacagac ggccagaatt 60

acctgtgggg gaaacaccat tggaagtaaa agtgtgcact ggtaccagca gagaccaggc 120

caggcccctg tgctggtcgt ctatgatgat agcgaccggc cctcagggat ccctgagcga 180

ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240

gatgaggccg actattactg tcaggtgtgg gatagtagta gtgatcgggt ggtattcggc 300

ggagggacca agctgaccgt cctag 325

<210> 74

<211> 108

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 74

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Thr Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp Arg

85 90 95

Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 75

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 75

Gly Phe Thr Phe Asp Asp Tyr Ala

1 5

<210> 76

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 76

Val Ser Trp Asn Ser Gly Thr Ile

1 5

<210> 77

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 77

Ala Arg Glu Val Gly Gly Thr Phe Gly Val Leu Ile Ser Arg Glu Gly

1 5 10 15

Gly Leu Asp Tyr

20

<210> 78

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 78

Thr Ile Gly Ser Lys Ser

1 5

<210> 79

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 79

Asp Asp Ser

1

<210> 80

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 80

Gln Val Trp Asp Ser Ser Ser Asp Arg Val Val

1 5 10

<210> 81

<211> 385

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 81

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60

atctgcactg tctctggtgg ctccgtcagc agtggtaatt tctactggag ctggatccgg 120

cagcccccag ggaagggact ggagtggatt ggatctatct attacactgg gagccccaac 180

tacaacccct ccctcaagag tcgagtcacc atatccctag acacgtccaa gaaccagttc 240

tccctgaagc tgagctctgt gaccgctgcg gacacggccg tgtattactg tgcgagagag 300

atctattatt atgatagaag tggttcttac aactctgatg cttttgatat ctggggccaa 360

gggacaatgg tcaccgtctc ttcag 385

<210> 82

<211> 128

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 82

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Ile Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly

20 25 30

Asn Phe Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Ser Ile Tyr Tyr Thr Gly Ser Pro Asn Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Leu Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Glu Ile Tyr Tyr Tyr Asp Arg Ser Gly Ser Tyr Asn Ser

100 105 110

Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 83

<211> 325

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 83

gatatcgtga tgactcagtc tccagccacc ctgtctgtgt ctccagggga aagaggcacc 60

ctctcctgca gggccagtca gagtgttagc agcaacttag cctggtacca gcagaaaccg 120

ggccaggctc ccaggctcct catctatggt gcatccacga gggccactgg tatcccagcc 180

aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct 240

gaagattttg cagtttatta ctgccagcag tataataact ggcctccgct cactttcggc 300

ggagggacca aagtggatat caaac 325

<210> 84

<211> 108

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 84

Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Gly Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Pro

85 90 95

Leu Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys

100 105

<210> 85

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 85

Gly Gly Ser Val Ser Ser Gly Asn Phe Tyr

1 5 10

<210> 86

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 86

Ile Tyr Tyr Thr Gly Ser Pro

1 5

<210> 87

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 87

Ala Arg Glu Ile Tyr Tyr Tyr Asp Arg Ser Gly Ser Tyr Asn Ser Asp

1 5 10 15

Ala Phe Asp Ile

20

<210> 88

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 88

Gln Ser Val Ser Ser Asn

1 5

<210> 89

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 89

Gly Ala Ser

1

<210> 90

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 90

Gln Gln Tyr Asn Asn Trp Pro Pro Leu Thr

1 5 10

<210> 91

<211> 381

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 91

caggtgcagc tggtggagtc tgggggaggc gtggttcagc ctgggaggtc cctgagactc 60

tcctgtgcag cctctggatt caccttcaat aactatcctt tgcactgggt ccgccaggct 120

ccaggcaagg ggccggagtg ggtggcagtt atttcacagg atggaggcaa taaatactac 180

gtagactccg tgaagggccg attcaccatc tccagagaca attccaagaa caccctgtat 240

ctgcaaatga acaacctgag agctgaggac acggctctgt attactgtgc gagagatgtt 300

gtagtggtgg tagctgctag gaaccactac tacaacggta tggacgtctg gggccaaggg 360

accacggtca ccgtctcctc a 381

<210> 92

<211> 127

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 92

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Tyr

20 25 30

Pro Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val

35 40 45

Ala Val Ile Ser Gln Asp Gly Gly Asn Lys Tyr Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Asp Val Val Val Val Val Ala Ala Arg Asn His Tyr Tyr Asn

100 105 110

Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 93

<211> 322

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 93

gacatccagt tgacccagtc tccatcttcc gtgtctgcat ctgtaggaga cagagtcacc 60

atcacttgtc gggcgagtca gggtattagc agctggttag cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gtatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttacta ttgtcaacag gctaagagtt tccctttcac tttcggccct 300

gggaccaagg tggagattaa ac 322

<210> 94

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 94

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Val Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Lys Ser Phe Pro Phe

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys

100 105

<210> 95

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 95

Gly Phe Thr Phe Asn Asn Tyr Pro

1 5

<210> 96

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 96

Ile Ser Gln Asp Gly Gly Asn Lys

1 5

<210> 97

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 97

Ala Arg Asp Val Val Val Val Val Ala Ala Arg Asn His Tyr Tyr Asn

1 5 10 15

Gly Met Asp Val

20

<210> 98

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 98

Gln Gly Ile Ser Ser Trp

1 5

<210> 99

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 99

Ala Val Ser

1

<210> 100

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 100

Gln Gln Ala Lys Ser Phe Pro Phe Thr

1 5

<210> 101

<211> 381

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 101

cagctgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagt agtggtagtt ataattggac ctggatccgg 120

cagcccgccg ggaagggact ggagtggatt gggcgtatat ataatagtgg gagcaccaac 180

tacaacccct ccctcaagag tcgagtcacc atatcagtag acacgtccaa gaaccagttg 240

tccctgaagg tgaggtctgt gaccgccgca gacacggccg tgtattactg tgcgagacat 300

tgcagtggtg gtacctgcta cccgaagtac tactacggta tggacgtctg gggccaaggg 360

accacggtca ccgtctcctc a 381

<210> 102

<211> 127

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 102

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Ser Tyr Asn Trp Thr Trp Ile Arg Gln Pro Ala Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Arg Ile Tyr Asn Ser Gly Ser Thr Asn Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Leu

65 70 75 80

Ser Leu Lys Val Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg His Cys Ser Gly Gly Thr Cys Tyr Pro Lys Tyr Tyr Tyr

100 105 110

Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 103

<211> 331

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 103

caatctgccc tgactcagcc accctcggtg tctgaagccc ccaggcagag ggtcaccatc 60

tcctgttctg gaagcagctc caacatcgga aataatgctg taaactggta ccagcagttc 120

ccaggaaagg ctcccaaact cctcatctat tatgatgatc tgctgccctc aggggtctct 180

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tggggtccag 240

tctgaggatg aggctgatta ttactgtgca gcatgggatg acagcctgaa tgtcgtggta 300

ttcggcggag ggaccaagct gaccgtccta g 331

<210> 104

<211> 110

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 104

Gln Ser Ala Leu Thr Gln Pro Pro Ser Val Ser Glu Ala Pro Arg Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Ala Val Asn Trp Tyr Gln Gln Phe Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Tyr Asp Asp Leu Leu Pro Ser Gly Val Ser Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Val Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Asn Val Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 105

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 105

Gly Gly Ser Ile Ser Ser Gly Ser Tyr Asn

1 5 10

<210> 106

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 106

Ile Tyr Asn Ser Gly Ser Thr

1 5

<210> 107

<211> 19

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 107

Ala Arg His Cys Ser Gly Gly Thr Cys Tyr Pro Lys Tyr Tyr Tyr Gly

1 5 10 15

Met Asp Val

<210> 108

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 108

Ser Ser Asn Ile Gly Asn Asn Ala

1 5

<210> 109

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 109

Tyr Asp Asp

1

<210> 110

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 110

Ala Ala Trp Asp Asp Ser Leu Asn Val Val Val

1 5 10

<210> 111

<211> 370

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 111

caggtgcagc tggtggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtaatagtt acttctgggg ctggatccgc 120

cagcccccag ggacggggct ggagtggatt gggaatatct attatactgg gagcacctac 180

tacaacccgt cgttcgagag tcgagtcacc atgtccgtag acacgtcgaa gaaccagttc 240

tccctgaggc tgagctctgt gaccgccgca gacacggctg tgtattactg tgcgagacat 300

gtcagggcct acgactatga tgcccctttt gatatctggg gccaagggac aatggtcacc 360

gtctcttcag 370

<210> 112

<211> 123

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 112

Gln Val Gln Leu Val Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Asn

20 25 30

Ser Tyr Phe Trp Gly Trp Ile Arg Gln Pro Pro Gly Thr Gly Leu Glu

35 40 45

Trp Ile Gly Asn Ile Tyr Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Phe Glu Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg His Val Arg Ala Tyr Asp Tyr Asp Ala Pro Phe Asp Ile

100 105 110

Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 113

<211> 322

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 113

gtcatctgga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 120

gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacacaa ttcactctca caatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtctacag attaatagtt atccgctcac tttcggcgga 300

gggaccaagg tggaaatcaa ac 322

<210> 114

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 114

Val Ile Trp Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Ile Asn Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 115

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 115

Gly Gly Ser Ile Ser Ser Asn Ser Tyr Phe

1 5 10

<210> 116

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 116

Ile Tyr Tyr Thr Gly Ser Thr

1 5

<210> 117

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 117

Ala Arg His Val Arg Ala Tyr Asp Tyr Asp Ala Pro Phe Asp Ile

1 5 10 15

<210> 118

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 118

Gln Gly Ile Arg Asn Asp

1 5

<210> 119

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 119

Ala Ala Ser

1

<210> 120

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 120

Leu Gln Ile Asn Ser Tyr Pro Leu Thr

1 5

<210> 121

<211> 349

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 121

caggtacagc tgcagcagtg gggcgcagga ctgttgaagc cttcggagac cctgtccctc 60

acctgcgctg tctatggtgg gtccttcagt ggttactact ggagctggat ccgccagccc 120

ccagggaagg ggctggagtg gattggggaa atcaatcata gtggaagcac caactacaac 180

ccgtccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240

aagctgagtt ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag aactgattac 300

tatgatagta tagactgggg ccagggaacc ctggtcaccg tctcctcag 349

<210> 122

<211> 116

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 122

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Thr Asp Tyr Tyr Asp Ser Ile Asp Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 123

<211> 328

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 123

cagtctgtgc tgactcagga gccctcactg actgtgtccc caggagggac agtcactctc 60

acctgtggct ccagcactgg agctgtcacc agtggtcatt atccctactg gttccagcag 120

aagcctggcc aagtccccag gacactgatt tatgatacaa ggaacaaaca ctcctggacc 180

cctgcccggt tctcaggctc cctccttggg ggcaaagctg ccctgaccct ttcgggtgcg 240

cagcctgagg atgaggctga atattactgc ttgctctcct ctagtggtgc tcgggtgttc 300

ggcggaggga ccaagctgac cgtcctag 328

<210> 124

<211> 109

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 124

Gln Ser Val Leu Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly

20 25 30

His Tyr Pro Tyr Trp Phe Gln Gln Lys Pro Gly Gln Val Pro Arg Thr

35 40 45

Leu Ile Tyr Asp Thr Arg Asn Lys His Ser Trp Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Leu Leu Ser Ser Ser Gly

85 90 95

Ala Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 125

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 125

Gly Gly Ser Phe Ser Gly Tyr Tyr

1 5

<210> 126

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 126

Ile Asn His Ser Gly Ser Thr

1 5

<210> 127

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 127

Ala Arg Thr Asp Tyr Tyr Asp Ser Ile Asp

1 5 10

<210> 128

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 128

Thr Gly Ala Val Thr Ser Gly His Tyr

1 5

<210> 129

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 129

Asp Thr Arg

1

<210> 130

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 130

Leu Leu Ser Ser Ser Gly Ala Arg Val

1 5

<210> 131

<211> 358

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 131

gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt caccttcagt acctacgaca tccactgggt ccgccaagct 120

acaggaaaag gtctggagtg ggtctcagct attggtactg ctggtgacac atactattca 180

ggctccgtga agggccgatt caccatctcc agagaaaatg ccaagaactc cttgtatctt 240

caaatgaaca gcctgagagc cggggacacg gctgtgtatt actgtgcaag gggtagtggg 300

acctacttct actactttga ctactggggc cagggaaccc tggtcaccgt ctcctcag 358

<210> 132

<211> 119

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 132

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Asp Ile His Trp Val Arg Gln Ala Thr Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Gly Thr Ala Gly Asp Thr Tyr Tyr Ser Gly Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Ser Gly Thr Tyr Phe Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 133

<211> 325

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 133

gacatcgtga tgactcagtc tccatcctcc ctgtctgcat ctgtaggaga cagaatcacc 60

atcacttgcc gggcaagtca gagcattaac aactatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatcccgtt tgcaaactgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat tccactctca ccatcaacac tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagtg cccctccgtg gacgttcggc 300

caagggacca aagtggatat caaac 325

<210> 134

<211> 108

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 134

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Ile Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Arg Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Ser Thr Leu Thr Ile Asn Thr Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ala Pro Pro

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys

100 105

<210> 135

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 135

Gly Phe Thr Phe Ser Thr Tyr Asp

1 5

<210> 136

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 136

Ile Gly Thr Ala Gly Asp Thr

1 5

<210> 137

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 137

Ala Arg Gly Ser Gly Thr Tyr Phe Tyr Tyr Phe Asp Tyr

1 5 10

<210> 138

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 138

Gln Ser Ile Asn Asn Tyr

1 5

<210> 139

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 139

Ala Ala Ser

1

<210> 140

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 140

Gln Gln Ser Tyr Ser Ala Pro Pro Trp Thr

1 5 10

<210> 141

<211> 366

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 141

caggtgcagc tggtggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctctggtgg ctcgatcagc agttcttact actggggctg gatccgccag 120

cccccaggga aggggctgga gtggattggg agtgtctatt atagtgggag cacctactac 180

aacccgtccc tcaagagtcg agtcaccata tccgtggaca cgtccaagaa ccagttctcc 240

ctgaggctga gctctgtgac cgccgcagac acggctgtgt attattgtgc gaggctgatg 300

accacggaag actactactc cggtatggac gtctggggcc aagggaccac ggtcaccgtc 360

tcctca 366

<210> 142

<211> 122

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 142

Gln Val Gln Leu Val Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser

20 25 30

Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Ser Val Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Met Thr Thr Glu Asp Tyr Tyr Ser Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 143

<211> 322

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 143

gccatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgtc gggcgagtca gggcattagc gattatttag cctggtttca gcagaaacca 120

gggaaagccc ctaagtccct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aagttcagcg gcggtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttatta ctgccaacag tatcatagtt acccgatcac cttcggccaa 300

gggacacgac tggagattaa ac 322

<210> 144

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 144

Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asp Tyr

20 25 30

Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Lys Phe Ser Gly

50 55 60

Gly Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr His Ser Tyr Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 145

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 145

Gly Gly Ser Ile Ser Ser Ser Tyr Tyr

1 5

<210> 146

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 146

Val Tyr Tyr Ser Gly Ser Thr

1 5

<210> 147

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 147

Ala Arg Leu Met Thr Thr Glu Asp Tyr Tyr Ser Gly Met Asp Val

1 5 10 15

<210> 148

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 148

Gln Gly Ile Ser Asp Tyr

1 5

<210> 149

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 149

Ala Ala Ser

1

<210> 150

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 150

Gln Gln Tyr His Ser Tyr Pro Ile Thr

1 5

<210> 151

<211> 351

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 151

caggtgcagc tggtggagtc tggaggaggc ttgatccagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggggt caccgtcagt agcaactaca tgagttgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcaatt atttatagtg gtggtaccac atactacgca 180

gactccgtga agggccgatt caccatctcc agagactctt ccatgaacac gctgtatctt 240

caaatgaaca gcctgagagc cgaggacacg gccgtgtatt actgtgcgag agatctgatg 300

gtgtacggta tagacgtctg gggccaaggg accacggtca ccgtctcctc a 351

<210> 152

<211> 117

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 152

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Thr Val Ser Ser Asn

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ile Ile Tyr Ser Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Ser Ser Met Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Leu Met Val Tyr Gly Ile Asp Val Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 153

<211> 328

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 153

gaaatagtga tgacgcagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggccagtca gggcattagc agttatttag cctggtatca gcaaaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccactt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtcaacag cttgatagtt acccccccgg gtacactttt 300

ggccagggga ccaaagtgga tatcaaac 328

<210> 154

<211> 109

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 154

Glu Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asp Ser Tyr Pro Pro

85 90 95

Gly Tyr Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys

100 105

<210> 155

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 155

Gly Val Thr Val Ser Ser Asn Tyr

1 5

<210> 156

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 156

Ile Tyr Ser Gly Gly Thr Thr

1 5

<210> 157

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 157

Ala Arg Asp Leu Met Val Tyr Gly Ile Asp Val

1 5 10

<210> 158

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 158

Gln Gly Ile Ser Ser Tyr

1 5

<210> 159

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 159

Ala Ala Ser

1

<210> 160

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 160

Gln Gln Leu Asp Ser Tyr Pro Pro Gly Tyr Thr

1 5 10

<210> 161

<211> 351

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 161

gaggtgcagc tgttggagtc tggaggagac ttgatccagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggggt caccgtcagt agcaactaca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcaatt atttatcccg gtgggagcac attctacgca 180

gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctt 240

caaatgcaca gcctgagagc cgaggacacg gccgtgtatt actgtgcgag agatcttggc 300

tcaggggaca tggacgtctg gggcaaaggg accacggtca ccgtctcctc a 351

<210> 162

<211> 117

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 162

Glu Val Gln Leu Leu Glu Ser Gly Gly Asp Leu Ile Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Thr Val Ser Ser Asn

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ile Ile Tyr Pro Gly Gly Ser Thr Phe Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met His Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Leu Gly Ser Gly Asp Met Asp Val Trp Gly Lys Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 163

<211> 322

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 163

gacatcgtga tgactcagtc tccatccttc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggccagtca gggcattagc agttatttag cctggtatca gcaaaaaccg 120

gggaaagccc ctaagctcct gatccaagct gcatccactt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtcaacag cttaatagtt accggtacac ttttggccag 300

gggaccaagg tggagatcaa ac 322

<210> 164

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 164

Asp Ile Val Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Gln Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr Arg Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 165

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 165

Gly Val Thr Val Ser Ser Asn Tyr

1 5

<210> 166

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 166

Ile Tyr Pro Gly Gly Ser Thr

1 5

<210> 167

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 167

Ala Arg Asp Leu Gly Ser Gly Asp Met Asp Val

1 5 10

<210> 168

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 168

Gln Gly Ile Ser Ser Tyr

1 5

<210> 169

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 169

Ala Ala Ser

1

<210> 170

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 170

Gln Gln Leu Asn Ser Tyr Arg Tyr Thr

1 5

<210> 171

<211> 369

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 171

gaggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cctctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcactt atatcatatg atggaggtaa tagatactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagactgag agctgaagac acggctatgt attactgtgc gaaagatcgt 300

gatgatgggt gggattggta ctacttcatg gacgtctggg gcaaagggac cacggtcacc 360

gtctcctca 369

<210> 172

<211> 123

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 172

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Leu Ile Ser Tyr Asp Gly Gly Asn Arg Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Arg Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Lys Asp Arg Asp Asp Gly Trp Asp Trp Tyr Tyr Phe Met Asp Val

100 105 110

Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 173

<211> 322

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 173

gacatccagt tgacccagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtattagc ggcaactact tagcctggta ccagcataaa 120

cctggccagg ctcccagact cctcatctat ggtgcatcca ccagggccac tggcatccca 180

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240

cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcgtacac ttttggccag 300

gggaccaagg tggagatcaa ac 322

<210> 174

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 174

Asp Ile Gln Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Gly Asn

20 25 30

Tyr Leu Ala Trp Tyr Gln His Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 175

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 175

Gly Phe Thr Phe Ser Ser Tyr Gly

1 5

<210> 176

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 176

Ile Ser Tyr Asp Gly Gly Asn Arg

1 5

<210> 177

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 177

Ala Lys Asp Arg Asp Asp Gly Trp Asp Trp Tyr Tyr Phe Met Asp Val

1 5 10 15

<210> 178

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 178

Gln Ser Ile Ser Gly Asn Tyr

1 5

<210> 179

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 179

Gly Ala Ser

1

<210> 180

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 180

Gln Gln Tyr Gly Ser Ser Tyr Thr

1 5

<210> 181

<211> 370

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 181

caggttcagc tggtgcagtc tgggcctgag gtgaagaagc ctgggacctc agtgaaggtc 60

tcctgcaagg cttctggatt cacctttact agctctgctg tgcagtgggt gcgacaggct 120

cgtggacagc gccttgagtg gataggatgg atcgtcgttg gcagtggtaa cacaaactac 180

gcacagaagt tccaggaaag cgtcaccatt accagggaca tgtccacaag cacagcctac 240

atggagctga gcagcctgag atccgaggac acggccgtgt attactgtgc ggccccacat 300

tgtattggtg gtagctgcca tgatgctttt gatatctggg gccaagggac aatggtcacc 360

gtctcttcag 370

<210> 182

<211> 123

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 182

Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Ser Ser

20 25 30

Ala Val Gln Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Trp Ile Val Val Gly Ser Gly Asn Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Glu Ser Val Thr Ile Thr Arg Asp Met Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Pro His Cys Ile Gly Gly Ser Cys His Asp Ala Phe Asp Ile

100 105 110

Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 183

<211> 325

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 183

gatatcgtga tgacccagtc tccaggcacc ctatctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttaga agcagctact tagcctggta ccagcagaaa 120

cctggccagg ctcccaggct cctcatctat ggtgcatcca ggaggggcac tggcatccca 180

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240

cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcaccctg gacgttcggc 300

caagggacca aggtggaaat caaac 325

<210> 184

<211> 108

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 184

Asp Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Arg Arg Gly Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 185

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 185

Gly Phe Thr Phe Thr Ser Ser Ala

1 5

<210> 186

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 186

Ile Val Val Gly Ser Gly Asn Thr

1 5

<210> 187

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 187

Ala Ala Pro His Cys Ile Gly Gly Ser Cys His Asp Ala Phe Asp Ile

1 5 10 15

<210> 188

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 188

Gln Ser Val Arg Ser Ser Tyr

1 5

<210> 189

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 189

Gly Ala Ser

1

<210> 190

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 190

Gln Gln Tyr Gly Ser Ser Pro Trp Thr

1 5

<210> 191

<211> 352

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 191

caggtgcagc tggtggagtc aggagcagag gtgaaaaagc ccggggagtc tctgaagatc 60

tcctgtaagg gttctggata cagctttacc agctactgga tcgtctgggt gcgccagatg 120

cccgggaaag gcctggagtg gatggggatc atctatcctg gtgactctga taccaaatac 180

agtccgtcct tccaaggcca ggtcagcatc tcagccgaca agcccatcag caccgcctac 240

ctgcagtgga gcaggctgaa ggcctcggac accgccatgt attactgtgc gagactaggg 300

aattggctgg tggactactg gggccaggga accctggtca ccgtctcctc ag 352

<210> 192

<211> 117

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 192

Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Ile Val Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Lys Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Ser Ile Ser Ala Asp Lys Pro Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Arg Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Leu Gly Asn Trp Leu Val Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 193

<211> 343

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 193

gatattgtga tgactcagtc tcctctctct ctgtccgtca cccctggaca gccggcctcc 60

atctcctgca agtctagtca gagcctcctg catagtgatg gaaagaccta tttgtattgg 120

tacctgcaga agccaggcca gcctccacag ctcctgatgt atgaagtttc caaccggttc 180

tctggagtgc cagataggtt cagtggcagc gggtcaggga cagacttcac acttaaaatc 240

agccgggtgg agtctgagga tgttggggtt tattactgca tgcaaagtat acagcttcct 300

cgcgggatca ccttcggcca agggacacga ctggagatta aac 343

<210> 194

<211> 114

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 194

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asp Gly Lys Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Pro

35 40 45

Pro Gln Leu Leu Met Tyr Glu Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ser Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ser

85 90 95

Ile Gln Leu Pro Arg Gly Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu

100 105 110

Ile Lys

<210> 195

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 195

Gly Tyr Ser Phe Thr Ser Tyr Trp

1 5

<210> 196

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 196

Ile Tyr Pro Gly Asp Ser Asp Thr

1 5

<210> 197

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 197

Ala Arg Leu Gly Asn Trp Leu Val Asp Tyr

1 5 10

<210> 198

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 198

Gln Ser Leu Leu His Ser Asp Gly Lys Thr Tyr

1 5 10

<210> 199

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 199

Glu Val Ser

1

<210> 200

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 200

Met Gln Ser Ile Gln Leu Pro Arg Gly Ile Thr

1 5 10

<210> 201

<211> 343

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 201

gaggtgcagc tggtggagtc tggaggaggc ttgatccagc ctggggggtc cctgagactc 60

tcctgtgcag cctctgggct caccgtcagt cgcaattaca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcactt atttatagcg gtggtagcac atactacgca 180

gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctt 240

caaatgaaca gcctgagagc cgaggacacg gccgtgtatt actgtgcgag agatctacgc 300

ggagaagtct ggggccaagg gacaatggtc accgtctctt cag 343

<210> 202

<211> 114

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 202

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Val Ser Arg Asn

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Leu Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Leu Arg Gly Glu Val Trp Gly Gln Gly Thr Met Val Thr Val

100 105 110

Ser Ser

<210> 203

<211> 322

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 203

gccatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc aggcgagtca ggacattagc aactttttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctacgat gcatccaatt tggaaacagg ggtcccatca 180

aggttcagtg gaagtggatc tgggacagat tttactttca ccatcagtag cctgcagcct 240

gaagatattg caacatatta ctgtcaccag tatgataatc tccctcgaac gttcggccaa 300

gggaccaaag tggatatcaa ac 322

<210> 204

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 204

Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Phe

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys His Gln Tyr Asp Asn Leu Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys

100 105

<210> 205

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 205

Gly Leu Thr Val Ser Arg Asn Tyr

1 5

<210> 206

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 206

Ile Tyr Ser Gly Gly Ser Thr

1 5

<210> 207

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 207

Ala Arg Asp Leu Arg Gly Glu Val

1 5

<210> 208

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 208

Gln Asp Ile Ser Asn Phe

1 5

<210> 209

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 209

Asp Ala Ser

1

<210> 210

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 210

His Gln Tyr Asp Asn Leu Pro Arg Thr

1 5

<210> 211

<211> 372

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 211

gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt caccttcagt aactacgaca tgcactgggt ccgccaagct 120

acaggaaaag gtctggagtg ggtctcactt attggtactg ctggtgacac atactatcca 180

gactccgtga agggccgatt caccatctcc agagaaaatg ccaagaactc cttgtatctt 240

caaatgaaca gcctgagagc cggggacacg gctgtgtatt actgtgcaag agggcaacac 300

actcaaatcg gtcactacta ctactactac atggacgtct ggggcaaagg gaccacggtc 360

accgtctcct ca 372

<210> 212

<211> 124

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 212

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Asp Met His Trp Val Arg Gln Ala Thr Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Leu Ile Gly Thr Ala Gly Asp Thr Tyr Tyr Pro Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Gln His Thr Gln Ile Gly His Tyr Tyr Tyr Tyr Tyr Met Asp

100 105 110

Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 213

<211> 328

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 213

gccatccgga tgacccagtc tccatcgtcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctttgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat tccactctaa ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta accctccgga gggcagtttt 300

ggccagggga ccaaagtgga gattaaac 328

<210> 214

<211> 109

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 214

Ala Ile Arg Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Phe Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Ser Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Asn Pro Pro

85 90 95

Glu Gly Ser Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 215

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 215

Gly Phe Thr Phe Ser Asn Tyr Asp

1 5

<210> 216

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 216

Ile Gly Thr Ala Gly Asp Thr

1 5

<210> 217

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 217

Ala Arg Gly Gln His Thr Gln Ile Gly His Tyr Tyr Tyr Tyr Tyr Met

1 5 10 15

Asp Val

<210> 218

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 218

Gln Ser Ile Ser Ser Tyr

1 5

<210> 219

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 219

Ala Ala Ser

1

<210> 220

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 220

Gln Gln Ser Tyr Ser Asn Pro Pro Glu Gly Ser

1 5 10

<210> 221

<211> 361

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 221

gaagtgcagc tggtggagac tggaggaggc ttgatccagc ctggggggtc cctgagactc 60

tcctgtgcag cctctgggtt caccgtcagt agcaactaca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagtt gtttatggcg gtggtaccac atactacgca 180

gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctt 240

caaatgaaca gcctgagagc cgaggacacg gccgtatatt actgtgcgac tgacaatgga 300

tacagctatg gtttttcatt tgactactgg ggccagggaa ccctggtcat cgtctcctca 360

g 361

<210> 222

<211> 120

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 222

Glu Val Gln Leu Val Glu Thr Gly Gly Gly Leu Ile Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Val Val Tyr Gly Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Thr Asp Asn Gly Tyr Ser Tyr Gly Phe Ser Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Ile Val Ser Ser

115 120

<210> 223

<211> 331

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 223

cagtctgtgc tgactcagcc tgcctccatg tctgggtctc ctggacagtc gatcaccatc 60

tcctgcactg gaaccagcag tgatgttggg ggttataacc ttgtctcctg gtaccaacag 120

cacccaggca aagcccccaa actcatgatt tatgagggca gtaagcggcc ctcaggggtt 180

tctaatcgct tctctggctc caagtctggc aacacggcct ccctgacaat ctctgggctc 240

caggctgagg acgaggctga ttattactgc tgctcatatg caggtagtag taattgggtg 300

ttcggcggag ggaccaagct gaccgtccta g 331

<210> 224

<211> 110

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 224

Gln Ser Val Leu Thr Gln Pro Ala Ser Met Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Leu Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Gly Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Cys Ser Tyr Ala Gly Ser

85 90 95

Ser Asn Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 225

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 225

Gly Phe Thr Val Ser Ser Asn Tyr

1 5

<210> 226

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 226

Val Tyr Gly Gly Gly Thr Thr

1 5

<210> 227

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 227

Ala Thr Asp Asn Gly Tyr Ser Tyr Gly Phe Ser Phe Asp Tyr

1 5 10

<210> 228

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 228

Ser Ser Asp Val Gly Gly Tyr Asn Leu

1 5

<210> 229

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 229

Glu Gly Ser

1

<210> 230

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 230

Cys Ser Tyr Ala Gly Ser Ser Asn Trp Val

1 5 10

<210> 231

<211> 373

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 231

caggtgcagc tggtggagtc tggggctgag gtggagaagc ctggggcctc agtgaaggtc 60

tcctgcaagg cttctggata caccttcacc ggctactata tgcactgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggatgg atcaacccta tcagtggtgg cacaaactat 180

gcacagaagt ttcagggcag ggtcaccatg accagggaca cgtccatcag cacagcctac 240

atggacctga gcaggctgag atctgacgac acggccgtgt attactgtgc gagaggaacg 300

tattactatg atagtagtgg ttacatccca tttgactact ggggccaggg aaccctggtc 360

accgtctcct cag 373

<210> 232

<211> 124

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 232

Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Glu Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Ile Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Asp Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Tyr Tyr Tyr Asp Ser Ser Gly Tyr Ile Pro Phe Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 233

<211> 331

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 233

cagtctgtgc tgactcagcc tgcctccgta tctgggtctc ctggacagtc gatcaccatc 60

tcctgcactg gaaccagcag tgatgttggg agttataacc ttgtctcctg gtaccaacag 120

cacccaggca aagcccccaa actcatgatt tatgagggca gtaagcggcc ctcaggggtt 180

tctaatcgct tctctggctc caagtctggc aacacggcct ccctgacaat ctctgggctc 240

caggctgagg acgaggctga ttattactgc tgctcatatg caggtagtag cactttggta 300

ttcggcggag ggaccaagct gaccgtccta g 331

<210> 234

<211> 110

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 234

Gln Ser Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr

20 25 30

Asn Leu Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Gly Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Cys Ser Tyr Ala Gly Ser

85 90 95

Ser Thr Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 235

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 235

Gly Tyr Thr Phe Thr Gly Tyr Tyr

1 5

<210> 236

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 236

Ile Asn Pro Ile Ser Gly Gly Thr

1 5

<210> 237

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 237

Ala Arg Gly Thr Tyr Tyr Tyr Asp Ser Ser Gly Tyr Ile Pro Phe Asp

1 5 10 15

Tyr

<210> 238

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 238

Ser Ser Asp Val Gly Ser Tyr Asn Leu

1 5

<210> 239

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 239

Glu Gly Ser

1

<210> 240

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 240

Cys Ser Tyr Ala Gly Ser Ser Thr Leu Val

1 5 10

<210> 241

<211> 385

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 241

caggttcagc tggtgcagtc tgggtctgag ttgaagaagc ctggggcctc agtgaaggtt 60

tcctgcaagg cttctggata caccttcagt agctatgcta tgacttgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggatgg atcaacacca acactgggaa cccaacgtat 180

gcccagggct tcacaggacg gtttgtcttc tccttggaca cctctgtcag cacggcatat 240

ctgcagatca gcagcctaaa ggctgaggac actgccgtgt attactgtgc gagagctctg 300

ggatattgta gtagtaccag ctgctatccc gcttgggctg cttttgatat ctggggccaa 360

gggacaatgg tcaccgtctc ttcag 385

<210> 242

<211> 128

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 242

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Ala Met Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Asn Thr Gly Asn Pro Thr Tyr Ala Gln Gly Phe

50 55 60

Thr Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Leu Gly Tyr Cys Ser Ser Thr Ser Cys Tyr Pro Ala Trp

100 105 110

Ala Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 243

<211> 316

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 243

tcctatgagc tgactcagcc actctcagtg tcagtggccc tgggacagac ggccagtatt 60

acctgtgggg gaaacaacat tggaagtaaa aatgtgcact ggtaccagca gaagccaggc 120

caggcccctg tgctggtcat ctatagggat agcaaccggc cctctgggat ccctgagcga 180

ttctctggct ccaactcggg gaacacggcc accctgacca tcagcagagc ccaagccggg 240

gatgaggctg actataactg tcaggtgtgg gacagcagcg tggtattcgg cggagggacc 300

aagctgaccg tcctag 316

<210> 244

<211> 105

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 244

Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Ala Ser Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Asn Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Arg Asp Ser Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Asn Cys Gln Val Trp Asp Ser Ser Val Val Phe

85 90 95

Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 245

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 245

Gly Tyr Thr Phe Ser Ser Tyr Ala

1 5

<210> 246

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 246

Ile Asn Thr Asn Thr Gly Asn Pro

1 5

<210> 247

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 247

Ala Arg Ala Leu Gly Tyr Cys Ser Ser Thr Ser Cys Tyr Pro Ala Trp

1 5 10 15

Ala Ala Phe Asp Ile

20

<210> 248

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 248

Asn Ile Gly Ser Lys Asn

1 5

<210> 249

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 249

Arg Asp Ser

1

<210> 250

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 250

Gln Val Trp Asp Ser Ser Val Val

1 5

<210> 251

<211> 358

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 251

gaggtgcagc tggtggagtc tggaggaggc ttgatccagc cgggggggtc cctgagactc 60

tcctgtgcag cctctgggct caccgtcagt agcaactaca tgagttgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagtt atttatagtg gtggtagcac gttctacgca 180

gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctt 240

caaatgaaca gcctgggagc cgaggacacg gccgtgtatt actgtgcgag aggagaaggt 300

agtcctggaa actggttcga cccctggggc cagggaaccc tggtcaccgt ctcctcag 358

<210> 252

<211> 119

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 252

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Val Ser Ser Asn

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Val Ile Tyr Ser Gly Gly Ser Thr Phe Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Gly Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Glu Gly Ser Pro Gly Asn Trp Phe Asp Pro Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 253

<211> 322

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 253

gatgttgtga tgactcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttccc agcagctact tagcctggta ccagcagaaa 120

cctggccagg ctcccaggct cctcatctat ggtgcatcca ccagggccac tggcatccca 180

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240

cctgaggatt ttgcagtgta ttactgtcag cactatgata cctcaccccg tttcggcgga 300

gggaccaaag tggatatcaa ac 322

<210> 254

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 254

Asp Val Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Pro Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Asp Thr Ser Pro

85 90 95

Arg Phe Gly Gly Gly Thr Lys Val Asp Ile Lys

100 105

<210> 255

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 255

Gly Leu Thr Val Ser Ser Asn Tyr

1 5

<210> 256

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 256

Ile Tyr Ser Gly Gly Ser Thr

1 5

<210> 257

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 257

Ala Arg Gly Glu Gly Ser Pro Gly Asn Trp Phe Asp Pro

1 5 10

<210> 258

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 258

Gln Ser Val Pro Ser Ser Tyr

1 5

<210> 259

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 259

Gly Ala Ser

1

<210> 260

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 260

Gln His Tyr Asp Thr Ser Pro Arg

1 5

<210> 261

<211> 370

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 261

caggtccagc tggtacagtc tgggcctgag gtgaagaagc ctgggacctc agtgaaggtc 60

tcctgcaagg cttctggatt cacctttact acctctgctg tgcagtgggt gcgacaggct 120

cgtggacaac gccttgagtg gataggatgg atcgtcgttg gcagtggtaa cacaaactac 180

gcacagaagt tccaggaaag agtcaccatt accagggaca tgtccacaac cacagcctac 240

atggagctga gcagcctgag atccgaggac acggccgtgt atttctgtgc ggcgcctcat 300

tgtaatagta ccagctgcta tgacgctttt gatatctggg gccaagggac aatggtcacc 360

gtctcttcag 370

<210> 262

<211> 123

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 262

Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Thr Ser

20 25 30

Ala Val Gln Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Trp Ile Val Val Gly Ser Gly Asn Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Glu Arg Val Thr Ile Thr Arg Asp Met Ser Thr Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Ala Pro His Cys Asn Ser Thr Ser Cys Tyr Asp Ala Phe Asp Ile

100 105 110

Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 263

<211> 325

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 263

gacatccaga tgacccagtc tccaggcacc ctgtctttgt ctccagggga aggagccacc 60

ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120

cctggccagg ctcccaggct cctcatctat ggtgcatcta gtggggccac tggcatccca 180

gacagattca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240

cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcacctta cacttttggc 300

caggggacca aggtggaaat caaac 325

<210> 264

<211> 108

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 264

Asp Ile Gln Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Gly Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Gly Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 265

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 265

Gly Phe Thr Phe Thr Thr Ser Ala

1 5

<210> 266

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 266

Ile Val Val Gly Ser Gly Asn Thr

1 5

<210> 267

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 267

Ala Ala Pro His Cys Asn Ser Thr Ser Cys Tyr Asp Ala Phe Asp Ile

1 5 10 15

<210> 268

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 268

Gln Ser Val Ser Ser Ser Tyr

1 5

<210> 269

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 269

Gly Ala Ser

1

<210> 270

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 270

Gln Gln Tyr Gly Ser Ser Pro Tyr Thr

1 5

<210> 271

<211> 352

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 271

caggtgcagc tggtggagtc tggaggaggc ttgatccagc ctggggggtc cctgagactc 60

tcctgtgcag cctctgggct caccgtcaat aggaactaca tgagctggat ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagtt atttatagcg gtggtagtac attttacgca 180

gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac actgtctctt 240

caaatgaaca gcctgagagc cgaggacacg gccatttatt actgtgcgag agacttctac 300

gagggttctt ttgatatctg gggccaaggg acaatggtca ccgtctcttc ag 352

<210> 272

<211> 117

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 272

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Val Asn Arg Asn

20 25 30

Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Val Ile Tyr Ser Gly Gly Ser Thr Phe Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Ser Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Asp Phe Tyr Glu Gly Ser Phe Asp Ile Trp Gly Gln Gly Thr Met

100 105 110

Val Thr Val Ser Ser

115

<210> 273

<211> 325

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 273

gccatccagt tgacccagtc tccttccttc ctgtctgcat ctataggaga cagagtcacc 60

atcacttgcc gggccagtca gggcattagc agttatttag cctggtatca gcaaaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccactt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240

gaagattttg catcttatta ctgtcaacag cttaatagtt accccgctcc ggttttcggc 300

cctgggacca aagtggatat caaac 325

<210> 274

<211> 108

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 274

Ala Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Ile Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Ser Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr Pro Ala

85 90 95

Pro Val Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105

<210> 275

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 275

Gly Leu Thr Val Asn Arg Asn Tyr

1 5

<210> 276

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 276

Ile Tyr Ser Gly Gly Ser Thr

1 5

<210> 277

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 277

Ala Arg Asp Phe Tyr Glu Gly Ser Phe Asp Ile

1 5 10

<210> 278

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 278

Gln Gly Ile Ser Ser Tyr

1 5

<210> 279

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 279

Ala Ala Ser

1

<210> 280

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 280

Gln Gln Leu Asn Ser Tyr Pro Ala Pro Val

1 5 10

<210> 281

<211> 370

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 281

caggtacagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60

tcctgcaagg cttctggtta catctttatc agatatggta ttagctgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggatgg atcagcgcta acaatggtta cacaaactat 180

gcacagaagc tccagggcag agtcaccatg accacagaca catccacgag cacagcctac 240

atggagctga ggagcctgag atctgacgac acggccgtgt attactgtgc gagagatggg 300

ggtattttga ctggttatct cgactacttt gaccactggg gccagggaac cctggtcacc 360

gtctcctcag 370

<210> 282

<211> 123

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 282

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Ile Arg Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Ser Ala Asn Asn Gly Tyr Thr Asn Tyr Ala Gln Lys Leu

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Gly Gly Ile Leu Thr Gly Tyr Leu Asp Tyr Phe Asp His

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 283

<211> 325

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 283

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagactcacc 60

atcacttgcc gggcaagtca gagcattgcc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacca ctgtcaacag agttacagta ccctcggaat cactttcggc 300

cctgggacca aagtggatat caaac 325

<210> 284

<211> 108

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 284

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Leu Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ala Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr His Cys Gln Gln Ser Tyr Ser Thr Leu Gly

85 90 95

Ile Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105

<210> 285

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 285

Gly Tyr Ile Phe Ile Arg Tyr Gly

1 5

<210> 286

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 286

Ile Ser Ala Asn Asn Gly Tyr Thr

1 5

<210> 287

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 287

Ala Arg Asp Gly Gly Ile Leu Thr Gly Tyr Leu Asp Tyr Phe Asp His

1 5 10 15

<210> 288

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 288

Gln Ser Ile Ala Ser Tyr

1 5

<210> 289

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 289

Ala Ala Ser

1

<210> 290

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 290

Gln Gln Ser Tyr Ser Thr Leu Gly Ile Thr

1 5 10

<210> 291

<211> 367

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 291

caggtgcagc tggtggagtc tgggggaggc ttggtccagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt cccctttagt atctattgga tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtggccaac ataaagcaag atggaagtga gaaatactat 180

gtggactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat 240

ctgcacatga acagcctgag aggcgaggac acggctgtgt attactgtgc gagccgatat 300

tacgattttc gaccggaggc ttggtttgac tactggggcc agggaaccct ggtcaccgtc 360

tcctcag 367

<210> 292

<211> 122

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 292

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Ser Ile Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu His Met Asn Ser Leu Arg Gly Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Arg Tyr Tyr Asp Phe Arg Pro Glu Ala Trp Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 293

<211> 343

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 293

gatattgtga tgacccagac tccactctcc tcacctgtca cccttggaca gccggcctcc 60

atctcctgca ggtctagtca aagcctcgta cacagggatg gaaacaccta cttgagctgg 120

cttcagcaga ggccaggcca gcctccaaga ctcctaattt ataagatttc taaccggttc 180

tctggggtcc cagacagatt cagtggcagt ggggcaggga cagatttcac actgaaaatc 240

agcagggtgg aagctgagga tgtcggggtt tattactgca tgcaagctac acaatttcct 300

catgggtaca cttttggcca ggggaccaag gtggagatca aac 343

<210> 294

<211> 114

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 294

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Arg

20 25 30

Asp Gly Asn Thr Tyr Leu Ser Trp Leu Gln Gln Arg Pro Gly Gln Pro

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Ile Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Thr Gln Phe Pro His Gly Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu

100 105 110

Ile Lys

<210> 295

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 295

Gly Phe Pro Phe Ser Ile Tyr Trp

1 5

<210> 296

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 296

Ile Lys Gln Asp Gly Ser Glu Lys

1 5

<210> 297

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 297

Ala Ser Arg Tyr Tyr Asp Phe Arg Pro Glu Ala Trp Phe Asp Tyr

1 5 10 15

<210> 298

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 298

Gln Ser Leu Val His Arg Asp Gly Asn Thr Tyr

1 5 10

<210> 299

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 299

Lys Ile Ser

1

<210> 300

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 300

Met Gln Ala Thr Gln Phe Pro His Gly Tyr Thr

1 5 10

<210> 301

<211> 355

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 301

caggtgcagc tgcaggagtc tgggggaggc ttggtccagc ctggggggtc cctgagactc 60

tcctgttcag cctctggatt caccgtcagt agcaactaca tgacctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagtt atttatagcg gtggtagcac attctacgca 180

gactccgtga agggcagatt caccatctcc agagacaatt ccaagaacac gctgtatctt 240

caaatgaaca gcctgagagc cgaggacacc gctgtgtatt actgtgcgag agatctggaa 300

gaggccgggg gatttgacta ctggggccag ggaaccctgg tcaccgtctc ctcag 355

<210> 302

<211> 118

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 302

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Val Ser Ser Asn

20 25 30

Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Val Ile Tyr Ser Gly Gly Ser Thr Phe Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Leu Glu Glu Ala Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 303

<211> 325

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 303

gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aaaagtcacc 60

ctctcctgca gggccagtca gagtgttagc agcacctact tagcctggta ccagcagaaa 120

cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcgtccca 180

gacaggttcc gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240

cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcgctgta cacttttggc 300

caggggacca aagtggatat caaac 325

<210> 304

<211> 108

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 304

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Thr

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Val Pro Asp Arg Phe Arg

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Leu

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys

100 105

<210> 305

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 305

Gly Phe Thr Val Ser Ser Asn Tyr

1 5

<210> 306

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 306

Ile Tyr Ser Gly Gly Ser Thr

1 5

<210> 307

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 307

Ala Arg Asp Leu Glu Glu Ala Gly Gly Phe Asp Tyr

1 5 10

<210> 308

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 308

Gln Ser Val Ser Ser Thr Tyr

1 5

<210> 309

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 309

Gly Ala Ser

1

<210> 310

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 310

Gln Gln Tyr Gly Ser Ser Leu Tyr Thr

1 5

<210> 311

<211> 385

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 311

cagctgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctccggtga ctccgtcagt aattactact ggagctggat ccggcagccc 120

gccgggaagg gactggagtg gattgggcgt atctatacca gtgggagcac caactacaac 180

ccctccctca agagtcgagt caccatgtca gtagacacgt ccaagaacca gttctccctg 240

aagctgagct ctgtgaccgc cgcggacacg gccgtgtatt actgtgcgag agatcaccgg 300

gcttcccggt atagcagtgg ctggtacgaa tggtggaact gcttcgaccc ctggggccag 360

ggaaccctgg tcaccgtctc ctcag 385

<210> 312

<211> 128

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 312

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Val Ser Asn Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Ala Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Tyr Thr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp His Arg Ala Ser Arg Tyr Ser Ser Gly Trp Tyr Glu Trp Trp

100 105 110

Asn Cys Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 313

<211> 325

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 313

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccgtca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcaacag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta cccccgcgct cactttcggc 300

ggagggacca aagtggatat caaac 325

<210> 314

<211> 108

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 314

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Ala

85 90 95

Leu Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys

100 105

<210> 315

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 315

Gly Asp Ser Val Ser Asn Tyr Tyr

1 5

<210> 316

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 316

Ile Tyr Thr Ser Gly Ser Thr

1 5

<210> 317

<211> 22

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 317

Ala Arg Asp His Arg Ala Ser Arg Tyr Ser Ser Gly Trp Tyr Glu Trp

1 5 10 15

Trp Asn Cys Phe Asp Pro

20

<210> 318

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 318

Gln Ser Ile Ser Ser Tyr

1 5

<210> 319

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 319

Ala Ala Ser

1

<210> 320

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 320

Gln Gln Ser Tyr Ser Thr Pro Ala Leu Thr

1 5 10

<210> 321

<211> 367

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 321

caggttcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60

tcctgcaagg cttctggata caccttcacc ggctactata tgcactgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggatgg atcaacccta acagtggtgg cacaaactat 180

acacagaagt ttcagggcag ggtcaccatg accagggaca cgtccatcag cacagcctac 240

atggagctga gcaggctgag atctgacgac acggccgtgt attcctgtgc gagagatatg 300

gcgtttagta tggttcgggg ttcctttgac tactggggcc agggaaccct ggtcaccgtc 360

tcctcag 367

<210> 322

<211> 122

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 322

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Thr Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Ser Cys

85 90 95

Ala Arg Asp Met Ala Phe Ser Met Val Arg Gly Ser Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 323

<211> 331

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 323

caggctgtgc tgactcagcc tccctccgcg tccgggtctc ctggacagtc agtcaccatc 60

tcctgcactg gaaccagcag tgacgttggt ggttataact atgtctcctg gtaccaacag 120

cacccaggca aagcccccaa actcatgatt tatgaggtca gtaagcggcc ctcaggggtc 180

cctgatcgct tctctggctc caagtctggc aacacggcct ccctgaccgt ctctgggctc 240

caggctgagg atgaggctga ttattactgc agctcatatg caggcagcaa ccattgggtg 300

ttcggcggag ggaccaagct gaccgtccta g 331

<210> 324

<211> 110

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 324

Gln Ala Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Ala Gly Ser

85 90 95

Asn His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 325

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 325

Gly Tyr Thr Phe Thr Gly Tyr Tyr

1 5

<210> 326

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 326

Ile Asn Pro Asn Ser Gly Gly Thr

1 5

<210> 327

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 327

Ala Arg Asp Met Ala Phe Ser Met Val Arg Gly Ser Phe Asp Tyr

1 5 10 15

<210> 328

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 328

Ser Ser Asp Val Gly Gly Tyr Asn Tyr

1 5

<210> 329

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 329

Glu Val Ser

1

<210> 330

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 330

Ser Ser Tyr Ala Gly Ser Asn His Trp Val

1 5 10

<210> 331

<211> 358

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 331

caggtgcagc tggtgcagtc tgggcctgag gtgaagaagc ctgggacctc agtgaaggtc 60

tcctgcaagg cgtctggatt cacccttact agctctgcta tgcagtgggt gcgacaggct 120

cgtggacaac gccttgagtg gataggatgg atcgtcgttg gcagtggcaa cacaaactac 180

gcacagaagt tccaggaaag agtcaccatt accagggaca tgtccacaag cacagcctac 240

atggagctga gcagcctgag atccgaggac acggccgtgt attattgtgc ggccggccgt 300

ggctacaatt cggactttga ctactggggc cagggaaccc tggtcaccgt ctcctcag 358

<210> 332

<211> 119

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 332

Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Leu Thr Ser Ser

20 25 30

Ala Met Gln Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Trp Ile Val Val Gly Ser Gly Asn Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Glu Arg Val Thr Ile Thr Arg Asp Met Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Gly Tyr Asn Ser Asp Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 333

<211> 325

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 333

gccatccgga tgacccagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaga 120

cctggccagg ctcccaggct cctcatctat ggtacatcca gcagggccac tggcatccca 180

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240

cctgaagatt ttgcagtgta ttactgtcag cagtatggtt actcagtgta cacttttggc 300

caggggacca aagtggatat caaac 325

<210> 334

<211> 108

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 334

Ala Ile Arg Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Thr Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Tyr Ser Val

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys

100 105

<210> 335

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 335

Gly Phe Thr Leu Thr Ser Ser Ala

1 5

<210> 336

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 336

Ile Val Val Gly Ser Gly Asn Thr

1 5

<210> 337

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 337

Ala Ala Gly Arg Gly Tyr Asn Ser Asp Phe Asp Tyr

1 5 10

<210> 338

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 338

Gln Ser Val Ser Ser Ser Tyr

1 5

<210> 339

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 339

Gly Thr Ser

1

<210> 340

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 340

Gln Gln Tyr Gly Tyr Ser Val Tyr Thr

1 5

<210> 341

<211> 367

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 341

caggtgcagc tggtggagtc tgaggctgag gtgaagaagc ctggggcctc agtgaaggtt 60

tcctgcaagg catctggata caccttcacc agctactata tgcactgggt gcgacaggcc 120

cctggacaag ggcttcagtg gatgggaata atcaacccta gtgctggtag cacaagctac 180

gcacagaagt tccagggcag agtcaccatg accacggaca cgtccacgac cacagtctac 240

atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagagattct 300

gtactagtac cagctgctaa tgcttttgat atctggggcc aagggacaat ggtcaccgtc 360

tcttcag 367

<210> 342

<211> 122

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 342

Gln Val Gln Leu Val Glu Ser Glu Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Gln Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Ala Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Thr Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Ser Val Leu Val Pro Ala Ala Asn Ala Phe Asp Ile Trp

100 105 110

Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 343

<211> 322

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 343

gaaatagtga tgacgcagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttagt agctacttag cctggtacca acagaaacct 120

ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc 180

aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct 240

gaagattttg cagtttatta ctgtcagcag cgtcgcaact ggctattcac tttcggccct 300

gggaccaaag tggatatcaa ac 322

<210> 344

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 344

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Arg Asn Trp Leu Phe

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105

<210> 345

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 345

Gly Tyr Thr Phe Thr Ser Tyr Tyr

1 5

<210> 346

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 346

Ile Asn Pro Ser Ala Gly Ser Thr

1 5

<210> 347

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 347

Ala Arg Asp Ser Val Leu Val Pro Ala Ala Asn Ala Phe Asp Ile

1 5 10 15

<210> 348

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 348

Gln Ser Val Ser Ser Tyr

1 5

<210> 349

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 349

Asp Ala Ser

1

<210> 350

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 350

Gln Gln Arg Arg Asn Trp Leu Phe Thr

1 5

<210> 351

<211> 358

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 351

caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60

tcctgcaagg cttctggaga caccttcact agctatactc tgcattgggt gcgccaggcc 120

cccggacaaa ggcttgagtg gatgggatgg atcaacgctg gcaatggtta cacaaaatat 180

tcacagaagt tccagggcag agtcaccatt accagggaca catccgcgag cacagcctac 240

atggagctga gcagcctgag atctgaagac acggctgtgt attactgtgc gaaatgtact 300

atgatagtag actactttga ctactggggc cagggaaccc tggtcaccgt ctcctcag 358

<210> 352

<211> 119

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 352

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Asp Thr Phe Thr Ser Tyr

20 25 30

Thr Leu His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Ala Gly Asn Gly Tyr Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Cys Thr Met Ile Val Asp Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 353

<211> 322

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 353

gccatccgga tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggccagtca gagtattagt ggctggttgg cctggtatca gcagaaacca 120

gagaaagccc ctaagctcct gatctatgat gcctccaatt tggaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcaacag cctgcagcct 240

gatgattttg caacttatta ctgccaacag tataatagtt acccgtggac gttcggccaa 300

gggaccaaag tggatatcaa ac 322

<210> 354

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 354

Ala Ile Arg Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Gly Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys

100 105

<210> 355

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 355

Gly Asp Thr Phe Thr Ser Tyr Thr

1 5

<210> 356

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 356

Ile Asn Ala Gly Asn Gly Tyr Thr

1 5

<210> 357

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 357

Ala Lys Cys Thr Met Ile Val Asp Tyr Phe Asp Tyr

1 5 10

<210> 358

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 358

Gln Ser Ile Ser Gly Trp

1 5

<210> 359

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 359

Asp Ala Ser

1

<210> 360

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 360

Gln Gln Tyr Asn Ser Tyr Pro Trp Thr

1 5

<210> 361

<211> 357

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 361

gaggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60

tcctgcaagg cttctgggta caccttcacc agttatgata tcaactgggt gcgacaggcc 120

actggacaag ggcttgagtg gatgggatgg atgaaccctc acagtgatac cacaggctat 180

gcacagaagt tccagggcag agtcaccatg accaggaaca cctccataac cacagcctac 240

atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc tcagggaccc 300

atagcagtga actacatgga cgtctggggc aaagggacca cggtcaccgt ctcctca 357

<210> 362

<211> 119

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 362

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asp Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Met Asn Pro His Ser Asp Thr Thr Gly Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asn Thr Ser Ile Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Gln Gly Pro Ile Ala Val Asn Tyr Met Asp Val Trp Gly Lys Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 363

<211> 325

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 363

cagcctgtgc tgactcagcc accctcagtg tcagtggccc caggaaagac ggccaggatt 60

acctgtgggg gaagcaacat tggaagtaaa agtgtgcact ggtaccagca gaagccaggc 120

caggcccctg tgctgatcat ctattatgat agcgaccggc cctcagggat ccctgagcga 180

ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240

gatgaggccg acttttactg tcaggtgtgg gatagtagta ctgatcatgt ggtattcggc 300

ggggggacca agctgaccgt cctag 325

<210> 364

<211> 108

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 364

Gln Pro Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Lys

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Ser Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Ile Ile Tyr

35 40 45

Tyr Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Phe Tyr Cys Gln Val Trp Asp Ser Ser Thr Asp His

85 90 95

Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 365

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 365

Gly Tyr Thr Phe Thr Ser Tyr Asp

1 5

<210> 366

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 366

Met Asn Pro His Ser Asp Thr Thr

1 5

<210> 367

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 367

Ala Gln Gly Pro Ile Ala Val Asn Tyr Met Asp Val

1 5 10

<210> 368

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 368

Asn Ile Gly Ser Lys Ser

1 5

<210> 369

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 369

Tyr Asp Ser

1

<210> 370

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 370

Gln Val Trp Asp Ser Ser Thr Asp His Val Val

1 5 10

<210> 371

<211> 376

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 371

gaggtgcagc tggtggagtc tgggggaggc ttggtcaagc ctggagagtc cctgagactc 60

tcctgtgcag cctctggatt caccttcagt gactactaca tgacctggat ccgccaggct 120

ccagggaagg ggctggagtg ggtttcatac attaggagta gtggtcatac tatatactac 180

gcagactctg tgaagggccg attcaccatc tccagggaca acgccaagaa ctcactgtat 240

ctacaaatga acagcctgag agtcgaggac acggccgtgt attactgtgc gagaggaggg 300

gttttacgat ttttggagtg gcctctcaat gcttttgata tctggggcca agggacaatg 360

gtcaccgtct cttcag 376

<210> 372

<211> 125

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 372

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Glu

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Thr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Arg Ser Ser Gly His Thr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Val Leu Arg Phe Leu Glu Trp Pro Leu Asn Ala Phe

100 105 110

Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 373

<211> 322

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 373

gacatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcgagtca gggcattagc aattatttag cctggtatca gcagaaacca 120

gggaaagttc ctaagctcct gatctatgct gcatccactt tgcaatcagg ggtcccatct 180

cggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagatgttg caacttatta ctgtcaaaag tataacaatg ccctcgggac gttcggccaa 300

gggaccaagg tggagatcaa ac 322

<210> 374

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 374

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Asn Asn Ala Leu Gly

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 375

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 375

Gly Phe Thr Phe Ser Asp Tyr Tyr

1 5

<210> 376

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 376

Ile Arg Ser Ser Gly His Thr Ile

1 5

<210> 377

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 377

Ala Arg Gly Gly Val Leu Arg Phe Leu Glu Trp Pro Leu Asn Ala Phe

1 5 10 15

Asp Ile

<210> 378

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 378

Gln Gly Ile Ser Asn Tyr

1 5

<210> 379

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 379

Ala Ala Ser

1

<210> 380

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 380

Gln Lys Tyr Asn Asn Ala Leu Gly Thr

1 5

<210> 381

<211> 364

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 381

gaggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60

tcctgcaagg cttctggata caccttcacc ggctactata tgcactgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggatgg atcagcccta acagtggtgg cacaaactat 180

gcacagaagt ttcagggcag ggtcaccatg accagggaca cgtccatcac cacagcctac 240

atggacctga gcaggctgag atctgacgac acggccgtgt attactgtgc gagaggttat 300

tactatgaag ccctcgatgc ttttgatatc tggggccaag ggacaatggt caccgtctct 360

tcag 364

<210> 382

<211> 121

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 382

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Ser Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Thr Thr Ala Tyr

65 70 75 80

Met Asp Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Tyr Tyr Glu Ala Leu Asp Ala Phe Asp Ile Trp Gly

100 105 110

Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 383

<211> 331

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 383

cagtctgtcg tgacgcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60

tcctgcactg gaaccagcag tgacgttggt ggttataact ttgtctcctg gtaccaacag 120

cacccaggca aagcccccaa actcatgatt tatgaggtca gtaatcggcc ctcaggggtt 180

tctaatcgct tctctggctc caagtctggc atcacggcct ccctgaccat ctctgggctc 240

caggctgagg acgaggctga ttattactgc aactcatata caagcaacag tactcgggta 300

ttcggcggag ggaccaagct gaccgtccta g 331

<210> 384

<211> 110

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 384

Gln Ser Val Val Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Phe Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Ile Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Tyr Thr Ser Asn

85 90 95

Ser Thr Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 385

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 385

Gly Tyr Thr Phe Thr Gly Tyr Tyr

1 5

<210> 386

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 386

Ile Ser Pro Asn Ser Gly Gly Thr

1 5

<210> 387

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 387

Ala Arg Gly Tyr Tyr Tyr Glu Ala Leu Asp Ala Phe Asp Ile

1 5 10

<210> 388

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 388

Ser Ser Asp Val Gly Gly Tyr Asn Phe

1 5

<210> 389

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 389

Glu Val Ser

1

<210> 390

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 390

Asn Ser Tyr Thr Ser Asn Ser Thr Arg Val

1 5 10

<210> 391

<211> 382

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 391

caggtgcagc tggtggagtc tgggggaggc ttggtccagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt caccgtcagt agcaactaca tgacctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagtt atttatagcg gtggtagcac atactacgca 180

gactccgtga agggcagatt caccatctcc agagacaatt ccaagaacac gctatatctt 240

caaatgaaca gcctgagagc cgacgacacg gctgtatatt actgtgcgag agactctaca 300

gccgattacg atttttggag tggttattat gtaggtgctt ttcatatctg gggccaaggg 360

acaatggtca ccgtctcttc ag 382

<210> 392

<211> 127

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 392

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn

20 25 30

Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Ser Thr Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Tyr Val Gly

100 105 110

Ala Phe His Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 393

<211> 331

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 393

cagactgtgc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60

tcctgcactg gaaccagcag tgacgttggt ggttacaact atgtctcctg gtaccaacag 120

cacccaggca aagcccccaa actcatgatt tatgaggtca ctaagcggcc ctcaggggtc 180

cctgatcgct tctctggctc caagtctggc aacacggcct ccctgaccgt ctctgggctc 240

caggctgagg atgaggctga ttattactgc agctcatatg caggcagcaa caattgggtg 300

ttcggcggag ggaccaagct gaccgtccta g 331

<210> 394

<211> 110

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 394

Gln Thr Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Val Thr Lys Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Ala Gly Ser

85 90 95

Asn Asn Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 395

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 395

Gly Phe Thr Val Ser Ser Asn Tyr

1 5

<210> 396

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 396

Ile Tyr Ser Gly Gly Ser Thr

1 5

<210> 397

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 397

Ala Arg Asp Ser Thr Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Tyr Val

1 5 10 15

Gly Ala Phe His Ile

20

<210> 398

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 398

Ser Ser Asp Val Gly Gly Tyr Asn Tyr

1 5

<210> 399

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 399

Glu Val Thr

1

<210> 400

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 400

Ser Ser Tyr Ala Gly Ser Asn Asn Trp Val

1 5 10

<210> 401

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<220>

<221> feature not yet classified

<222> (6)..(6)

<223> Xaa can be any naturally occurring amino acid

<400> 401

Gly Phe Thr Phe Thr Xaa Ser Ala

1 5

<210> 402

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 402

Ile Val Val Gly Ser Gly Asn Thr

1 5

<210> 403

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<220>

<221> feature not yet classified

<222> (4)..(4)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> feature not yet classified

<222> (6)..(7)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> modified residue

<222> (8)..(8)

<223> S or T

<220>

<221> feature not yet classified

<222> (10)..(10)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> feature not yet classified

<222> (12)..(12)

<223> Xaa can be any naturally occurring amino acid

<400> 403

Ala Ala Pro Xaa Cys Xaa Xaa Xaa Cys Xaa Asp Xaa Phe Asp Ile

1 5 10 15

<210> 404

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<220>

<221> feature not yet classified

<222> (4)..(4)

<223> Xaa can be any naturally occurring amino acid

<400> 404

Gln Ser Val Xaa Ser Ser Tyr

1 5

<210> 405

<400> 405

000

<210> 406

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<220>

<221> feature not yet classified

<222> (8)..(8)

<223> Xaa can be any naturally occurring amino acid

<400> 406

Gln Gln Tyr Gly Ser Ser Pro Xaa Thr

1 5

<210> 407

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<220>

<221> feature not yet classified

<222> (2)..(2)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> feature not yet classified

<222> (5)..(6)

<223> Xaa can be any naturally occurring amino acid

<400> 407

Gly Xaa Thr Cys Xaa Xaa Asn Tyr

1 5

<210> 408

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<220>

<221> feature not yet classified

<222> (3)..(3)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> feature not yet classified

<222> (6)..(6)

<223> Xaa can be any naturally occurring amino acid

<400> 408

Ile Tyr Xaa Gly Gly Xaa Thr

1 5

<210> 409

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 409

Arg Arg Ala Arg

1

<210> 410

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 410

Gly Ser Ala Ser

1

<210> 411

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 411

His His His His His His His His

1 5

<210> 412

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 412

Arg Arg Lys Arg

1

<210> 413

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 413

Arg Arg Ser Arg

1

<210> 414

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 414

Arg Ser Val Gly

1

<210> 415

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 415

Ala Ser Val Gly

1

<210> 416

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 416

Arg Ser Ala Arg

1

<210> 417

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 417

atgctgcaat cgtgctacaa 20

<210> 418

<211> 17

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 418

gactgccgcc tctgctc 17

<210> 419

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> feature not yet classified

<222> (1)..(1)

<223> 56-FAM

<220>

<221> feature not yet classified

<222> (9)..(10)

<223> ZEN

<220>

<221> feature not yet classified

<222> (20)..(20)

<223> 3IABkFQ

<400> 419

tcaaggaaca acattgccaa 20

<210> 420

<211> 41

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 420

ctacggcttt cagcccacat acggtgtggg ctaccagcct t 41

<210> 421

<211> 41

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 421

aaggctggta gcccacaccg tatgtgggct gaaagccgta g 41

<210> 422

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 422

cagctcctgg gcaacgtgct 20

<210> 423

<211> 19

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 423

cgtaaaagga gcaacatag 19

<210> 424

<211> 27

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 424

gggcagaccg gcacgatcgc cgactac 27

<210> 425

<211> 27

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 425

gtagtcggcg atcgtgccgg tctgccc 27

<210> 426

<211> 35

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 426

cagggcagac cggcaatatc gccgactaca attac 35

<210> 427

<211> 35

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 427

gtaattgtag tcggcgatat tgccggtctg ccctg 35

<210> 428

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 428

Cys Ala Ala Pro His Cys Asn Ser Thr Ser Cys Tyr Asp Ala Phe Asp

1 5 10 15

Ile Trp

<210> 429

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 429

Cys Ala Ala Pro Ala Cys Gly Thr Ser Cys Ser Asp Ala Phe Asp Ile

1 5 10 15

Trp

<210> 430

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 430

Cys Ala Ala Pro His Cys Ile Gly Gly Ser Cys His Asp Ala Phe Asp

1 5 10 15

Ile Trp

<210> 431

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 431

Cys Gln Gln Tyr Gly Ser Ser Phe Tyr Thr Phe

1 5 10

<210> 432

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 432

Cys Gln Gln Tyr Gly Ser Ser Pro Trp Thr Phe

1 5 10

<210> 433

<211> 58

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 433

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ser Ser

1 5 10 15

Met Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

20 25 30

Ala Val Tyr Tyr Cys Ala Arg Asp Leu Met Val Tyr Gly Ile Asp Val

35 40 45

Trp Gly Gln Gly Thr Thr Val Thr Val Ser

50 55

<210> 434

<211> 59

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 434

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

1 5 10 15

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

20 25 30

Ala Val Tyr Tyr Cys Ala Arg Asp Leu Asp Val Ser Gly Gly Met Asp

35 40 45

Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

50 55

<210> 435

<211> 56

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 435

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg His Asn Ser

1 5 10 15

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Glu Ala Glu Asp Thr

20 25 30

Ala Val Tyr Tyr Cys Ala Arg Glu Ala Tyr Gly Met Asp Val Trp Gly

35 40 45

Gln Gly Thr Thr Val Thr Val Ser

50 55

<210> 436

<211> 58

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 436

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

1 5 10 15

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

20 25 30

Ala Val Tyr His Cys Ala Arg Asp Leu Val Val Tyr Gly Met Asp Val

35 40 45

Trp Gly Gln Gly Thr Thr Val Thr Val Ser

50 55

<210> 437

<211> 58

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 437

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

1 5 10 15

Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr

20 25 30

Ala Val Tyr Tyr Cys Ala Arg Asp Leu Gly Pro Tyr Gly Met Asp Val

35 40 45

Trp Gly Gln Gly Thr Thr Val Thr Val Ser

50 55

<210> 438

<211> 59

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 438

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser

1 5 10 15

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

20 25 30

Ala Val Tyr Tyr Cys Ala Arg Asp Leu Gly Glu Ala Gly Gly Met Asp

35 40 45

Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

50 55

<210> 439

<211> 58

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 439

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

1 5 10 15

Lys Asn Thr Leu Tyr Leu Gln Met His Ser Leu Arg Ala Glu Asp Thr

20 25 30

Ala Val Tyr Tyr Cys Ala Arg Asp Leu Gly Ser Gly Asp Met Asp Val

35 40 45

Trp Gly Lys Gly Thr Thr Val Thr Val Ser

50 55

<210> 440

<211> 59

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 440

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

1 5 10 15

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

20 25 30

Ala Val Tyr Tyr Cys Ala Arg Asp Leu Glu Arg Ala Gly Gly Met Asp

35 40 45

Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

50 55

<210> 441

<211> 58

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 441

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

1 5 10 15

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

20 25 30

Ala Val Tyr Tyr Cys Ala Arg Asp Leu Asp Val Tyr Gly Leu Asp Val

35 40 45

Trp Gly Gln Gly Thr Thr Val Thr Val Ser

50 55

<210> 442

<211> 55

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 442

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

1 5 10 15

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

20 25 30

Ala Val Tyr Tyr Cys Ala Arg Asp Leu Arg Gly Glu Val Trp Gly Gln

35 40 45

Gly Thr Met Val Thr Val Ser

50 55

<210> 443

<211> 58

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 443

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

1 5 10 15

Lys Ser Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr

20 25 30

Ala Val Tyr Tyr Cys Ala Arg Asp Phe Gly Asp Phe Tyr Phe Asp Tyr

35 40 45

Trp Gly Gln Gly Thr Leu Val Thr Val Ser

50 55

<210> 444

<211> 58

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 444

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

1 5 10 15

Lys Asn Thr Leu Asn Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

20 25 30

Ala Val Tyr Tyr Cys Ala Arg Asp Tyr Gly Asp Tyr Tyr Phe Asp Tyr

35 40 45

Trp Gly Gln Gly Thr Leu Val Thr Val Ser

50 55

<210> 445

<211> 60

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 445

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

1 5 10 15

Met Asn Thr Leu Phe Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

20 25 30

Ala Val Tyr Tyr Cys Ala Arg Val Leu Pro Met Tyr Gly Asp Tyr Leu

35 40 45

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

50 55 60

<210> 446

<211> 59

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 446

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

1 5 10 15

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr

20 25 30

Ala Val Tyr Tyr Cys Ala Arg Asp Leu Gln Glu Leu Gly Ser Leu Asp

35 40 45

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

50 55

<210> 447

<211> 60

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 447

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

1 5 10 15

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Gly Ala Glu Asp Thr

20 25 30

Ala Val Tyr Tyr Cys Ala Arg Gly Glu Gly Ser Pro Gly Asn Trp Phe

35 40 45

Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser

50 55 60

<210> 448

<211> 58

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 448

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

1 5 10 15

Lys Asn Thr Leu Ser Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

20 25 30

Ala Ile Tyr Tyr Cys Ala Arg Asp Phe Tyr Glu Gly Ser Phe Asp Ile

35 40 45

Trp Gly Gln Gly Thr Met Val Thr Val Ser

50 55

<210> 449

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 449

Cys Ala Ala Ser Gly Val Thr Val Ser Ser Asn Tyr Met Ser Trp

1 5 10 15

<210> 450

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 450

Trp Val Ser Ile Ile Tyr Ser Gly Gly Thr Thr Tyr Tyr Ala

1 5 10

<210> 451

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 451

Cys Ala Arg Asp Leu Met Val Tyr Gly Ile Asp Val Trp

1 5 10

<210> 452

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 452

Cys Ala Ala Ser Gly Val Thr Val Ser Ser Asn Tyr Met Ser Trp

1 5 10 15

<210> 453

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 453

Trp Val Ser Ile Ile Tyr Pro Gly Gly Ser Thr Phe Tyr Ala

1 5 10

<210> 454

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 454

Cys Ala Arg Asp Leu Gly Ser Gly Asp Met Asp Val Trp

1 5 10

<210> 455

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 455

Cys Ala Ala Ser Gly Leu Thr Val Ser Ser Asn Tyr Met Ser Trp

1 5 10 15

<210> 456

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 456

Trp Val Ser Val Ile Tyr Ser Gly Gly Ser Thr Phe Tyr Ala

1 5 10

<210> 457

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 457

Cys Ala Arg Gly Glu Gly Ser Pro Gly Asn Trp Phe Asp Pro Trp

1 5 10 15

<210> 458

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 458

Cys Ala Ala Ser Gly Leu Thr Val Asn Arg Asn Tyr Met Ser Trp

1 5 10 15

<210> 459

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 459

Trp Val Ser Val Ile Tyr Ser Gly Gly Ser Thr Phe Tyr Ala

1 5 10

<210> 460

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 460

Cys Ala Arg Asp Phe Tyr Glu Gly Ser Phe Asp Ile Trp

1 5 10

<210> 461

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 461

Cys Ala Ala Ser Gly Leu Thr Val Ser Arg Asn Tyr Met Ser Trp

1 5 10 15

<210> 462

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 462

Trp Val Ser Leu Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala

1 5 10

<210> 463

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 463

Cys Ala Arg Asp Leu Arg Gly Glu Val Trp

1 5 10

<210> 464

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 464

Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr Leu Ala Trp

1 5 10

<210> 465

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 465

Cys Gln Gln Leu Asp Ser Tyr Pro Pro Gly Tyr Thr Phe

1 5 10

<210> 466

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 466

Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr Leu Ala Trp

1 5 10

<210> 467

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 467

Cys Gln Gln Leu Asn Ser Tyr Arg Tyr Thr Phe

1 5 10

<210> 468

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 468

Cys Arg Ala Ser Gln Ser Val Pro Ser Ser Tyr Leu Ala Trp

1 5 10

<210> 469

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 469

Cys Gln His Tyr Asp Thr Ser Pro Arg Phe

1 5 10

<210> 470

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 470

Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr Leu Ala Trp

1 5 10

<210> 471

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 471

Cys Gln Gln Leu Asn Ser Tyr Pro Ala Pro Val Phe

1 5 10

<210> 472

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 472

Cys Gln Ala Ser Gln Asp Ile Ser Asn Phe Leu Asn Trp

1 5 10

<210> 473

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 473

Cys His Gln Tyr Asp Asn Leu Pro Arg Thr Phe

1 5 10

Claims

1. An antibody capable of binding to spike protein of coronavirus SARS-CoV-2, wherein the antibody:

(a) At least three CDRs comprising antibody 222 or any one of the 41 antibodies in table 1; and/or

(b) Binds to the same epitope as antibodies 159, 45 or 384 or competes with these antibodies.

2. The antibody of claim 1, comprising:

(a) At least four, five, or all six CDRs of an antibody in table 1;

(b) A heavy chain variable domain having at least 80% sequence identity to a heavy chain variable domain of an antibody in table 1;

(c) A light chain variable domain having at least 80% sequence identity to a light chain variable domain of an antibody in table 1; and/or

(d) A heavy chain variable domain and a light chain variable domain having at least 80% identity to the heavy chain variable domain and the light chain variable domain, respectively, of an antibody in table 1.

3. The antibody of claim 1 or claim 2, wherein the antibody in table 1 is:

(a) 222, 253H55L, 253H165L, 318, 253, 55, 165, 384, 159, 88, 40 or 316; or alternatively

(b) 58, 222, 253 or 253H55L.

4. The antibody according to any one of the preceding claims, comprising:

(a) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 having the amino acid sequences shown in SEQ ID NOs 265, 266, 267, 68, 69 and 70, respectively;

(b) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 having the amino acid sequences shown in SEQ ID NOs 265, 266, 267, 188, 189 and 190, respectively;

(c) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 having the amino acid sequences shown in SEQ ID NOs 255 to 260, respectively;

(d) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 having the amino acid sequences shown in SEQ ID NOs 335 to 340, respectively;

(e) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 having the amino acid sequences shown in SEQ ID NOs 65 to 70, respectively;

(f) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 having the amino acid sequences shown in SEQ ID NOs 185 to 190, respectively;

(g) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 having the amino acid sequences shown in SEQ ID NOs 265 to 270, respectively;

(h) CDRH1, CDRH2 and CDRH3 having the amino acid sequences shown in SEQ ID NOs 175 to 177, respectively;

(i) CDRH2, CDRH3, CDRL1 and CDRL3 having the amino acid sequences shown in SEQ ID NOs 376, 377, 378 and 380, respectively;

(j) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 having the amino acid sequences shown in SEQ ID NOs 105 to 110, respectively; or alternatively

(k) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, respectively, having the amino acid sequences shown in SEQ ID NOs 25 to 30.

5. The antibody according to claim 1 or 2, comprising a heavy chain variable domain amino acid sequence having at least 80% sequence identity to a heavy chain variable domain from a first antibody in table 1 and a light chain variable domain amino acid sequence having at least 80% sequence identity to a light chain variable domain from a second antibody in table 1, wherein the first and second antibodies are derived from the same germline heavy chain v region, optionally wherein the heavy chain v region is IGHV3-53, IGHV1-58 or IGHV3-66; for example, wherein:

(a) The first antibody is 150 and the second antibody is 222;

(b) The first antibody is 253 and the second antibody is 55;

(c) The first antibody is 253 and the second antibody is 165; or alternatively

(d) The second antibody is 222.

6. The antibody according to any one of the preceding claims, wherein the antibody binds to an epitope that:

(a) Defined by residues 144-147, 155-158 and 250-253 of the N-terminal domain of the spike protein of coronavirus SARS-CoV-2; or alternatively

(b) Defined by residues F104, L105, L455, F456 and G482 to F486 of the receptor binding domain of the spike protein of coronavirus SARS-CoV-2.

7. The antibody of claim 1 or claim 2, wherein the antibody in table 1 is 1, 88, 132, 253, 263, 316, 337, or 382, and wherein the N-glycosylation sequence of the antibody in table 1 is retained.

8. The antibody according to any one of the preceding claims, which is, for example, a full length antibody comprising an IgG1 constant region.

9. The antibody according to one of the preceding claims, comprising an Fc region comprising at least one modification such that serum half-life is prolonged.

10. An antibody combination comprising two or more antibodies according to any one of claims 1 to 9.

11. The antibody combination according to claim 10, comprising two, three or four antibodies according to any one of claims 1 to 9.

12. The antibody combination according to claim 10 or claim 11, comprising:

(a) A combination of two antibodies listed in the row of table 4;

(b) A combination of two antibodies listed in the row of table 5;

(c) Combinations of the three antibodies listed in the rows of table 4;

(d) A combination of two antibodies listed in the row of table 4 and antibody 159;

(e) A combination of two antibodies listed in the row of table 5 and antibody 159; or alternatively

(f) Three antibodies listed in the row of table 5, as well as combinations of antibodies 159.

13. One or more polynucleotides encoding the antibody of any one of claims 1 to 9, one or more vectors comprising said polynucleotides, or a host cell comprising said vectors.

14. A method for producing an antibody capable of binding to the spike protein of coronavirus SARS-CoV-2, the method comprising culturing the host cell of claim 13 and isolating the antibody from said culturing.

15. A pharmaceutical composition comprising: (a) The antibody of any one of claims 1 to 9 or the antibody combination of any one of claims 10 to 12, and (b) at least one pharmaceutically acceptable diluent or carrier.

16. The antibody according to any one of claims 1 to 9, the combination according to any one of claims 10 to 12 or the pharmaceutical composition according to claim 15 for use in a method of treatment of the human or animal body by therapy.

17. The antibody according to any one of claims 1 to 9, the combination according to any one of claims 10 to 12 or the pharmaceutical composition according to claim 15 for use in a method of treating or preventing a coronavirus infection or a disease or complication associated with a coronavirus infection.

18. A method of treating a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody according to any one of claims 1 to 9, a combination according to any one of claims 10 to 12, or a pharmaceutical composition according to claim 15.

19. The method according to claim 17 or 18, wherein the method is for treating SARS-CoV-2 infection or a disease or complication associated therewith.

20. A method of identifying the presence of a coronavirus or a protein or protein fragment thereof in a sample, the method comprising:

(i) Contacting the sample with an antibody according to any one of claims 1 to 9 or a combination according to any one of claims 10 to 12, and

(ii) Detecting the presence or absence of an antibody-antigen complex,

wherein the presence of the antibody-antigen complex is indicative of the presence of coronavirus or a protein or protein fragment thereof in the sample.

21. The method of claim 20, wherein the antibody is antibody 45 or the combination comprises antibody 45.

22. A method of treating or preventing a coronavirus infection or a disease or complication associated therewith in a subject, the method comprising identifying the presence of coronavirus in a sample according to the method of claim 20 or claim 21 and treating the subject with an antiviral or anti-inflammatory agent.

23. The antibody according to any one of claims 1 to 9, the combination according to any one of claims 10 to 12 or the pharmaceutical composition according to claim 15 for use in: (i) Preventing, treating and/or diagnosing a coronavirus infection or a disease or complication associated therewith, or (ii) identifying the presence of coronavirus or a protein or protein fragment thereof in the sample.

24. Use of an antibody according to any one of claims 1 to 9, a combination according to any one of claims 10 to 12 or a pharmaceutical composition according to claim 15 for the manufacture of a medicament for the treatment or prevention of a coronavirus infection or a disease or complication associated therewith.

25. The antibody according to any one of claims 1 to 9, the combination according to any one of claims 10 to 12 or the pharmaceutical composition according to claim 15, for use in a method of preventing, treating or diagnosing a coronavirus infection caused by a strain of SARS-CoV-2, the strain of SARS-CoV-2 comprising a substitution at positions 417, 484 and/or 501 in the spike protein relative to the spike protein of the strain of hCoV-19/Wuhan/WIV04/2019, e.g. it is a member of lineage b.1.1.7, b.1.351, p.1 or b.1.1.529.

26. A method of preventing, treating or diagnosing a coronavirus infection by a SARS-CoV-2 strain in a subject, wherein the method comprises administering to the subject an antibody according to any one of claims 1 to 9, a combination according to any one of claims 10 to 12 or a pharmaceutical composition according to claim 15, wherein the SARS-CoV-2 strain comprises a mutation at position 417, 484 and/or 501 in the spike protein relative to the spike protein of the hCoV-19/Wuhan/WIV04/2019 strain, e.g. it is a member of lineage b.1.1.7, b.1.351, p.1 or b.1.1.529.

27. Use of an antibody according to any one of claims 1 to 9, a combination according to any one of claims 10 to 12 or a pharmaceutical composition according to claim 15 for the manufacture of a medicament for the prevention, treatment or diagnosis of coronavirus infection caused by a strain of SARS-CoV-2, the strain of SARS-CoV-2 comprising a substitution at positions 417, 484 and/or 501 in the spike protein relative to the spike protein of the strain of hCoV-19/Wuhan/WIV04/2019, e.g. it is a member of lineage b.1.1.7, b.1.351, p.1 or b.1.1.529.