CN115315442B - SARS-COV-2 antibody and its application - Google Patents

SARS-COV-2 antibody and its application Download PDF

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CN115315442B
CN115315442B CN202080099668.XA CN202080099668A CN115315442B CN 115315442 B CN115315442 B CN 115315442B CN 202080099668 A CN202080099668 A CN 202080099668A CN 115315442 B CN115315442 B CN 115315442B
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CN115315442A (en
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顾春银
邓俗俊
王宗达
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Shanghai Jiyu Pharmaceutical Technology Co ltd
Jiangxi Jemincare Group Co Ltd
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Abstract

A SARS-CoV-2 antibody and its application are disclosed. The SARS-CoV-2 antibody can block the RBD of S protein of SARS-CoV-2 or the mutant thereof from combining with human ACE 2; blocking the binding of RBD of S protein of SARS-CoV to human ACE 2; at an extremely low K D An RBD or mutant thereof that specifically binds to the S protein of SARS-CoV-2; the affinity of RBD with S protein of SARS-CoV-2 is stronger than that of human ACE2-Fc; RBD that specifically binds to S protein of SARS-CoV; has hydrophilicity; and is beneficial to preparation and purification; the thermal stability is good; has the activity of neutralizing SARS-CoV-2. Also provided are methods of making and uses related to the SARS-CoV-2 antibodies.

Description

SARS-CoV-2 antibody and its application
Technical Field
The application relates to the field of biological medicine, in particular to a SARS-CoV-2 antibody and application thereof.
Background
The novel coronavirus (SARS-CoV-2) has long incubation period, high concealment and strong infectivity, and becomes a sudden public health event in the world health field. At the moment of such severe epidemic prevention situation, no specific medicine aiming at the novel coronavirus exists, so that the development of specific antibody medicine for the novel coronavirus epidemic prevention is urgent.
The novel coronavirus is an enveloped virus of positive RNA genome, belonging to the genus beta of the subgenera Nidovirales of the family coronaviridae. At present, a plurality of cases of clinically treating critical patients by utilizing specific plasma of recovered patients and having remarkable effects exist. However, after all, the sources of the plasma products are limited, and the plasma products can be used clinically through a strict blood biosafety detection method, and the current epidemic prevention and treatment requirements are not fully met.
Disclosure of Invention
The present application provides SARS-CoV-2 antibody and its application. The SARS-CoV-2 antibodies described herein can have one or more of the following properties: blocking the binding of RBD of S protein of SARS-CoV-2 or mutant thereof to human ACE 2; blocking the binding of RBD of S protein of SARS-CoV to human ACE 2; at an extremely low K D An RBD or mutant thereof that specifically binds to the S protein of SARS-CoV-2; the affinity of RBD with S protein of SARS-CoV-2 is stronger than that of human ACE2-Fc; RBD that specifically binds to S protein of SARS-CoV; has hydrophilicity; is beneficial to preparation and purification; the stability, especially the thermal stability is good; has the activity of neutralizing SARS-CoV-2. The application also provides a preparation method and application of the SARS-CoV-2 antibody.
In one aspect, the present application provides an isolated antigen binding protein having one or more of the following properties: blocking the binding of RBD of S protein of SARS-CoV-2 or mutant thereof to human ACE 2; blocking the binding of RBD of S protein of SARS-CoV to human ACE 2; in the Octet assay, about 5.0 x 10 -8 K below M D Value RBD that specifically binds to S protein of SARS-CoV-2; in the Octet assay, about 6.0 x 10 -10 K below M D Mutants of RBD that specifically bind to the S protein of SARS-CoV-2; the affinity of RBD with S protein of SARS-CoV-2 is stronger than that of human ACE2-Fc; RBD that specifically binds to S protein of SARS-CoV; hydrophilic; the charge heterogeneity analysis main peak is about 45% -85%; in a Thermal shift assay, the Tm is at least about 75deg.CThe method comprises the steps of carrying out a first treatment on the surface of the Has the activity of neutralizing SARS-CoV-2.
In certain embodiments, the isolated antigen binding protein comprises at least one CDR in a light chain variable region VL comprising the amino acid sequence of SEQ ID NO 124 or SEQ ID NO 125.
In certain embodiments, the isolated antigen binding protein comprises at least one CDR in a heavy chain variable region VH comprising an amino acid sequence as set forth in any one of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:17, SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:40, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:71 and SEQ ID NO: 73.
In certain embodiments, the isolated antigen binding protein comprises an antibody or antigen binding fragment thereof.
In certain embodiments, the antigen binding fragment comprises a Fab, fab ', F (ab) 2, fv fragment, F (ab') 2, scFv, di-scFv, and/or dAb.
In certain embodiments, the VL comprises LCDR1, LCDR2 and LCDR3, and the LCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NO: 122-123.
In certain embodiments, the LCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NO. 3, SEQ ID NO. 13, SEQ ID NO. 20, SEQ ID NO. 28, SEQ ID NO. 36, SEQ ID NO. 43, and SEQ ID NO. 61.
In certain embodiments, the LCDR1 comprises an amino acid sequence set forth in any one of SEQ ID NOS: 118-119.
In certain embodiments, the LCDR1 comprises an amino acid sequence set forth in any one of SEQ ID NO. 1, SEQ ID NO. 11, SEQ ID NO. 18, SEQ ID NO. 26, SEQ ID NO. 34, SEQ ID NO. 41, SEQ ID NO. 59, and SEQ ID NO. 111.
In certain embodiments, the LCDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOS: 120-121.
In certain embodiments, the LCDR2 comprises an amino acid sequence set forth in any one of SEQ ID NO. 2, SEQ ID NO. 12, SEQ ID NO. 19, SEQ ID NO. 27, SEQ ID NO. 35, SEQ ID NO. 42, SEQ ID NO. 60, and SEQ ID NO. 112.
In certain embodiments, the VH comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprising an amino acid sequence set forth in any one of SEQ ID NO 5, SEQ ID NO 22, SEQ ID NO 30, SEQ ID NO 45, SEQ ID NO 51, SEQ ID NO 55, SEQ ID NO 63 and SEQ ID NO 114.
In certain embodiments, the HCDR2 comprises an amino acid sequence set forth in any one of SEQ ID NO. 6, SEQ ID NO. 9, SEQ ID NO. 15, SEQ ID NO. 23, SEQ ID NO. 31, SEQ ID NO. 38, SEQ ID NO. 46, SEQ ID NO. 52, SEQ ID NO. 56, SEQ ID NO. 64, SEQ ID NO. 67, and SEQ ID NO. 115.
In certain embodiments, the HCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NO:7, SEQ ID NO:16, SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:39, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:65, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, and SEQ ID NO: 116.
In certain embodiments, the VL comprises framework regions L-FR1, L-FR2, L-FR3, and L-FR4.
In certain embodiments, the C-terminus of the L-FR1 is directly or indirectly linked to the N-terminus of the LCDR1, and the L-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NOS: 74-75.
In certain embodiments, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises an amino acid sequence set forth in any one of SEQ ID NO:76, SEQ ID NO:77, and SEQ ID NO: 78.
In certain embodiments, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises an amino acid sequence set forth in any one of SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, and SEQ ID NO: 82.
In certain embodiments, the N-terminus of the L-FR4 is directly or indirectly linked to the C-terminus of the LCDR3 and the L-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85 and SEQ ID NO: 86.
In certain embodiments, the VL comprises the amino acid sequence set forth in SEQ ID NO. 124 or SEQ ID NO. 125.
In certain embodiments, the VL comprises an amino acid sequence set forth in any one of SEQ ID NO. 4, SEQ ID NO. 14, SEQ ID NO. 21, SEQ ID NO. 29, SEQ ID NO. 37, SEQ ID NO. 44, SEQ ID NO. 62, and SEQ ID NO. 113.
In certain embodiments, the isolated antigen binding protein comprises an antibody light chain constant region, and the antibody light chain constant region comprises a human igκ constant region or a human igλ constant region.
In certain embodiments, the VH comprises framework regions H-FR1, H-FR2, H-FR3, and H-FR4.
In certain embodiments, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, and SEQ ID NO: 94.
In certain embodiments, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises an amino acid sequence set forth in any one of SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO: 98.
In certain embodiments, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises an amino acid sequence set forth in any one of SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, and SEQ ID NO: 106.
In certain embodiments, the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the HCDR3 and the H-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109 and SEQ ID NO: 110.
In certain embodiments, the VH comprises an amino acid sequence as set forth in any one of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:17, SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:40, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:71 and SEQ ID NO: 73.
In certain embodiments, the isolated antigen binding protein comprises an antibody heavy chain constant region, and the antibody heavy chain constant region comprises a human IgG constant region.
In certain embodiments, the isolated antigen binding protein comprises an antibody heavy chain constant region, and the antibody heavy chain constant region comprises a human IgG1 constant region.
In another aspect, the present application provides a bispecific antigen binding protein that specifically binds to the RBD of the S protein of SARS-CoV-2 and the RBD of the S protein of SARS-CoV.
In certain embodiments, the bispecific antigen binding protein comprises a first targeting moiety of an RBD that specifically binds to the S protein of SARS-CoV-2, wherein the first targeting moiety comprises an isolated antigen binding protein described herein.
In certain embodiments, the bispecific antigen binding protein comprises a second targeting moiety that specifically binds to RBD of S protein of SARS-CoV, wherein the second targeting moiety comprises an isolated antigen binding protein described herein.
In certain embodiments, the bispecific antigen binding protein comprises an antibody.
In certain embodiments, the first targeting moiety comprises a first heavy chain and a first light chain, and the second targeting moiety comprises a second heavy chain and a second light chain, wherein the first light chain and the second light chain are identical.
In certain embodiments, the first light chain and the second light chain comprise the amino acid sequence set forth in SEQ ID NO. 44.
In certain embodiments, the VH of the first heavy chain comprises the amino acid sequence shown in SEQ ID NO. 48.
In certain embodiments, the VH of the second heavy chain comprises the amino acid sequence shown as SEQ ID NO. 50.
In certain embodiments, the VH of the first heavy chain comprises the amino acid sequence shown in SEQ ID NO. 48 and the VH of the second heavy chain comprises the amino acid sequence shown in SEQ ID NO. 50.
In another aspect, the present application provides an isolated nucleic acid molecule or molecules encoding an isolated antigen binding protein described herein, and/or a bispecific antigen binding protein described herein.
In another aspect, the present application provides a vector comprising a nucleic acid molecule as described herein.
In another aspect, the present application provides a cell comprising a nucleic acid molecule described herein or a vector described herein.
In another aspect, the present application provides a method of making an isolated antigen binding protein described herein, a bispecific antigen binding protein described herein, comprising culturing a cell described herein under conditions such that the isolated antigen binding protein described herein and/or the bispecific antigen binding protein described herein is expressed.
In another aspect, the present application provides a pharmaceutical composition comprising an isolated antigen binding protein described herein, a bispecific antigen binding protein described herein, a nucleic acid molecule described herein, a vector described herein and/or a cell described herein, and optionally a pharmaceutically acceptable adjuvant.
In another aspect, the present application provides the use of an isolated antigen binding protein described herein, a bispecific antigen binding protein described herein, a nucleic acid molecule described herein, a vector described herein, a cell described herein and/or a pharmaceutical composition described herein in the manufacture of a medicament for preventing, alleviating and/or treating an infection by a coronavirus.
In certain embodiments, the infection with a coronavirus comprises a covd-19.
In another aspect, the present application provides a method of blocking the binding of an RBD of the S protein of SARS-CoV-2 or a mutant thereof to human ACE2 comprising the step of administering an isolated antigen binding protein described herein, a bispecific antigen binding protein described herein, a nucleic acid molecule described herein, a vector described herein, a cell described herein and/or a pharmaceutical composition described herein.
In another aspect, the present application provides a method of blocking the binding of RBD of S protein of SARS-CoV to human ACE2 comprising the step of administering an isolated antigen binding protein described herein, a bispecific antigen binding protein described herein, a nucleic acid molecule described herein, a vector described herein, a cell described herein and/or a pharmaceutical composition described herein.
Other aspects and advantages of the present application will become readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will recognize, the present disclosure enables one skilled in the art to make modifications to the disclosed embodiments without departing from the spirit and scope of the invention as described herein. Accordingly, the drawings and descriptions herein are to be regarded as illustrative in nature and not as restrictive.
Drawings
The specific features of the invention related to this application are set forth in the appended claims. The features and advantages of the invention that are related to the present application will be better understood by reference to the exemplary embodiments and the drawings that are described in detail below. The brief description of the drawings is as follows:
FIGS. 1A-1D show the results of detection of the rate of dissociation of the binding of an antibody described herein to SARS-CoV-2S 1.
FIG. 2 shows that the antibodies described herein block SARS-CoV-2S1 binding to hACE 2-Fc.
FIG. 3 shows that the antibodies described in the present application block SARS-CoV S1 binding to hACE 2.
FIG. 4 shows the hydrophilicity coefficients of the antibodies described herein.
FIG. 5 shows the results of the charge heterogeneity analysis of antibodies described herein.
FIG. 6 shows the results of the charge heterogeneity profile analysis of antibodies described herein.
FIG. 7 shows the melting temperature of antibodies (Fab) described herein.
FIGS. 8A-8B show the results of the antibody virus neutralization activity assay described herein.
FIGS. 9A-9B show the results of the antibody virus neutralization activity assay described herein.
FIG. 10 shows the results of half-maximal neutralization concentration determination of the antibodies described herein.
FIG. 11 shows the results of the detection of the rate of binding dissociation of the antibodies described in this application to each mutant of SARS-CoV-2S1 RBD.
FIGS. 12A-12E show curves and ICs of neutralizing antibodies described herein blocking SARS-CoV-2S1 RBD and its mutants binding to hACE2 50 Values.
Detailed Description
Further advantages and effects of the invention of the present application will become apparent to those skilled in the art from the disclosure of the present application, from the following description of specific embodiments.
Definition of terms
In this application, the term "SARS-CoV" generally refers to the SARS coronavirus, a severe acute respiratory syndrome coronavirus (all English called Severe acute respiratory syndrome coronavirus), which belongs to the genus Coronaviridae (Coronaviridae) B coronavirus (Betacorovirus) Sha Bei virus subgenera (Sarbecovirus).
In this application, the term "SARS-CoV-2" refers generally to severe acute respiratory syndrome coronavirus type 2, designated by the full English name Severe Acute Respiratory Syndrome Coronavirus 2.SARS-CoV-2 belongs to the Coronaviridae (Coronaviridae) genus B coronavirus (Betacorovirus) Sha Bei subgenera (Sarbecovirus). SARS-CoV-2 is a enveloped, non-segmented, positive-stranded single-stranded RNA virus. SARS-CoV-2 can cause a new form of coronavirus pneumonia (COVID-19). In the present application, the SARS-CoV-2 can comprise an S protein (spike protein).
In the present application, the term "S protein of coronavirus" generally refers to spike protein (spike protein) of coronaprotein. The S proteins can be combined into trimers, which contain about 1300 amino acids. The S protein may belong to a first class of membrane fusion proteins (Class I viral fusion protein). The S protein may typically contain two subunits (subt), S1 and S2. S1 mainly comprises a receptor binding region (receptor binding domain RBD), which may be responsible for recognizing the receptor of the cell. S2 contains the essential elements required for the membrane fusion process, including an intrinsic membrane fusion peptide (HR), two 7 peptide repeats (heptde repeat), an aromatic amino acid-rich membrane proximal region (membrane proximal external region, MPER), and a transmembrane region (TM). The S1 protein can be further divided into two regions (domains), namely an N-terminal domain (NTD) and a C-terminal domain (CTD). The S protein can determine the host range and specificity of the virus (e.g., coronavirus SARS-CoV-2), can also be an important site of action for neutralizing antibodies in the host, and/or can be a critical target for vaccine design. The S protein may be the S protein of SARS-CoV-2, for example, the structure of which can be found in Daniel Wrapp et al, cryo-EM structure of the 2019-nCoV spike in the prefusion conformation, science.
In the present application, the term "ACE2" generally refers to Angiotensin converting enzyme II (Angiotenin-converting enzyme 2) or a functional fragment thereof. The angiotensin converting enzyme II may catalyze the conversion of angiotensin I to angiotensin- (1-9) or angiotensin II to angiotensin- (1-7) exopeptidase. The ACE2 may include an N-terminal PD region (peptidase domain) and a C-terminal CLD region (collector-like domain). The angiotensin converting enzyme II may be a receptor for SARS coronavirus (SARS-CoV) or severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), e.g., the extracellular domain of ACE2 (e.g., the PD region of ACE 2) may bind RBD of the S protein of coronavirus. Human angiotensin converting enzyme II has accession number Q9BYF1 in the UniProt database. The human ACE2 gene may contain 18 exons, see Table 1 of Tipnis, S.R., hooper, N.M., hyde, R., karran, E., christie, G., turner, A.J. A human homolog of angiotensin-converting enzyme: cloning and functional expression as a captopril-fundamental carboxypepidase.J.biol.chem.275: 33238-33243,2000. In the present application, a functional fragment of the ACE2 protein may comprise a truncate or variant of the complete ACE2 protein, provided that the functional fragment still functions as a coronavirus (e.g. SARS-CoV and/or SARS-CoV-2) receptor.
In the present application, the term "ACE2-Fc" generally refers to a fusion protein comprising a binding protein of the S protein described herein or a functional fragment thereof and an Fc region of an IgG antibody. In this application, the binding protein of the S protein or a functional fragment thereof is directly or indirectly linked to the Fc region of the IgG antibody. For example, the Fc region of the IgG antibody may be located at the C-terminus of the ACE2-Fc fusion protein. In this application, the IgG may be IgG1, for example, human IgG1.
In this application, the term "Coronavirus" generally refers to a virus belonging to the genus Coronaviruse (Coronavirus) of the family Coronaviridae (Nidovirales) of the order Coronavirales. The coronavirus is a linear single-stranded positive strand RNA virus. The coronavirus may include an envelope having a spinous process. The genome of the coronavirus may have a methylated cap structure at the 5 'end and a poly (A) tail at the 3' end, the genome being approximately 27-32kb in length. In this application, the coronavirus includes severe acute respiratory syndrome-associated coronaviruses, i.e., severe acute respiratory syndrome-related coronavirus, which is a species of coronaviridae, genus b. In this application, the coronavirus may cause a cold as well as Middle East Respiratory Syndrome (MERS), severe Acute Respiratory Syndrome (SARS) and/or novel coronavirus pneumonia (covd-19).
In the present application, the term "mutant" generally refers to a mutated amino acid sequence by deletion, insertion or substitution of one or more selected amino acids. In the present application, the mutant can comprise an amino acid sequence that has at least about 90% (e.g., at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) identity as compared to the amino acid sequence of the RBD of the S protein of SARS-CoV-2. For example, the amino acid sequence of the RBD mutant of the S protein of SARS-CoV-2 can be found at RBD mutations from circulating SARS-CoV-2strains enhance the structure stability and infectivity of the spike protein,bioRxiv,2020.
In this application, the term "covd-19" generally refers to coronavirus disease 2019, which is a respiratory disease caused by the SARS-CoV-2 virus. Common symptoms of covd-19 include fever, cough, fatigue, shortness of breath, and loss of smell and taste, some of which may progress to viral pneumonia, multiple organ failure or cytokine storm. The disease is transmitted primarily upon intimate contact from person to person, for example, by small droplets produced by coughing, sneezing and speaking. The world health organization announced that the outbreak of COVID-19 was pandemic (pandemic) on day 3 and 11 of 2020. There is currently no vaccine or specific treatment available against covd-19.
In the present application, the term "antigen binding protein" generally refers to a protein comprising an antigen binding moiety, and optionally a scaffold or backbone moiety that allows the antigen binding moiety to adopt a conformation that facilitates binding of the antigen binding protein to an antigen. Examples of antigen binding proteins include, but are not limited to, antibodies, antigen binding fragments (Fab, fab', F (ab) 2 Fv fragment, F (ab') 2 scFv, di-scFv and/or dAb), immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., so long as they exhibit the desired antigen-binding activity.
In the present application, the term "Fab" generally refers to a fragment containing a heavy chain variable domain and a light chain variable domain, and also contains the constant domain of the light chain and the first constant domain of the heavy chain (CH 1); the term "Fab'" generally refers to a fragment that differs from Fab by the addition of a small number of residues (including one or more cysteines from the antibody hinge region) at the carboxy terminus of the heavy chain CH1 domain; the term "F (ab') 2 "generally refers to a dimer of Fab', an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region. The term "Fv" generally refers to the smallest antibody fragment that contains the complete antigen recognition and binding site. In some cases, the fragment may be composed of a heavy chain variable region and a light chain variable region to be closely non-consensus A valence-bound dimer composition; the term "dsFv" generally refers to disulfide stabilized Fv fragments in which the linkage between a single light chain variable region and a single heavy chain variable region is disulfide. The term "dAb fragment" generally refers to an antibody fragment consisting of a VH domain. In the present application, the term "scFv" generally refers to a monovalent molecule formed by the covalent linkage of one heavy chain variable domain and one light chain variable domain of an antibody to each other via a flexible peptide linker; such scFv molecules may have the general structure: NH (NH) 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH.
In this application, the term "antibody" is used in its broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies comprising two light chains and two heavy chains), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single domain antibodies. An "antibody" may generally comprise a protein of at least two Heavy Chains (HC) and two Light Chains (LC), or antigen-binding fragments thereof, interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region. In certain naturally occurring IgG, igD, and IgA antibodies, the heavy chain constant region comprises three domains, CH1, CH2, and CH3. In certain naturally occurring antibodies, each light chain comprises a light chain variable region (VL) and a light chain constant region. The light chain constant region comprises one domain, CL. VH and VL regions can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), alternating with regions of greater conservation termed Framework Regions (FR). Each VH and VL comprises three CDRs and four Framework Regions (FR), arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable domains of the natural heavy and light chains each comprise four FR regions (H-FR 1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR 4), mostly in the β -sheet configuration, connected by three CDRs, forming a loop connection, and in some cases forming part of a β -sheet structure. The CDRs in each chain are in close proximity by the FR region and form together with the CDRs from the other chain an antigen binding site of the antibody. The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).
In the present application, the term "variable" generally refers to the fact that certain parts of the sequence of the variable domain of an antibody vary strongly, which results in the binding and specificity of various specific antibodies for their specific antigens. However, variability is not evenly distributed throughout the variable regions of antibodies. It focuses on three segments in the light and heavy chain variable regions, known as Complementarity Determining Regions (CDRs) or hypervariable regions (HVRs). The more highly conserved parts in the variable domain are called Frameworks (FR). In the art, CDRs of antibodies can be defined by a variety of methods, such as Kabat definition rules based on sequence variability (see, kabat et al, immunological protein sequences, fifth edition, national institutes of health, besseda, maryland (1991)), chothia definition rules based on structural loop region position (see, A1-Lazikani et al, jmol Biol 273:927-48,1997), and KABAT definition rules based on the concept of the IMGT ONTOLOGY (IMGT-ONTOLOGY) and the rule of the IMGT Scientific chart.
IMGT refers to the International ImMunogenetics information System, a global reference database of ImMunoGeneTics and immunoinformatics (http:// www.imgt.org). IMGT specifically studies Immunoglobulins (IG) or antibodies, T cell receptors (TR), major Histocompatibility (MH) from humans and other vertebrates, as well as immunoglobulin superfamily (IgSF), MH superfamily (MhSF) and immune system Related Proteins (RPI) from vertebrates and non-vertebrates.
In the present application, the term "isolated" antigen binding protein generally refers to an antigen binding protein that has been recognized, isolated and/or recovered from components of its production environment (e.g., natural or recombinant). The environmental pollution components that they produce are typically substances that interfere with their research, diagnostic or therapeutic uses and may include enzymes, hormones and other proteinaceous or non-proteinaceous solutes. An isolated antigen binding protein or antibody will typically be prepared by at least one purification step.
In the present application, the term "monoclonal antibody" generally refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies in the population are identical except for the small number of natural mutations that may be present. Monoclonal antibodies are generally highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which typically have different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized by hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" refers to the characteristics of the antibody as obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies as used herein may be prepared in hybridoma cells or may be prepared by recombinant DNA methods.
In this application, the term "chimeric antibody" generally refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species. Typically, the variable region is derived from an antibody of an experimental animal such as a rodent ("parent antibody") and the constant region is derived from a human antibody such that the resulting chimeric antibody has a reduced likelihood of eliciting an adverse immune response in a human individual as compared to the parent (e.g., mouse-derived) antibody.
In the present application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR regions of a non-human antibody (e.g., a mouse antibody) are replaced with the corresponding amino acids derived from a human immunoglobulin. Small additions, deletions, insertions, substitutions or modifications of amino acids in the CDR regions may also be permissible, provided that they still retain the ability of the antibody to bind to a particular antigen. The humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region. "humanized antibodies" retain antigen specificity similar to the original antibody. A "humanized" form of a non-human (e.g., murine) antibody may minimally comprise chimeric antibodies derived from sequences of non-human immunoglobulins. In some cases, CDR region residues in a human immunoglobulin (recipient antibody) may be replaced with CDR region residues of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired properties, affinity and/or capability. In some cases, the FR region residues of the human immunoglobulin may be replaced with corresponding non-human residues. In addition, the humanized antibody may comprise amino acid modifications that are not in the recipient antibody or in the donor antibody. These modifications may be made to further improve the properties of the antibody, such as binding affinity.
In the present application, the term "fully human antibody" generally refers to an antibody expressed by an animal by transferring a gene encoding a human antibody into a genetically engineered antibody gene-deleted animal. All parts of an antibody (including the variable and constant regions of an antibody) are encoded by genes of human origin. The fully human antibody can greatly reduce the immune side reaction of the heterologous antibody to human body. Methods for obtaining fully human antibodies in the art can include phage display technology, transgenic mouse technology, ribosome display technology, RNA-polypeptide technology, and the like.
In this application, the terms "binding," "specific binding," or "specific for …" generally refer to a measurable and reproducible interaction, such as binding between an antigen and an antibody, which can determine the presence of a target in the presence of a heterogeneous population of molecules (including biological molecules). For example, an antibody binds to an epitope through its antigen binding domain, and this binding requires some complementarity between the antigen binding domain and the epitope. For example, an antibody that specifically binds to a target (which may be an epitope) is one that binds to that target with greater affinity, avidity, more readily, and/or for a greater duration than it binds to other targets. An antibody is said to "specifically bind" to an epitope when it will bind to the epitope more readily through its antigen binding domain than it will bind to a random, unrelated epitope. An "epitope" refers to a particular atomic group (e.g., sugar side chain, phosphoryl, sulfonyl) or amino acid on an antigen that binds to an antigen binding protein (e.g., an antibody).
In the present application, the terms "KD", "K D "interchangeably used, generally refers to an equilibrium dissociation constant," KD "is the dissociation rate constant (kdis), also known as" off-rate "or" KD ")Ratio to the binding rate constant (kon, also known as "binding rate (kon)" or "ka"). Binding rate constant (kon), dissociation rate constant (kdis) and equilibrium dissociation constant (K) can be used D ) Represents the binding affinity of an antigen binding protein (e.g., an antibody) to an antigen. Methods for determining the association and dissociation rate constants are well known in the art and include, but are not limited to, biofilm interference techniques (BLI), radioimmunoassay (RIA), equilibrium dialysis, surface Plasmon Resonance (SPR), fluorescence Resonance Energy Transfer (FRET), co-immunoprecipitation (Co-IP) and protein chip techniques. If measured under different conditions (e.g., salt concentration, pH), the affinity of a particular protein-protein interaction measured may be different.
In the present application, the term "reference antibody" generally refers to an antibody with which the antigen binding proteins described herein compete for binding to an antigen (e.g., RBD of S protein of SARS-CoV-2).
In this application, the term "CDC" generally refers to a process initiated by the binding of complement factor C1q to the Fc portion of most IgG antibody subclasses. Binding of C1q to the antibody may be caused by defined protein-protein interactions of the binding site of the Fc portion. The binding sites of these Fc portions may comprise amino acids L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbering according to EU index of Kabat). Antibodies of the IgG1, igG2, and IgG3 subtypes may generally exhibit complement activation including C1q and C3 binding, while IgG4 does not activate the complement system and may not bind to C1q and/or C3.
In the present application, the term "ADCC" or "antibody-dependent cell-mediated cytotoxicity" refers generally to a form of cytotoxicity in which some secreted immunoglobulins bind to Fc receptors (fcrs) on certain cytotoxic effector cells (e.g., NK cells, neutrophils and macrophages) so that these cytotoxic effector cells are able to specifically bind to antigen-bearing target cells, which are then killed with cytotoxins. Major cells mediating ADCC, such as NK cells, express fcyriii only, whereas monocytes express fcyri, fcyrii and feyriii (see Ravetch and Kinet, annu. Rev. Immunol.9:457-92 (1991) page table 464, 3). In vitro and/or in vivo cytotoxicity assays may be performed to assess ADCC activity of the molecule of interest, e.g., in vitro ADCC assays may be performed as described in U.S. Pat. No.5,500,362 or No.5,821,337 or U.S. Pat. No.6,737,056 (Presta). Effector cells useful in such assays include PBMCs and NK cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo, for example in an animal model, such as that disclosed in Clynes et al, PNAS (USA) 95:652-656 (1998). For example, an Fc receptor (FcR) binding assay may be performed to ensure that the antibody lacks fcγr binding (and thus potentially ADCC activity), but retains FcRn binding capacity.
ADCC activity may be reduced by modification of the Fc region. In some cases, the sites that affect binding to Fc receptors may be removed, e.g., sites that are not salvage receptor binding sites. In some cases, the Fc region may be modified to remove ADCC sites. ADCC sites are known in the art, see, e.g., sarama et al (1992) molecular immunol.29 (5): 633-9 for ADCC sites for IgG 1.
In the present application, the term "between … …" generally means that the C-terminus of a certain amino acid fragment is directly or indirectly linked to the N-terminus of a first amino acid fragment, and that its N-terminus is directly or indirectly linked to the C-terminus of a second amino acid fragment. In the light chain, for example, the N-terminus of the L-FR2 is directly or indirectly linked to the C-terminus of the LCDR1, and the C-terminus of the L-FR2 is directly or indirectly linked to the N-terminus of the LCDR 2. For another example, the N-terminus of the L-FR3 is directly or indirectly linked to the C-terminus of the LCDR2, and the C-terminus of the L-FR3 is directly or indirectly linked to the N-terminus of the LCDR 3. In the heavy chain, for example, the N-terminus of the H-FR2 is directly or indirectly linked to the C-terminus of the HCDR1, and the C-terminus of the H-FR2 is directly or indirectly linked to the N-terminus of the HCDR 2. For another example, the N-terminus of the H-FR3 is directly or indirectly linked to the C-terminus of the HCDR2, and the C-terminus of the H-FR3 is directly or indirectly linked to the N-terminus of the HCDR 3. In the present application, the "first amino acid fragment" and the "second amino acid fragment" may be any one of the same or different amino acid fragments.
In the present application, the term "isolated" antigen binding protein generally refers to an antigen binding protein that has been recognized, isolated and/or recovered from components of its production environment (e.g., natural or recombinant). The environmental pollution components that they produce are typically substances that interfere with their research, diagnostic or therapeutic uses and may include enzymes, hormones and other proteinaceous or non-proteinaceous solutes. An isolated antigen binding protein or antibody will typically be prepared by at least one purification step.
In this application, the term "isolated nucleic acid molecule" or "isolated polynucleotide" refers generally to DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or a certain combination thereof, that is not associated with all or a portion of a polynucleotide found in nature, or that is linked to a polynucleotide to which it is not linked in nature.
In the present application, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells. The vector may include a vector mainly used for inserting DNA or RNA into a cell, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of transcription and/or translation of DNA or RNA. The carrier also includes a carrier having a plurality of functions as described above. The vector may be a polynucleotide capable of transcription and translation into a polypeptide when introduced into a suitable host cell. Typically, the vector will produce the desired expression product by culturing a suitable host cell comprising the vector.
In the present application, the term "cell" generally refers to an individual cell, cell line or cell culture that may or has contained a plasmid or vector comprising a nucleic acid molecule as described herein, or that is capable of expressing an antibody or antigen binding fragment thereof as described herein. The cell may comprise progeny of a single host cell. The daughter cells may not necessarily be identical in morphology or in genome to the original parent cells due to natural, unexpected or deliberate mutation, but are capable of expressing the antibodies or antigen-binding fragments thereof described herein. The cells may be obtained by transfecting the cells in vitro using the vectors described herein. The cells may be prokaryotic cells (e.g., E.coli) or eukaryotic cells (e.g., yeast cells, e.g., COS cells, chinese Hamster Ovary (CHO) cells, heLa cells, HEK293 cells, COS-1 cells, NS0 cells, or myeloma cells). In some cases, the cell may be a mammalian cell. For example, the mammalian cell may be a CHO-K1 cell. In the present application, the term "recombinant cell" generally refers to a cell into which a recombinant expression vector is introduced. The recombinant host cell includes not only a particular cell but also the progeny of such a cell.
In this application, the term "pharmaceutically acceptable adjuvant" generally includes pharmaceutically acceptable carriers, excipients or stabilizers which are non-toxic to the cells or mammals to which they are exposed at the dosages and concentrations employed. Typically, the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers can include buffers, antioxidants, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming counter-ionic and/or nonionic surfactants.
As used herein, the terms "administering" and "treating" refer to the application of an exogenous drug, therapeutic, diagnostic, or composition to an animal, human, subject, cell, tissue, organ, or biological fluid. "administration" and "treatment" may refer to therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of a cell includes contacting a reagent with the cell, contacting a reagent with a fluid, and contacting a fluid with the cell. "administration" and "treatment" also mean in vitro and ex vivo treatment by an agent, diagnosis, binding composition, or by another cell. "treatment" when applied to a human, animal or study subject refers to therapeutic treatment, prophylactic or preventative measures, study and diagnosis; including contacting a coronavirus (e.g., SARS-CoV-2) with a human or animal, subject, cell, tissue, physiological compartment, or physiological fluid.
As used herein, the term "treatment" refers to the administration of an internally or externally used therapeutic agent, including any of the SARS-CoV-2 antigen binding proteins of the present application, and compositions thereof, to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the patient is administered an amount of the therapeutic agent (therapeutically effective amount) effective to alleviate one or more symptoms of the disease. Desirable effects of treatment include reducing the rate of disease progression, improving or alleviating the disease state, and regression or improved prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with cancer are reduced or eliminated, including, but not limited to, reducing (or destroying) cancer cell proliferation, reducing symptoms derived from the disease, improving the quality of life of those individuals suffering from the disease, reducing the dosage of other drugs required to treat the disease, delaying the progression of the disease, and/or prolonging survival of the individual.
In this application, the terms "comprises," "comprising," and "includes" are used in their plain, inclusive, and open-ended meaning. In some cases, the meaning of "as", "consisting of … …" is also indicated.
In this application, the term "about" generally means ranging from 0.5% to 10% above or below the specified value, e.g., ranging from 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.
Detailed Description
Antigen binding proteins
In one aspect, the present application provides an isolated antigen binding protein having one or more of the following properties:
blocking the binding of RBD of S protein of SARS-CoV-2 or mutant thereof to human ACE 2;
blocking the binding of RBD of S protein of SARS-CoV to human ACE 2;
in the Octet assay, about 5.0 x 10 -8 M or less (e.g., may be about 5.0 x 10 -8 M or less, about 2.0 x 10 -8 M or less, about 1.0 x 10 -8 Below M, about 9.0 x 10 -9 Below M, about 8.0 x 10 -9 Below M, about 7.0 x 10 -9 M or less, about 6.0 x 10 -9 Below M, about 5.0 x 10 -9 M is less than or equal to,About 4.0 x 10 -9 M or less, about 3.0 x 10 -9 M or less, about 2.0 x 10 -9 M or less, about 1.0 x 10 -10 Below M, about 9.0 x 10 -10 Below M, about 8.0 x 10 -10 Below M, about 7.0 x 10 -10 M or less, about 6.0 x 10 -10 Below M, about 5.0 x 10 -10 Below M, about 4.0 x 10 -10 M or less, about 3.0 x 10 -10 M or less, about 2.0 x 10 -10 M or less or about 1.0 x 10 -10 M or less) K D Value RBD that specifically binds to S protein of SARS-CoV-2;
in the Octet assay, about 6.0 x 10 -10 M or less (e.g., may be about 6.0 x 10 -10 Below M, about 5.5 x 10 - 10 Below M, about 5.0 x 10 -10 Below M about 4.5 x 10 -10 Below M, about 4.0 x 10 -10 M or less, about 3.5 x 10 -10 M or less, about 3.0 x 10 -10 Below M, about 2.5 x 10 -10 M or less, about 2.0 x 10 -10 M or less or about 1.5 x 10 -10 M or less) K D Mutants of RBD that specifically bind to the S protein of SARS-CoV-2;
the affinity of RBD with S protein of SARS-CoV-2 is stronger than that of human ACE2-Fc; RBD that specifically binds to S protein of SARS-CoV;
has hydrophilicity;
the charge heterogeneity analysis major peak is about 45% -85% (e.g., may be about 45% -about 85%, about 45% -about 84%, about 45% -about 83%, about 45% -about 81%, about 45% -about 80%, about 45% -about 79%, about 45% -about 78%, about 45% -about 77%, about 45% -about 76%, about 45% -about 75%, or about 45% -about 74%);
in a Thermal shift assay, the Tm is at least about 75 ℃ (e.g., can be at least about 75 ℃, at least about 76 ℃, at least about 77 ℃, at least about 78 ℃, at least about 79 ℃, at least about 80 ℃, at least about 81 ℃, at least about 82 ℃, at least about 83 ℃, or at least about 84 ℃);
has the activity of neutralizing SARS-CoV-2.
The isolated antigen binding protein described herein is capable of competing with a reference antibody for the RBD of the S protein that binds SARS-CoV-2, wherein the reference antibody may comprise a heavy chain variable region and a light chain variable region, the heavy chain variable region of the reference antibody may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 5, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO. 6, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 7, the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 1, the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO. 2, and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 3.
The isolated antigen binding protein described herein is capable of competing with a reference antibody for the RBD that binds to the S protein of SARS-CoV-2, wherein the reference antibody may comprise a heavy chain variable region and a light chain variable region, the heavy chain variable region of the reference antibody may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence of SEQ ID NO:5, the HCDR2 may comprise the amino acid sequence of SEQ ID NO:9, and the HCDR3 may comprise the amino acid sequence of SEQ ID NO:7, the LCDR1 may comprise the amino acid sequence of SEQ ID NO:1, the LCDR2 may comprise the amino acid sequence of SEQ ID NO:2, and the LCDR3 may comprise the amino acid sequence of SEQ ID NO: 3.
The isolated antigen binding protein described herein is capable of competing with a reference antibody for the RBD of the S protein that binds SARS-CoV-2, wherein the reference antibody may comprise a heavy chain variable region and a light chain variable region, the heavy chain variable region of the reference antibody may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 5, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO. 15, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 16, the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 11, the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO. 12, and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 13.
The isolated antigen binding protein described herein is capable of competing with a reference antibody for the RBD of the S protein that binds SARS-CoV-2, wherein the reference antibody may comprise a heavy chain variable region and a light chain variable region, the heavy chain variable region of the reference antibody may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:22, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:23, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:24, the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:18, the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:19, and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 20.
The isolated antigen binding protein described herein is capable of competing with a reference antibody for binding to the RBD of the S protein of SARS-CoV-2, wherein said reference antibody may comprise a heavy chain variable region and a light chain variable region, said reference antibody heavy chain variable region may comprise HCDR1, HCDR2 and HCDR3, said HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:30, said HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:31, and said HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:32, said LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:26, said LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:27, and said LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 28.
The isolated antigen binding protein described herein is capable of competing with a reference antibody for binding to an RBD of an S protein of SARS-CoV-2, wherein the reference antibody may comprise a heavy chain variable region and a light chain variable region, the heavy chain variable region of the reference antibody may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence of SEQ ID NO:30, the HCDR2 may comprise the amino acid sequence of SEQ ID NO:38, and the HCDR3 may comprise the amino acid sequence of SEQ ID NO:39, the LCDR1 may comprise the amino acid sequence of SEQ ID NO:34, the LCDR2 may comprise the amino acid sequence of SEQ ID NO:35, and the LCDR3 may comprise the amino acid sequence of SEQ ID NO: 36.
The isolated antigen binding protein described herein is capable of competing with a reference antibody for binding to an RBD of an S protein of SARS-CoV-2, wherein the reference antibody may comprise a heavy chain variable region and a light chain variable region, the heavy chain variable region of the reference antibody may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence of SEQ ID No. 45, the HCDR2 may comprise the amino acid sequence of SEQ ID No. 46, and the HCDR3 may comprise the amino acid sequence of SEQ ID No. 47, the LCDR1 may comprise the amino acid sequence of SEQ ID No. 41, the LCDR2 may comprise the amino acid sequence of SEQ ID No. 42, and the LCDR3 may comprise the amino acid sequence of SEQ ID No. 43.
The isolated antigen binding protein described herein is capable of competing with a reference antibody for binding to the RBD of the S protein of SARS-CoV-2, wherein said reference antibody may comprise a heavy chain variable region and a light chain variable region, said reference antibody heavy chain variable region may comprise HCDR1, HCDR2 and HCDR3, said HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 5, said HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO. 9, and said HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 49, said LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 41, said LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO. 42, and said LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 43.
The isolated antigen binding protein described herein is capable of competing with a reference antibody for binding to the RBD of the S protein of SARS-CoV-2, wherein said reference antibody may comprise a heavy chain variable region and a light chain variable region, said reference antibody heavy chain variable region may comprise HCDR1, HCDR2 and HCDR3, said HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:51, said HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:52, and said HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:53, said LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:41, said LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:42, and said LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 43.
The isolated antigen binding protein described herein is capable of competing with a reference antibody for binding to the RBD of the S protein of SARS-CoV-2, wherein said reference antibody may comprise a heavy chain variable region and a light chain variable region, said reference antibody heavy chain variable region may comprise HCDR1, HCDR2 and HCDR3, said HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:55, said HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:56, and said HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:57, said LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:41, said LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:42, and said LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 43.
The isolated antigen binding protein described herein is capable of competing with a reference antibody for the RBD of the S protein that binds SARS-CoV-2, wherein the reference antibody may comprise a heavy chain variable region and a light chain variable region, the heavy chain variable region of the reference antibody may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:63, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:64, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:65, the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:59, the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:60, and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 61.
The isolated antigen binding protein described herein is capable of competing with a reference antibody for binding to the RBD of the S protein of SARS-CoV-2, wherein said reference antibody may comprise a heavy chain variable region and a light chain variable region, said reference antibody heavy chain variable region may comprise HCDR1, HCDR2 and HCDR3, said HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 5, said HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO. 67, and said HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 68, said LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 41, said LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO. 42, and said LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 43.
The isolated antigen binding protein described herein is capable of competing with a reference antibody for binding to an RBD of an S protein of SARS-CoV-2, wherein the reference antibody may comprise a heavy chain variable region and a light chain variable region, the heavy chain variable region of the reference antibody may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence of SEQ ID NO:55, the HCDR2 may comprise the amino acid sequence of SEQ ID NO:56, and the HCDR3 may comprise the amino acid sequence of SEQ ID NO:70, the LCDR1 may comprise the amino acid sequence of SEQ ID NO:41, the LCDR2 may comprise the amino acid sequence of SEQ ID NO:42, and the LCDR3 may comprise the amino acid sequence of SEQ ID NO: 43.
The isolated antigen binding protein described herein is capable of competing with a reference antibody for binding to the RBD of the S protein of SARS-CoV-2, wherein said reference antibody may comprise a heavy chain variable region and a light chain variable region, said reference antibody heavy chain variable region may comprise HCDR1, HCDR2 and HCDR3, said HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:55, said HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:56, and said HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:72, said LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:41, said LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:42, and said LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 43.
In this application, the isolated antigen binding protein may comprise at least one CDR in a light chain variable region VL which may comprise the amino acid sequence shown as SEQ ID NO. 124 or SEQ ID NO. 125.
In this application, the isolated antigen binding protein may comprise at least one CDR in a heavy chain variable region VH that may comprise the amino acid sequence shown in any one of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:17, SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:40, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:71 and SEQ ID NO: 73.
For example, the isolated antigen binding protein may comprise an antibody or antigen binding fragment thereof.
For example, the antigen binding fragment can comprise a Fab, fab ', F (ab) 2, fv fragment, F (ab') 2, scFv, di-scFv, and/or dAb.
In this application, the VL may comprise LCDR1, LCDR2 and LCDR3, and the LCDR3 may comprise an amino acid sequence set forth in any one of SEQ ID NOS: 122-123.
For example, wherein the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 122: QQSYSTPX 8 X 9 X 10 Wherein X is 8 Is Pro or Ser, X 9 Is Ile or Thr, X 10 Is Thr or absent. For example, the sequence may be according to KABThe AT defines a sequence determined by the rule.
For example, wherein the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 123; QQYX 4 X 5 X 6 PX 8 T, where X 4 Asp or Gly, X 5 Is Asn or Ser, X 6 Leu or Ser, X 8 Ile, leu, gln or Val. For example, the sequence may be a sequence determined according to KABAT definition rules.
For example, the LCDR3 may comprise an amino acid sequence set forth in any one of SEQ ID NO. 3, SEQ ID NO. 13, SEQ ID NO. 20, SEQ ID NO. 28, SEQ ID NO. 36, SEQ ID NO. 43, and SEQ ID NO. 61.
For example, the LCDR1 may comprise an amino acid sequence set forth in any one of SEQ ID NOS: 118-119.
For example, wherein the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 118: RASQX 5 ISX 8 X 9 LX 11 Wherein X is 5 Gly or Ser, X 8 Is Asn or Ser, X 9 Is Ser or Tyr, X 11 Is Ala or Asn. For example, the sequence may be a sequence determined according to KABAT definition rules.
For example, wherein the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 119; x is X 1 ASQX 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 Wherein X is 1 Is Gln or Arg, X 5 Is Asp, gly or Ser, X 6 Is Ile or Val, X 7 Is Asn or Ser, X 8 Gly, asn or Ser, X 9 Is Ser, trp or Tyr, X 10 Is Leu or Tyr, X 11 Is Ala, leu or Asn, X 12 Either Ala or absent. For example, the sequence may be a sequence determined according to KABAT definition rules.
For example, the LCDR1 may comprise an amino acid sequence set forth in any one of SEQ ID NO. 1, SEQ ID NO. 11, SEQ ID NO. 18, SEQ ID NO. 26, SEQ ID NO. 34, SEQ ID NO. 41, SEQ ID NO. 59, and SEQ ID NO. 111.
For example, the LCDR2 may comprise an amino acid sequence set forth in any one of SEQ ID NOS: 120-121.
For example, wherein the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO. 120: AASX 4 LX 6 S, wherein X 4 Arg or Ser, X 6 Is Glu or Gln. For example, the sequence may be a sequence determined according to KABAT definition rules.
For example, wherein the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 121; x is X 1 ASX 4 X 5 X 6 T, where X 1 Is Ala, asp or Gly, X 4 Is Asn, ser or Thr, X 5 Leu or Arg, X 6 Is Ala or Glu. For example, the sequence may be a sequence determined according to KABAT definition rules.
For example, the LCDR2 may comprise an amino acid sequence set forth in any one of SEQ ID NO. 2, SEQ ID NO. 12, SEQ ID NO. 19, SEQ ID NO. 27, SEQ ID NO. 35, SEQ ID NO. 42, SEQ ID NO. 60, and SEQ ID NO. 112.
For example, the VH may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1 may comprise an amino acid sequence as set forth in any one of SEQ ID NO 5, SEQ ID NO 22, SEQ ID NO 30, SEQ ID NO 45, SEQ ID NO 51, SEQ ID NO 55, SEQ ID NO 63 and SEQ ID NO 114.
For example, the HCDR2 can comprise an amino acid sequence set forth in any one of SEQ ID NO. 6, SEQ ID NO. 9, SEQ ID NO. 15, SEQ ID NO. 23, SEQ ID NO. 31, SEQ ID NO. 38, SEQ ID NO. 46, SEQ ID NO. 52, SEQ ID NO. 56, SEQ ID NO. 64, SEQ ID NO. 67, and SEQ ID NO. 115.
For example, the HCDR3 may comprise an amino acid sequence set forth in any one of SEQ ID NO:7, SEQ ID NO:16, SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:39, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:65, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, and SEQ ID NO: 116.
For example, the VL may comprise framework regions L-FR1, L-FR2, L-FR3, and L-FR4.
For example, the C-terminus of the L-FR1 can be directly or indirectly linked to the N-terminus of the LCDR1, and the L-FR1 can comprise the amino acid sequence set forth in any one of SEQ ID NOS: 74-75.
For example, the L-FR2 can be located between the LCDR1 and the LCDR2, and the L-FR2 can comprise an amino acid sequence set forth in any one of SEQ ID NO:76, SEQ ID NO:77, and SEQ ID NO: 78.
For example, the L-FR3 can be located between the LCDR2 and the LCDR3, and the L-FR3 can comprise an amino acid sequence set forth in any one of SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, and SEQ ID NO: 82.
For example, the N-terminus of the L-FR4 can be directly or indirectly linked to the C-terminus of the LCDR3, and the L-FR4 can comprise the amino acid sequence set forth in any one of SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, and SEQ ID NO: 86.
In this application, the VL may comprise the amino acid sequence shown as SEQ ID NO. 124 or SEQ ID NO. 125.
For example, wherein the VL may comprise the amino acid sequence shown in SEQ ID NO. 124: DIQQTQSPSSLSASVGDRVTITCRASQX 28 ISX 31 X 32 LX 34 WYQQKPGKAPKLLX 48 YAASX 53 LX 55 SGVPSRFSGSGSGTDX 71 TLTISSLQPEDFATYYCQQSYSTPX 96 X 97 TFGQGTX 104 X 105 EIK, where X 28 Gly or Ser, X 31 Is Asn or Ser, X 32 Is Ser or Tyr, X 34 Is Ala or Asn, X 48 Is Ile or Leu, X 53 Arg or Ser, X 55 Is Glu or Gln, X 71 Is Phe or Tyr, X 96 Is Pro or Ser, X 97 For Ile or absent, X 104 Is Lys or Arg, X 105 Leu or Val. For example, the sequence may be a sequence determined according to KABAT definition rules.
For example, wherein the VL may comprise the amino acid sequence shown as SEQ ID NO. 125; x is X 1 IX 3 X 4 TQSPX 9 X 10 LSX 13 SX 15 GX 17 RX 19 TX 21 X 22 CX 24 ASQX 28 X 29 X 30 X 31 X 32 X 33 LX 35 WYQQKPG X 43 APX 46 LLIYX 51 ASX 54 X 55 X 56 TGX 5 9 PX 61 RFSGSGSGTDFTX 74 TISX 78 LX 80 PEDX 84 AX 86 YYCQ QYX 93 X 94 X 95 PX 97 TFGX 101 GTX 104 X 105 EIK, where X 1 Is Asp or Glu, X 3 Is Gln or Val, X 4 Is Leu or Met, X 9 Gly or Ser, X 10 Is Ser or Thr, X 13 Is Ala or Leu, X 15 Is Pro or Val, X 17 Is Asp or Glu, X 19 Ala or Val, X 21 Is Ile or Leu, X 22 Is Ser or Thr, X 24 Is Gln or Arg, X 28 Is Asp, gly or Ser, X 29 Is Ile or Val, X 30 Is Asn or Ser, X 31 Gly, asn or Ser, X 32 For Ser or absent, X 33 Is Trp or Tyr, X 35 Is Ala or Asn, X 43 Is Lys or Gln, X 46 Is Lys or Arg, X 51 Is Ala, asp or Gly, X 54 Is Asn, ser or Thr, X 55 Leu or Arg, X 56 Is Ala or Glu, X 59 Is Ile or Val, X 61 Is Asp or Ser, X 74 Is Phe or Leu, X 78 Arg or Ser, X 80 Is Glu or Gln, X 84 Is Phe or Ile, X 86 Thr or Val, X 93 Asp or Gly, X 94 Is Asn or Ser, X 95 Leu or Ser, X 97 Ile, leu, gln or Val, X 101 Is Gly or Gln, X 104 Is Lys or Arg, X 105 Leu or Val. For example, the sequence may be a sequence determined according to KABAT definition rules.
For example, the VL may comprise an amino acid sequence set forth in any one of SEQ ID NO. 4, SEQ ID NO. 14, SEQ ID NO. 21, SEQ ID NO. 29, SEQ ID NO. 37, SEQ ID NO. 44, SEQ ID NO. 62, and SEQ ID NO. 113.
For example, the isolated antigen binding protein may comprise an antibody light chain constant region, and the antibody light chain constant region comprises a human igκ constant region or a human igλ constant region.
In this application, the gene encoding the human igκ constant region may be as shown in GenBank accession number 50802 of NCBI database; the gene encoding the human Ig lambda constant region may be as shown in GenBank accession No. 3535 of NCBI database.
For example, the VH can include framework regions H-FR1, H-FR2, H-FR3, and H-FR4.
For example, the C-terminus of the H-FR1 can be directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 can comprise the amino acid sequence set forth in any one of SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, and SEQ ID NO: 94.
For example, the H-FR2 can be located between the HCDR1 and the HCDR2, and the H-FR2 can comprise an amino acid sequence set forth in any one of SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO: 98.
For example, the H-FR3 can be located between the HCDR2 and the HCDR3, and the H-FR3 can comprise an amino acid sequence set forth in any one of SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, and SEQ ID NO: 106.
For example, the N-terminus of the H-FR4 can be directly or indirectly linked to the C-terminus of the HCDR3, and the H-FR4 can comprise the amino acid sequence set forth in any one of SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, and SEQ ID NO: 110.
For example, the VH may comprise an amino acid sequence as set out in any one of SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 17, SEQ ID NO. 25, SEQ ID NO. 33, SEQ ID NO. 40, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 54, SEQ ID NO. 58, SEQ ID NO. 66, SEQ ID NO. 69, SEQ ID NO. 71 and SEQ ID NO. 73.
For example, the isolated antigen binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region comprises a human IgG constant region.
For example, the isolated antigen binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region comprises a human IgG1 constant region.
In this application, the gene encoding the human IgG1 constant region may be as shown in GenBank accession number 3500 of NCBI database.
In this application, the isolated antigen binding protein may comprise antibody light chain variable region CDRs, LCDR1, LCDR2, and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID No. 1, the LCDR2 may comprise the amino acid sequence shown in SEQ ID No. 2, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID No. 3.
In this application, the isolated antigen binding protein may comprise antibody light chain variable region CDRs, LCDR1, LCDR2, and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID No. 11, the LCDR2 may comprise the amino acid sequence shown in SEQ ID No. 12, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID No. 13.
In this application, the isolated antigen binding protein may comprise antibody light chain variable region CDRs, LCDR1, LCDR2, and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID No. 18, the LCDR2 may comprise the amino acid sequence shown in SEQ ID No. 19, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID No. 20.
In this application, the isolated antigen binding protein may comprise the antibody light chain variable region CDRs, LCDR1, LCDR2, and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID No. 26, the LCDR2 may comprise the amino acid sequence shown in SEQ ID No. 27, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID No. 28.
In this application, the isolated antigen binding protein may comprise antibody light chain variable region CDRs, LCDR1, LCDR2, and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID No. 34, the LCDR2 may comprise the amino acid sequence shown in SEQ ID No. 35, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID No. 36.
In this application, the isolated antigen binding protein may comprise antibody light chain variable region CDRs, LCDR1, LCDR2, and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID No. 41, the LCDR2 may comprise the amino acid sequence shown in SEQ ID No. 42, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID No. 43.
In this application, the isolated antigen binding protein may comprise antibody light chain variable region CDRs, LCDR1, LCDR2, and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID No. 59, the LCDR2 may comprise the amino acid sequence shown in SEQ ID No. 60, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID No. 61.
In this application, the isolated antigen binding protein may comprise antibody heavy chain variable region CDRs, HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence of SEQ ID No. 5, the HCDR2 may comprise the amino acid sequence of SEQ ID No. 6, and the HCDR3 may comprise the amino acid sequence of SEQ ID No. 7.
In this application, the isolated antigen binding protein may comprise antibody heavy chain variable region CDRs, HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence of SEQ ID No. 5, the HCDR2 may comprise the amino acid sequence of SEQ ID No. 9, and the HCDR3 may comprise the amino acid sequence of SEQ ID No. 7.
In this application, the isolated antigen binding protein may comprise antibody heavy chain variable region CDRs, HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence of SEQ ID No. 5, the HCDR2 may comprise the amino acid sequence of SEQ ID No. 15, and the HCDR3 may comprise the amino acid sequence of SEQ ID No. 16.
In this application, the isolated antigen binding protein may comprise antibody heavy chain variable region CDRs, HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence of SEQ ID No. 22, the HCDR2 may comprise the amino acid sequence of SEQ ID No. 23, and the HCDR3 may comprise the amino acid sequence of SEQ ID No. 24.
In this application, the isolated antigen binding protein may comprise antibody heavy chain variable region CDRs, HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence of SEQ ID No. 30, the HCDR2 may comprise the amino acid sequence of SEQ ID No. 31, and the HCDR3 may comprise the amino acid sequence of SEQ ID No. 32.
In this application, the isolated antigen binding protein may comprise antibody heavy chain variable region CDRs, HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence of SEQ ID No. 30, the HCDR2 may comprise the amino acid sequence of SEQ ID No. 38, and the HCDR3 may comprise the amino acid sequence of SEQ ID No. 39.
In this application, the isolated antigen binding protein may comprise antibody heavy chain variable region CDRs, HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence of SEQ ID No. 45, the HCDR2 may comprise the amino acid sequence of SEQ ID No. 46, and the HCDR3 may comprise the amino acid sequence of SEQ ID No. 47.
In this application, the isolated antigen binding protein may comprise antibody heavy chain variable region CDRs, HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence of SEQ ID No. 5, the HCDR2 may comprise the amino acid sequence of SEQ ID No. 9, and the HCDR3 may comprise the amino acid sequence of SEQ ID No. 49.
In this application, the isolated antigen binding protein may comprise antibody heavy chain variable region CDRs, HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence of SEQ ID No. 51, the HCDR2 may comprise the amino acid sequence of SEQ ID No. 52, and the HCDR3 may comprise the amino acid sequence of SEQ ID No. 53.
In this application, the isolated antigen binding protein may comprise antibody heavy chain variable region CDRs, HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence of SEQ ID No. 55, the HCDR2 may comprise the amino acid sequence of SEQ ID No. 56, and the HCDR3 may comprise the amino acid sequence of SEQ ID No. 57.
In this application, the isolated antigen binding protein may comprise antibody heavy chain variable region CDRs, HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence of SEQ ID No. 63, the HCDR2 may comprise the amino acid sequence of SEQ ID No. 64, and the HCDR3 may comprise the amino acid sequence of SEQ ID No. 65.
In this application, the isolated antigen binding protein may comprise antibody heavy chain variable region CDRs, HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence of SEQ ID No. 5, the HCDR2 may comprise the amino acid sequence of SEQ ID No. 67, and the HCDR3 may comprise the amino acid sequence of SEQ ID No. 68.
In this application, the isolated antigen binding protein may comprise antibody heavy chain variable region CDRs, HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence of SEQ ID No. 55, the HCDR2 may comprise the amino acid sequence of SEQ ID No. 56, and the HCDR3 may comprise the amino acid sequence of SEQ ID No. 70.
In this application, the isolated antigen binding protein may comprise antibody heavy chain variable region CDRs, HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence of SEQ ID No. 55, the HCDR2 may comprise the amino acid sequence of SEQ ID No. 56, and the HCDR3 may comprise the amino acid sequence of SEQ ID No. 72.
In this application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 5, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 6, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 7, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 1, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 2, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 3.
In this application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID No. 5, the HCDR2 may comprise the amino acid sequence shown in SEQ ID No. 9, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID No. 7, the LCDR1 may comprise the amino acid sequence shown in SEQ ID No. 1, the LCDR2 may comprise the amino acid sequence shown in SEQ ID No. 2, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID No. 3.
In this application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 5, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 15, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 16, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 11, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 12, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 13.
In this application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 22, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 23, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 24, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 18, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 19, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 20.
In this application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, the HCDR1 may comprise the amino acid sequence depicted in SEQ ID NO. 30, the HCDR2 may comprise the amino acid sequence depicted in SEQ ID NO. 31, and the HCDR3 may comprise the amino acid sequence depicted in SEQ ID NO. 32, the LCDR1 may comprise the amino acid sequence depicted in SEQ ID NO. 26, the LCDR2 may comprise the amino acid sequence depicted in SEQ ID NO. 27, and the LCDR3 may comprise the amino acid sequence depicted in SEQ ID NO. 28.
In this application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:30, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:38, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:39, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:34, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:35, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 36.
In this application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, the HCDR1 may comprise the amino acid sequence depicted in SEQ ID NO. 45, the HCDR2 may comprise the amino acid sequence depicted in SEQ ID NO. 46, and the HCDR3 may comprise the amino acid sequence depicted in SEQ ID NO. 47, the LCDR1 may comprise the amino acid sequence depicted in SEQ ID NO. 41, the LCDR2 may comprise the amino acid sequence depicted in SEQ ID NO. 42, and the LCDR3 may comprise the amino acid sequence depicted in SEQ ID NO. 43.
In this application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 5, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 9, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 49, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 41, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 42, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 43.
In this application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, the HCDR1 may comprise the amino acid sequence depicted in SEQ ID NO:51, the HCDR2 may comprise the amino acid sequence depicted in SEQ ID NO:52, and the HCDR3 may comprise the amino acid sequence depicted in SEQ ID NO:53, the LCDR1 may comprise the amino acid sequence depicted in SEQ ID NO:41, the LCDR2 may comprise the amino acid sequence depicted in SEQ ID NO:42, and the LCDR3 may comprise the amino acid sequence depicted in SEQ ID NO: 43.
In this application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, the HCDR1 may comprise the amino acid sequence depicted in SEQ ID NO:55, the HCDR2 may comprise the amino acid sequence depicted in SEQ ID NO:56, and the HCDR3 may comprise the amino acid sequence depicted in SEQ ID NO:57, the LCDR1 may comprise the amino acid sequence depicted in SEQ ID NO:41, the LCDR2 may comprise the amino acid sequence depicted in SEQ ID NO:42, and the LCDR3 may comprise the amino acid sequence depicted in SEQ ID NO: 43.
In this application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, the HCDR1 may comprise the amino acid sequence depicted in SEQ ID NO:63, the HCDR2 may comprise the amino acid sequence depicted in SEQ ID NO:64, and the HCDR3 may comprise the amino acid sequence depicted in SEQ ID NO:65, the LCDR1 may comprise the amino acid sequence depicted in SEQ ID NO:59, the LCDR2 may comprise the amino acid sequence depicted in SEQ ID NO:60, and the LCDR3 may comprise the amino acid sequence depicted in SEQ ID NO: 61.
In this application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, the HCDR1 may comprise the amino acid sequence depicted in SEQ ID NO. 5, the HCDR2 may comprise the amino acid sequence depicted in SEQ ID NO. 67, and the HCDR3 may comprise the amino acid sequence depicted in SEQ ID NO. 68, the LCDR1 may comprise the amino acid sequence depicted in SEQ ID NO. 41, the LCDR2 may comprise the amino acid sequence depicted in SEQ ID NO. 42, and the LCDR3 may comprise the amino acid sequence depicted in SEQ ID NO. 43.
In this application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, the HCDR1 may comprise the amino acid sequence depicted in SEQ ID NO. 55, the HCDR2 may comprise the amino acid sequence depicted in SEQ ID NO. 56, and the HCDR3 may comprise the amino acid sequence depicted in SEQ ID NO. 70, the LCDR1 may comprise the amino acid sequence depicted in SEQ ID NO. 41, the LCDR2 may comprise the amino acid sequence depicted in SEQ ID NO. 42, and the LCDR3 may comprise the amino acid sequence depicted in SEQ ID NO. 43.
In this application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, the HCDR1 may comprise the amino acid sequence depicted in SEQ ID NO. 55, the HCDR2 may comprise the amino acid sequence depicted in SEQ ID NO. 56, and the HCDR3 may comprise the amino acid sequence depicted in SEQ ID NO. 72, the LCDR1 may comprise the amino acid sequence depicted in SEQ ID NO. 41, the LCDR2 may comprise the amino acid sequence depicted in SEQ ID NO. 42, and the LCDR3 may comprise the amino acid sequence depicted in SEQ ID NO. 43.
For example, the isolated antigen binding protein may comprise a heavy chain variable region VH, which may comprise the amino acid sequence shown in SEQ ID No. 8, and a light chain variable region VL, which may comprise the amino acid sequence shown in SEQ ID No. 4. The isolated antigen binding protein may be referred to as ab2001.02.
For example, the isolated antigen binding protein may comprise a heavy chain variable region VH, which may comprise the amino acid sequence shown in SEQ ID No. 10, and a light chain variable region VL, which may comprise the amino acid sequence shown in SEQ ID No. 4. The isolated antigen binding protein may be referred to as ab2001.03.
For example, the isolated antigen binding protein may comprise a heavy chain variable region VH, which may comprise the amino acid sequence shown in SEQ ID No. 17, and a light chain variable region VL, which may comprise the amino acid sequence shown in SEQ ID No. 14. The isolated antigen binding protein may be referred to as ab2001.04.
For example, the isolated antigen binding protein may comprise a heavy chain variable region VH, which may comprise the amino acid sequence shown in SEQ ID No. 25, and a light chain variable region VL, which may comprise the amino acid sequence shown in SEQ ID No. 21. The isolated antigen binding protein may be referred to as ab2001.08.
For example, the isolated antigen binding protein may comprise a heavy chain variable region VH, which may comprise the amino acid sequence shown in SEQ ID No. 33, and a light chain variable region VL, which may comprise the amino acid sequence shown in SEQ ID No. 29. The isolated antigen binding protein may be referred to as ab2001.09.
For example, the isolated antigen binding protein may comprise a heavy chain variable region VH, which may comprise the amino acid sequence shown in SEQ ID No. 40, and a light chain variable region VL, which may comprise the amino acid sequence shown in SEQ ID No. 37. The isolated antigen binding protein may be referred to as ab2001.10.
For example, the isolated antigen binding protein may comprise a heavy chain variable region VH, which may comprise the amino acid sequence shown in SEQ ID No. 48, and a light chain variable region VL, which may comprise the amino acid sequence shown in SEQ ID No. 44. The isolated antigen binding protein may be referred to as ab2001.11.
For example, the isolated antigen binding protein may comprise a heavy chain variable region VH, which may comprise the amino acid sequence shown in SEQ ID No. 50, and a light chain variable region VL, which may comprise the amino acid sequence shown in SEQ ID No. 44. The isolated antigen binding protein may be referred to as ab2001.12.
For example, the isolated antigen binding protein may comprise a heavy chain variable region VH, which may comprise the amino acid sequence shown in SEQ ID No. 54, and a light chain variable region VL, which may comprise the amino acid sequence shown in SEQ ID No. 44. The isolated antigen binding protein may be referred to as ab2001.13.
For example, the isolated antigen binding protein may comprise a heavy chain variable region VH, which may comprise the amino acid sequence shown in SEQ ID No. 58, and a light chain variable region VL, which may comprise the amino acid sequence shown in SEQ ID No. 44. The isolated antigen binding protein may be referred to as ab2001.14.
For example, the isolated antigen binding protein may comprise a heavy chain variable region VH, which may comprise the amino acid sequence shown in SEQ ID No. 66, and a light chain variable region VL, which may comprise the amino acid sequence shown in SEQ ID No. 62. The isolated antigen binding protein may be referred to as ab2001.19.
For example, the isolated antigen binding protein may comprise a heavy chain variable region VH, which may comprise the amino acid sequence shown in SEQ ID No. 69, and a light chain variable region VL, which may comprise the amino acid sequence shown in SEQ ID No. 44. The isolated antigen binding protein may be referred to as ab2001.23.
For example, the isolated antigen binding protein may comprise a heavy chain variable region VH, which may comprise the amino acid sequence shown in SEQ ID No. 71, and a light chain variable region VL, which may comprise the amino acid sequence shown in SEQ ID No. 44. The isolated antigen binding protein may be referred to as ab2001.24.
For example, the isolated antigen binding protein may comprise a heavy chain variable region VH, which may comprise the amino acid sequence shown in SEQ ID No. 73, and a light chain variable region VL, which may comprise the amino acid sequence shown in SEQ ID No. 44. The isolated antigen binding protein may be referred to as ab2001.25.
In this application, the VL of antibodies Ab2001.2, ab2001.3, ab2001.11, ab2001.12, ab2001.13, ab2001.14, ab2001.23, ab2001.24 and Ab2001.25 may comprise the amino acid sequence shown in SEQ ID NO: 124.
In this application, the VL of antibodies Ab2001.4, ab2001.8, ab2001.9, ab2001.10 and Ab2001.19 may comprise the amino acid sequence shown in SEQ ID NO. 125.
The antigen binding proteins described herein (e.g., SARS-CoV-2 antibodies) are capable of specifically binding the RBD of the S protein of SARS-CoV-2. Antigen binding proteins (e.g., antibodies) that "specifically bind" to SARS-CoV-2 antigen (e.g., RBD of S protein of SARS-CoV-2) can generally be at about 5.0 x 10 -8 K of M D A value of or greater (e.g., about 9.0 x 10 -9 Below M, about 8.0 x 10 -9 Below M) binds to the RBD of the S protein of SARS-CoV-2, but not to other proteins lacking the SARS-CoV-2 sequence.
Whether an antigen binding protein (e.g., an antibody) binds to a SARS-CoV-2 antigen (e.g., the RBD of the S protein of SARS-CoV-2) can be determined using any assay known in the art. Examples of assays known in the art to determine binding affinity include surface plasmon resonance (e.g., BIACORE) or similar techniques (e.g., kinExa or OCTET). In some cases, the SARS-CoV-2 antibodies described herein can also cross-react with SARS-CoV. For example, by flow assay techniques and enzyme-linked immunosorbent assay. As used herein, "cross-reactivity" refers to the ability of an antibody to react with homologous proteins from other species.
The antigen binding proteins described herein (e.g., SARS-CoV-2 antibodies) are capable of blocking the binding of RBD of the S protein of SARS-CoV-2 or mutants thereof to human ACE 2. Blocking assays can be performed using competition methods, e.g., mixing the antigen binding protein (e.g., SARS-CoV-2 antibody) with an antigen (or cells that express an antigen) and a ligand of the antigen (or cells that express a ligand), and reacting the ability of the antigen binding protein to competitively bind to the ligand of the antigen based on the intensity (e.g., fluorescence intensity or concentration) of the detectable label. In this application, the antigen binding protein (e.g., SARS-CoV-2 antibody) can also block the binding of RBD of S protein of SARS-CoV to human ACE 2.
Bispecific antigen binding proteins
In another aspect, the present application provides a bispecific antigen binding protein that specifically binds to the RBD of the S protein of SARS-CoV-2 and the RBD of the S protein of SARS-CoV.
In the present application, the bispecific antigen binding protein may comprise a first targeting moiety of an RBD that specifically binds to the S protein of SARS-CoV-2, wherein the first targeting moiety may comprise an isolated antigen binding protein as described herein.
For example, antibodies described herein, ab2001.2, ab2001.3, ab2001.11, ab2001.12, ab2001.13, ab2001.14, ab2001.23, ab2001.24, and ab2001.25; and antibodies Ab2001.4, ab2001.8, ab2001.9, ab2001.10 and Ab2001.19 described herein, as well as antigen binding fragments thereof (e.g., which may be VL, VH thereof), can specifically bind RBD of the S protein of SARS-CoV-2.
In the present application, the bispecific antigen binding protein may comprise a second targeting moiety of an RBD that specifically binds to the S protein of SARS-CoV, wherein the second targeting moiety may comprise an isolated antigen binding protein as described herein.
For example, antibodies described herein, ab2001.2, ab2001.3, ab2001.11, ab2001.14, ab2001.24, and ab2001.25, and antigen binding fragments thereof (e.g., which may be VL, VH thereof), can specifically bind RBD of the S protein of SARS-CoV.
For example, the bispecific antigen binding protein may comprise an antibody.
For example, the first targeting moiety may comprise a first heavy chain and a first light chain, and the second targeting moiety may comprise a second heavy chain and a second light chain, wherein the first light chain and the second light chain may be the same.
For example, the first light chain and the second light chain may comprise the amino acid sequence shown in SEQ ID NO. 44.
For example, the VH of the first heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 48.
For example, the VH of the second heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 50.
For example, the VH of the first heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 48, and the VH of the second heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 50.
In the present application, the bispecific antigen binding protein may be antibody ab2001.16, wherein the first light chain and the second light chain may comprise the amino acid sequence shown in SEQ ID No. 44; the VH of the first heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 48 and the VH of the second heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 50.
The protein, polypeptide and/or amino acid sequences referred to in this application are also understood to comprise at least the following ranges: variants or homologues having the same or similar function as the protein or polypeptide.
In the present application, the variant may be a protein or polypeptide in which one or more amino acids have been substituted, deleted or added in the amino acid sequence of the protein and/or the polypeptide (e.g., antigen binding protein described herein). For example, the functional variant may comprise a protein or polypeptide that has been altered in amino acids by at least 1, such as 1-30, 1-20, or 1-10, and yet another such as 1, 2, 3, 4, or 5 amino acid substitutions, deletions, and/or insertions. The functional variant may substantially retain the biological properties of the protein or the polypeptide prior to alteration (e.g., substitution, deletion, or addition). For example, the functional variant may retain at least 60%,70%,80%,90%, or 100% of the biological activity (e.g., antigen binding capacity) of the protein or the polypeptide prior to alteration. For example, the substitution may be a conservative substitution.
In the present application, a part of the amino acid sequence of the antigen binding protein may be homologous to a corresponding amino acid sequence in an antibody from a specific species, or belong to a specific class. For example, the variable and constant regions of an antibody may be derived from the variable and constant regions of an antibody of an animal species (e.g., human). In the present application, the homolog may be a protein or polypeptide having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology to the amino acid sequence of the protein and/or the polypeptide (e.g., the antigen binding protein described herein).
In this application, homology generally refers to similarity, similarity or association between two or more sequences. "percent sequence homology" can be calculated by: the two sequences to be aligned are compared in a comparison window, the number of positions in the two sequences where the same nucleobase (e.g., A, T, C, G) or the same amino acid residue (e.g., ala, pro, ser, thr, gly, val, leu, ile, phe, tyr, trp, lys, arg, his, asp, glu, asn, gln, cys and Met) is present is determined to give the number of matched positions, the number of matched positions is divided by the total number of positions in the comparison window (i.e., window size), and the result is multiplied by 100 to produce the percent sequence homology. Alignment to determine percent sequence homology can be accomplished in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length sequence being compared or over the region of the target sequence. The homology can also be determined by the following method: FASTA and BLAST. For a description of FASTA algorithm, see w.r.pearson and d.j.lipman, "improved tools for biological sequence comparison", proc.Natl. Acad.Sci., U.S. Proc., 85:2444-2448, 1988; "quick sensitive protein similarity search" by d.j.lipman and w.r.pearson, science,227:1435-1441, 1989. For a description of the BLAST algorithm, see "a basic local contrast (alignment) search tool", journal of molecular biology, 215:403-410, 1990.
Nucleic acids, vectors, cells and methods of making
In another aspect, the present application provides an isolated nucleic acid molecule or molecules encoding an isolated antigen binding protein described herein, and/or a bispecific antigen binding protein described herein.
In another aspect, the present application provides a vector that may comprise a nucleic acid molecule as described herein.
In another aspect, the present application provides a cell, which may comprise a nucleic acid molecule as described herein or a vector as described herein.
The nucleic acid molecules described herein may be isolated. For example, it may be produced or synthesized by: (i) amplified in vitro, e.g. by Polymerase Chain Reaction (PCR) amplification, (ii) produced by clonal recombination, (iii) purified, e.g. fractionated by cleavage and gel electrophoresis, or (iv) synthesized, e.g. by chemical synthesis. In certain embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA techniques.
In this application, nucleic acids encoding the antibodies, antigen binding fragments thereof, can be prepared by a variety of methods known in the art, including, but not limited to, overlap extension PCR using restriction fragment procedures or using synthetic oligonucleotides, see Sambrook et al, molecular Cloning, A Laboratory Manual, cold Spring Harbor Laboratory Press, cold Spring Harbor, n.y.,1989; and Ausube et al Current Protocols in Molecular Biology, greene Publishing and Wiley-Interscience, new York N.Y.,1993.
In another aspect, the present application provides one or more vectors comprising one or more nucleic acid molecules described herein. Each vector may comprise one or more of the nucleic acid molecules. In addition, other genes may be included in the vector, such as marker genes that allow selection of the vector in an appropriate host cell and under appropriate conditions. In addition, the vector may also contain expression control elements that allow for proper expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers and other control elements which regulate gene transcription or mRNA translation, and the like. In certain embodiments, the expression control sequence is a tunable element. The specific structure of the expression control sequences may vary depending on the species or cell type function, but typically comprises 5' non-transcribed and 5' and 3' non-translated sequences involved in transcription and translation initiation, respectively, such as TATA boxes, capping sequences, CAAT sequences, and the like. For example, a 5' non-transcriptional expression control sequence may comprise a promoter region that may comprise a promoter sequence for a transcriptional control functional attachment nucleic acid. The expression control sequences may also include enhancer sequences or upstream activator sequences. In this application, suitable promoters may include, for example, promoters for SP6, T3 and T7 polymerase, the human U6RNA promoter, the CMV promoter, and artificial hybrid promoters thereof (e.g., CMV), wherein a portion of the promoter may be fused to a portion of the promoter of other cellular proteins (e.g., human GAPDH, glyceraldehyde-3-phosphate dehydrogenase) gene, which may or may not comprise additional introns. One or more nucleic acid molecules described herein may be operably linked to the expression control element. The vector may include, for example, a plasmid, cosmid, virus, phage, or other vector commonly used in, for example, genetic engineering. For example, the vector is an expression vector.
In another aspect, the present application provides a host cell that can comprise one or more nucleic acid molecules described herein and/or one or more vectors described herein. In certain embodiments, each or each host cell may comprise one or more nucleic acid molecules or vectors described herein. In certain embodiments, each or each host cell may comprise a plurality (e.g., 2 or more) or a plurality (e.g., 2 or more) of the nucleic acid molecules or vectors described herein. For example, the vectors described herein may be introduced into such host cells, e.g., eukaryotic cells, such as cells from plants, fungal or yeast cells, and the like. The vectors described herein can be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamine transfection, and the like.
In another aspect, the present application provides a method of making an isolated antigen binding protein described herein, a bispecific antigen binding protein described herein, comprising culturing a cell described herein under conditions such that the isolated antigen binding protein described herein and/or the bispecific antigen binding protein described herein is expressed.
The method may comprise culturing the host cell described herein under conditions such that the antigen binding protein is expressed. For example, such methods are known to those of ordinary skill in the art by using an appropriate medium, an appropriate temperature, an appropriate incubation time, and the like.
Any method suitable for producing monoclonal antibodies can be used to produce the antigen binding proteins of the present application (e.g., SARS-CoV-2 antibodies).
Any suitable form of SARS-CoV-2 (e.g., RBD of S protein of SARS-CoV-2) can be used as an immunogen (antigen) for generating antibodies specific for SARS-CoV-2, and screening the antibodies for biological activity. The immunogens may be used alone or in combination with one or more immunogenicity enhancing agents known in the art. DNA encoding the immunogen may be expressed using suitable genetic vectors including, but not limited to, adenovirus vectors, adeno-associated virus vectors, baculovirus vectors, prime and non-viral vectors.
The humanized antibody may be selected from any class of immunoglobulins, including IgM, igD, igG, igA and IgE. In this application, the antibody is an IgG antibody, and an IgG1 subtype is used. Also, any type of light chain may be used in the compounds and methods herein. For example, kappa, lambda chains or variants thereof are suitable for use in the present application.
The sequence of the DNA molecule of the antigen binding protein or fragment thereof of the present application may be obtained by conventional techniques, such as by PCR amplification or genomic library screening. In addition, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.
Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. The nucleic acid molecule can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art.
The present application also relates to vectors comprising the above-described suitable nucleic acid molecules and suitable promoters or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein. The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. For example, the animal cells may include: CHO-S, CHO-K1 and/or HEK-293 cells.
The steps described herein for transforming a host cell with recombinant DNA may be performed using techniques well known in the art. The resulting transformants may be cultured by conventional methods, and the transformants express the polypeptide encoded by the nucleic acid molecules of the present application. Depending on the host cell used, it is cultivated in conventional medium under suitable conditions. Typically, the resulting host cells are cultured under conditions suitable for expression of the antigen binding proteins of the present application. The antigen binding proteins of the present application are then purified by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, using conventional separation and purification means well known to those skilled in the art.
The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined using immunoprecipitation or in vitro binding assays, such as flow cell sorting (FACS), radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA).
Pharmaceutical composition
In another aspect, the present application provides a pharmaceutical composition that may comprise an isolated antigen binding protein described herein, a bispecific antigen binding protein described herein, a nucleic acid molecule described herein, a vector described herein, and/or a cell described herein, and optionally a pharmaceutically acceptable adjuvant.
The pharmaceutical compositions described herein can be used directly for RBD binding to the S protein of SARS-CoV-2, and thus can be used for the prevention and treatment of diseases associated with coronavirus infection (e.g., COVID-19). In addition, other therapeutic agents may also be used simultaneously.
The pharmaceutical compositions of the present application may contain a safe and effective amount (e.g., 0.001-99wt%,0.01-90wt%, or 0.1-80 wt%) of the antigen binding proteins described herein, together with a pharmaceutically acceptable adjuvant (which may include a carrier or excipient). Such vectors may include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions described herein may be formulated for injection, for example, by conventional methods using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. In addition, the antigen binding proteins described herein may also be used with other therapeutic agents.
The antigen binding proteins or pharmaceutical compositions described herein may be formulated, administered, and administered in a manner consistent with good medical practice. Considerations in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the etiology of the disorder, the site of agent delivery, the method of administration, and other factors known to medical practitioners. Therapeutic agents (e.g., SARS-CoV-2 antibodies) need not be, but are optionally formulated and/or administered simultaneously with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of therapeutic agent (e.g., SARS-CoV-2 antibody) present in the formulation, the type of disorder or treatment, and other factors discussed above. These agents can generally be used in any dosage that is empirically/clinically determined to be appropriate and by any route that is empirically/clinically determined to be appropriate. The dosage of antibody administered in combination therapy may be reduced compared to single therapy. The progress of this therapy is readily monitored by conventional techniques.
Use of the same
In another aspect, the present application provides the use of an isolated antigen binding protein described herein, a bispecific antigen binding protein described herein, a nucleic acid molecule described herein, a vector described herein, a cell described herein and/or a pharmaceutical composition described herein in the manufacture of a medicament for preventing, alleviating and/or treating an infection by a coronavirus.
The present application provides a method of preventing, alleviating and/or treating an infection by a coronavirus comprising administering to a subject in need thereof an isolated antigen binding protein as described herein, a bispecific antigen binding protein as described herein, a nucleic acid molecule as described herein, a vector as described herein, a cell as described herein and/or a pharmaceutical composition as described herein.
The present application provides isolated antigen binding proteins, bispecific antigen binding proteins described herein, nucleic acid molecules described herein, vectors described herein, cells described herein, and/or pharmaceutical compositions described herein, which can prevent, alleviate and/or treat infection by coronaviruses.
In this application, the infection with coronavirus may include covd-19. The infection by the coronavirus may also include SARS.
In another aspect, the present application provides a method of blocking the binding of an RBD of the S protein of SARS-CoV-2 or a mutant thereof to human ACE2 comprising the step of administering an isolated antigen binding protein described herein, a bispecific antigen binding protein described herein, a nucleic acid molecule described herein, a vector described herein, a cell described herein and/or a pharmaceutical composition described herein.
In another aspect, the present application provides a method of blocking the binding of RBD of S protein of SARS-CoV to human ACE2 comprising the step of administering an isolated antigen binding protein described herein, a bispecific antigen binding protein described herein, a nucleic acid molecule described herein, a vector described herein, a cell described herein and/or a pharmaceutical composition described herein.
The antigen binding proteins of the present application can be used in detection applications, for example for detecting samples, thereby providing diagnostic information. For example, the antibodies and/or methods described herein can be used to detect a specimen (e.g., a pharyngeal swab test sample, such as serum, whole blood, sputum, oral/nasopharyngeal secretions or wash, urine, stool, pleuroperitoneal fluid, cerebrospinal fluid, and tissue specimens) of a subject (e.g., a patient suspected of being or having been infected with SARS-CoV-2 or SARS-CoV), as an indicator of efficacy observations and whether infectivity and isolation is desired. For example, the antibodies and/or methods described herein can provide a monitoring regimen for therapeutic intervention.
In the present application, a sample (specimen) is taken to include a cell, a tissue sample, and a biopsy specimen. The term "biopsy" as used herein shall include all kinds of biopsies known to a person skilled in the art. A biopsy as used in this application may thus include tissue samples prepared, for example, by endoscopic methods or by puncture or needle biopsy of an organ. For example, the sample may comprise a fixed or preserved cell or tissue sample.
The present application also provides a kit comprising the antigen binding proteins of the present application. In some cases, the kit may further comprise a container, instructions for use, buffers, and the like. For example, the pro-binding proteins of the present application may be immobilized to a detection plate.
Without intending to be limited by any theory, the following examples are presented in order to illustrate the fusion proteins, methods of preparation, uses, etc. of the present application and are not intended to limit the scope of the invention of the present application.
Examples
Example 1 antibody screening
1.1 construction and screening of Yeast display library
A common light chain phage library and a single-chain phage library are selected and used for SARS-CoV-2S1 RBD (sino biological product number): 40592-V05H) were subjected to two rounds of panning to obtain positive enrichment. Designing primers to perform Polymerase Chain Reaction (PCR) to amplify single-chain antibody (scFv) by taking plasmid after phage two rounds of panning as a template; the scFv gene fragment amplified by PCR is recovered and then co-transferred into a saccharomyces cerevisiae strain EBY100 (purchased from ATCC) together with a yeast display plasmid, and the scFv gene is inserted into the yeast display plasmid through homologous recombination of saccharomyces cerevisiae, so that single-chain antibodies are displayed on the surface of a yeast cell wall. The yeast display single chain antibody library and the common light chain antibody library are respectively named JYYDL056 and JYYDL057. Library JYYDL056, JYYDL057 were electrotransformed and incubated overnight at 30℃in 100mL SD-Trp medium (Clontech, cat# 630308), 225 rpm; 1.0X10 each 8 The bacterial load was resuspended in 20mL YPGP broth (2% galactose, 2% peptone, 1% yeast extract, 0.54% Na) 2 HPO 4 ，0.86％NaH 2 PO 4 ·H 2 O), culturing at 20 ℃ for 24 hours at 225 rpm, and placing in a refrigerator at 4 ℃ for standby.
After induction, measuring OD of the bacterial liquid 600 At 1OD of 1.0X10 7 Cell number was calculated, 4.0X10 each 7 Cells were flow stained sorted as follows: 1. wash once with 2ml of 1 XPBS (1 XPBS+1% BSA), centrifuge at 3000 rpm for 3 min (all conditions are given below) discard supernatant; 2. each tube was incubated with 500. Mu.L of 1 XPBS containing 100nM SARS-CoV-2S1 RBD mFc (Sino biological, cat# 40592-V05H) for 30 minutes at room temperature; 3. 1mL of 1 XPBS was added to each, and the supernatant was discarded by centrifugation; 4. mu.L of 300nM Human ACE2-biotin (Kactus, cat# ACE-HM 401) diluted with 1 XPBS were added each and incubated on ice for 20 minutes; 5. 1mL of 1 XPBS was added to each, and the supernatant was centrifuged off; 6. mu.L of 1 XPBS containing fluorescent antibody (SA-PE manufacturer eBioscience, cat# 12-4317-8, diluted 1:200; goat anti mouse-Alexa Fluor 647 manufacturer thermo-fisher, cat# A-21235, diluted 1:400) were added, and incubated for 20 minutes on light-protected ice; 7. step 5 was repeated, 2mL of 1 XPBS resuspended cells were added, and the cell population with strong fluorescent signal Alexa Fluor 647 and weak PE signal was collected for flow sorting. After sorting, the cells were cultured overnight at 30℃in 5mL SD-Trp liquid medium at 225 rpm; 1mL of the bacterial liquid is taken and added into 4mL of YPGP liquid culture medium, 2 Culturing at 0deg.C for 24 hr at 225 rpm, and placing in a refrigerator at 4deg.C for use. As with the first round of screening protocol, SARS-CoV-2S1 RBD mFc concentration was reduced to 50nM, and the second round of screening was performed, and the sorted cells were plated on SD-Trp solid medium (Clontech, cat# 630309) and cultured at 30℃for 3 days.
1.2 monoclonal Yeast colony sequencing and flow staining identification
Performing second-round screening products of JYYDL056 and JYYDL057, respectively picking 400 monoclone to sequence, and finally obtaining 34 unique single-chain antibody sequences from the second-round screening products of JYYDL056 library; the second round of screening of JYYDL057 library resulted in 42 unique common light chain antibody sequences. Flow staining of the corresponding Yeast monoclonal colonies was performed, 1X 10 in each case, by the staining procedure of reference example 2.1 6 Individual cells were stained as follows (table 1).
The intensity of the antibody blocking SARS-CoV-2-RBD and hACE2-Fc is reflected by the MFI value of Alexa Fluor 647;
similar protocol 2 allows assessment of antibody binding and blocking ability to SARS S1;
the MFI values of PE and Alexa Fluor 647 of scheme 3 reflect the display level and non-specific binding level, respectively, of single chain antibodies.
Table 1 monoclonal Yeast colony flow staining identification scheme
After staining each monoclonal with three staining protocols, flow analysis was performed using a GUAVA microcapillary cell analysis platform. According to the experimental results, clones in scheme 1 which bind strongly to SARS-CoV-2-RBD (MFI value of PE fluorescent signal is high) and block SARS-CoV-2RBD from binding strongly to hACE2-Fc (MFI value of Alexa Fluor 647 is low) were selected; while excluding the cloned sequences of scheme 3 that have high binding signals to unrelated antigens.
Comprehensively obtaining 8 monoclonal antibodies (Y28A 5, Y28B6, Y29B2, Y29F6, Y34H3, Y35F3, Y35G1 and Y36F 4) from JYYDL056 library; 7 common light chain monoclonal antibodies (Y28B 4, Y30B4, Y34B6, Y34G5, Y38A3, Y39B3, Y39G 2) were obtained from JYYDL057 library (the results are shown in Table 2). The common light chain monoclonal (Y28B 4 and Y34G 5) was selected to construct a common light chain bispecific antibody ab2001.16 by Knob and Hole mutation of the Fc region (see US5731168A for mutation modification), as shown in table 3. A total of 16 antibodies were submitted to contract outsourcing company for mammalian cell expression and antibody samples were prepared in small amounts.
TABLE 2 flow-through staining analysis results of Yeast monoclonal colonies after two rounds of screening of library
TABLE 3 expression combinations of Co-light chain bispecific antibodies
Antibody name Knob clone number Hole clone number Light chain
Ab2001.16 Y28B4 Y34G5 Identical light chain germline1-39
Example 2 antibody expression
The candidate antibody sequence codes and divides the CDR region by Kabat numbering method, and entrusts the plasmid construction and the expression and purification of the antibody to the Biotechnology Co., baiying, taizhou, and the corresponding information of the antibody sequence and expression are shown in Table 4.
30 ml HEK293 cells are transiently transformed, and candidate antibody proteins are obtained after expression and purification, and specific information is shown in Table 5.
TABLE 4 antibody sequence information
TABLE 5 antibody expression information
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Example 3 in vitro Activity Screen
3.1Octet Red detection of the binding dissociation Rate of candidate antibody to SARS-CoV-2S1
The binding dissociation rate of the candidate antibody to SARS-CoV-2S1 was measured, and the binding dissociation rate of the candidate antibody to SARS-CoV-2S1 (Sino Biological, cat# 40591-V08H) was measured by immobilizing the candidate antibody on an AHC sensor (for bio, cat# 18-0015). Each cycle comprises the steps of: 1) Regenerating a sensor; 2) Immersing in buffer (PBST, 10. Mu.L Tween 20 in 50mL PBS) for 60 seconds; 3) The 5. Mu.g/mL fully human antibody was immobilized on an AHC sensor for 40 seconds; 4) The sensor is immersed in the buffer for 180 seconds; 5) 100nM SARS-CoV-2S1 was conjugated to antibody for 180 seconds; 6) Dissociation of antigen-antibody takes 10 minutes. The results of the affinity were analyzed by Octet Data Analysis Software (fortebio) and are shown in Table 6.
TABLE 6
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As can be seen from Table 6, the candidate antibodies bound to SARS-CoV-2S1 antigen, except Ab2001.07. Wherein Ab2001.08, ab2001.10 and Ab2001.16 have higher affinity, and the binding dissociation rates of SARS-CoV-2S1 with different concentrations are further determined by selecting 5 concentrations of SARS-CoV-2S1 of 50nM, 25nM, 12.5nM, 6.3nM and 3.2nM, and comparing with hACE2-Fc ( organism, cat# ACE-HM 501) (see Table 7, FIG. 1)
TABLE 7
As can be seen from Table 7, the affinities of Ab2001.08, ab2001.10, ab2001.16, hACE2-Fc and SARS-CoV-2S1 were 2.93nM (FIG. 1A), 4.21nM (FIG. 1B), 2.39nM (FIG. 1C) and 11.1nM (FIG. 1D), respectively, and the affinities of the antibodies and SARS-CoV-2S1 were all stronger than those of hACE2-Fc.
3.2 candidate antibodies blocking SARS-CoV-2S1 binding to hACE2-Fc
According to the affinity data of candidate antibodies, ab2001.02, ab2001.03, ab2001.08, ab2001.09, ab2001.10, ab2001.11 and Ab2001.16 are selected for blocking experiments of SARS-CoV-2S1 binding to hACE 2; since the ab2001.16 bispecific antibody is composed of a combination of sequences of ab2001.11 and ab2001.12, comparison was made using the same amount of the ab2001.11+ab2001.12 combination.
The experiment was carried out as follows,
1. hACE2-Fc was diluted to 1. Mu.g/mL of coated ELISA plate with 1 XPBS, 100. Mu.l/well, sealed plate film, incubated overnight at 4℃and then plates were washed 3 times with wash solution (1 XPBS+0.05% TWEEN-20). Blocking solution (1 XPBS+0.05% TWEEN-20+2% BSA) was then prepared with washing solution, 300. Mu.l blocking solution was added to each well and blocked at 37℃for 1 hour. 2. Preparing biotinylated SARS-CoV-2S1 Fc ( organism, product number: COV-VM5S 1) with the blocking solution at a final concentration of 0.2 μg/mL; the antibody was then diluted with blocking solution at an initial final concentration of 15. Mu.g/mL, diluted 5-fold in a gradient (7 concentration spots +1 concentration spots of 0. Mu.g/mL), and then the anti-SARS-CoV-2 antibody and biotinylated SARS-CoV-2S1 Fc were incubated at 37℃for 1 hour. 3. The blocked ELISA plate in step 1 was removed, the plate was washed 3 times with washing solution, 100. Mu.l/well of the incubation solution in step 2 was added, and incubated at 37℃for 1 hour. 4. Washing the plate 3 times by using a washing liquid, wherein the volume ratio of the sealing liquid is 1: SA-HRP (sigma, S2438-250 UG) was diluted 5000 and incubated in 100. Mu.l/Kong Jiazhi ELISA plates for 1 hour at 37 ℃;5. the plate was washed 3 times with a washing solution, TMB developing solution (Biopanda, cat# TMB-S-003) was added at 100. Mu.l/well, reacted at room temperature in the dark for 10 minutes, and then the reaction was terminated by adding a termination solution (Solarbio, cat# C1058) at 50. Mu.l/well, and the absorbance was measured at a wavelength of 450 nm. The data were processed with GraphPad Prism software as shown in fig. 2. As is clear from FIG. 2, ab2001.08 and Ab2001.10 have a blocking effect about 15-fold stronger than that of hACE2-Fc.
3.3 identification of candidate antibodies and SARS-CoV-2 antigen binding epitopes
The candidate antibody and SARS-CoV-2S1 antigen binding epitope adopts In-tandem method, i.e. SARS-CoV-2S1 antigen is solidified on the sensor, then is interacted with first antibody and second antibody In turn, and the binding signal of second antibody is detected so as to judge that two antibodies can recognize identical epitope. In this experiment, each cycle comprised the following steps: 1) The sensor was first immersed in buffer (PBST, 10. Mu.L Tween 20 in 50mL PBS) for 30s; 2) 1 μg/ml Biotin human SARS-CoV-2RBD his ( organism, cat: COV-VM4 BD) was cured on the SA sensor (fortebio, cat No.: 18-0009) for 65 seconds; 3) The sensor was immersed in the buffer for 30 seconds; 4) 100nM of the first antibody binds to the antigen on the sensor surface for 3 minutes, saturating the binding site of the first antibody on the antigen; 5) 100nM of the second antibody was conjugated to antigen for 3 minutes. The results of epitope identification were analyzed by Octet Data Analysis Software (fortbio) and are shown in table 8.
Table 8 identification of candidate antibodies and SARS-CoV-2 antigen binding epitopes
The self-response signal was <20%, and the experimental data were valid. The judgment standard of the experimental result is as follows: 1) 60% -100%: completely uncompetitive; 2) 20% -60%: partial competition; 3) <20% >: complete competition, two antibodies are considered to be competing if one direction meets this criterion. The data show that antibody No. ab2001.10, 12, 13 is a binding epitope; four antibodies, ab2001.02, ab2001.03, ab2001.11, are another binding epitope.
The antibody of ab2001.10 was selected to determine whether the antigen binding epitopes of ab2001.08 and ab2001.16 are identical, and the results are shown in table 9. The results showed that ab2001.08, ab2001.10 are 2 different antigen binding epitopes, and ab2001.16 diabody contains two different antigen binding epitopes of ab2001.11 and ab2001.12.
Table 9 identification of candidate antibodies and SARS-CoV-2 antigen binding epitopes
3.4 candidate antibodies blocking SARS-CoV S1 binding to hACE2
As can be seen from Table 2, the MFI of SARS-CoV S1 to hACE2-Fc after the Y28B4 clone bound to SARS-CoV S1 was only 43.5, indicating that it may have the ability to block the binding of SARS-CoV S1 to hACE 2. CR3022 is a neutralizing antibody against SARS-CoV S1 reported in the literature, and can block the binding of SARS-CoV S1 to hACE2, and can be used as a positive control. The antibody corresponding to the Y28B4 clone was designated Ab2001.11, while bispecific antibody Ab2001.16 consisted of Ab2001.11, ab2001.12, so that the ability of candidate antibodies Ab2001.11, ab2001.12, ab2001.16 to block SARS CoV S1 binding to hACE2 was further evaluated. The experimental protocol was the same as in example 3.2, starting with a 5-gradient dilution of the antibody concentration from 200. Mu.g/mL for a total of 7 concentrations, adding one concentration point of 0. Mu.g/mL for the antibody, and the results are shown in FIG. 3.
As can be seen from FIG. 3, the control antibody has a strong blocking effect on the binding of SARS-CoV S1 to hACE2 Is the ability of IC 50 The value was at 6.21nM; ab2001.11 has weaker blocking ability, IC 50 The value was 303.87nM; the remaining antibodies had no blocking ability. Although Ab2001.11 has a weak blocking ability, an increase in affinity by affinity maturation might increase the blocking ability, and is expected to be a broad-spectrum neutralizing antibody that blocks both SARS-CoV-2S1 and SARS-CoV S1 binding to hACE 2.
EXAMPLE 4 evaluation of physicochemical Properties
Experimental materials
Sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium hydroxide, guanidine hydrochloride, sodium chloride, ammonium sulfate, hydrochloric acid, glycine, sodium hydroxide, ethanol and ammonium bicarbonate are purchased from Chinese medicinal reagents; 1% methylcellulose, CIEF Peptide Marker Kit, TTM solution, electrode solution, capillary wash, cIEF Cartridge FC-COATED are available from Protein Simple company; TSKG3000SWxl (7.8X105 mm,5 μm) was purchased from TOSOH; protein Thermal Shift TM Starter Kit, propac WCX-10 (4X 250mm,5 μm), MAbPacTMHIC-10 (4X 250mm,5 μm) were purchased from Thermo.
4.1 candidate antibody hydrophilicity analysis
As can be seen from fig. 3, candidate antibodies ab2001.02, ab2001.03, ab2001.08, ab2001.09, ab2001.10, ab2001.11, ab2001.12, and ab2001.16 have the ability to block the binding of SARS-CoV-2S1 to the human receptor protein ACE 2. The candidate antibodies were subjected to comparison of hydrophilicity with omalizumab, and the specific experimental procedure is as follows, and the experimental results are shown in fig. 4.
(1) The sample was diluted to 1mg/mL, mixed well, centrifuged at 12000 rpm for 5 minutes, the supernatant was transferred to a sample bottle and placed on an HPLC sample tray.
(2) Setting chromatographic conditions:
(3) Performing gradient elution analysis by using 50mM phosphate buffer solution/1M ammonium sulfate, wherein pH 7.0 is used as a mobile phase A, 50mM phosphate buffer solution and pH 7.0 are used as a mobile phase B, performing data analysis by chromatographic software, dividing the peak-out retention time of a reference antibody with good hydrophilicity by the peak-out retention time of a candidate antibody, and calculating the hydrophilicity coefficient of each sample; a hydrophilic coefficient greater than 0.5 is defined internally as an acceptable standard line based on past experience.
As can be seen from fig. 4, the candidate antibodies all have a hydrophilicity of greater than 0.5 and meet the internal pharmaceutical standards, wherein the candidate antibodies AB2001.02, AB2001.03, AB2001.09, AB2001.10 exhibit excellent hydrophilicity.
4.2 analysis of candidate antibody Charge heterogeneity
The charge heterogeneity analysis was performed on candidate antibodies ab2001.02, ab2001.03, ab2001.08, ab2001.09, ab2001.10, ab2001.11, ab2001.12, ab2001.16 using imaging capillary isoelectric focusing (iCIEF), as follows:
(1) The sample solution was taken and added to the following system, which had been thoroughly mixed: 1% Methylcellulose (MC) 70. Mu.L, urea 4M, 8. Mu.L ampholyte Pharmalyte pH 3-10, pI markers 5.5 and 9.5 each 2. Mu.L. Proper volume of ultrapure water was added to 200. Mu.L, and the mixture was homogenized.
(2) All samples were centrifuged at 6000 rpm for 3 minutes to remove air bubbles, and then the supernatant was transferred to a sample bottle, placed on a sample tray, and the sample position was recorded.
(3) And (3) starting up, after analysis, introducing the result file into the Chrom Perfect software to perform map integration processing, and calculating isoelectric points of the peaks and the percentage of the peaks.
As shown in fig. 5, the results of the candidate antibody charge heterogeneity analysis show that the candidate antibodies have good charge heterogeneity except for the main peak of ab2001.09, which is higher than 70%. Through charge heterogeneity pattern analysis, as shown in fig. 6, the ab2001.16 bispecific antibody was purified in one step by Protein a, the main peak purity had reached 74.4%, the heterodimer ratio was close to 93%, or the production and purification process could be co-used with monoclonal antibodies.
4.3 candidate antibody thermal stability Studies
Thermal stability studies were performed on candidate antibodies ab2001.02, ab2001.03, ab2001.08, ab2001.09, ab2001.10, ab2001.11, ab2001.12, ab2001.16.
The method comprises the following specific steps: 1. diluting a sample to be tested to 1mg/mL with a sample buffer solution; 2. according to Protein Thermal Shift TM Starter Kit instruction, taking 13 mu L of the sample solution to be tested and adding the 13 mu L of the sample solution into a PCR tube; 3. after adding 5. Mu. L Protein Thermal shift TM buffer, 2. Mu.L of 10 Xstaining solution was added thereto to give a reaction volume of 20. Mu.L; 4. after uniform mixing, centrifuging for 5 minutes at 12000 r to remove bubbles; 5. the sample was placed in a PCR apparatus, the sample was analyzed, and the Fab melting temperature (Fab Tm) of the sample was recorded, and the result is shown in FIG. 7.
In combination, candidate antibodies ab2001.02, ab2001.03, ab2001.08, ab2001.10, ab2001.11, ab2001.12, ab2001.16 have good physicochemical properties.
Example 5 Virus neutralization assay
Combining the experimental results of examples 3 and 4, candidate antibodies ab2001.02, ab2001.03, ab2001.08, ab2001.09, ab2001.10, ab2001.11, ab2001.12, ab2001.13, ab2001.16 were selected; ab2001.07 as a negative control, the ability of the candidate antibodies to neutralize the infection of monkey kidney cells (Vero E6 cells) by the novel coronavirus was assessed. As live viruses are used, experiments were carried out by the Chinese academy of sciences of biological safety third-level laboratory (BSL 3) with the following specific experimental steps:
reagent and consumable:
reagent: DMEM (Thermo Fisher), FBS (Gibco), methylcellulose (Millipore) antibody diluent: PBS (Thermo Fisher)
Viral RNA extraction: QIAamp 96Virus QIAcube HT Kit,Qiagen company, CAT#57731;
viral nucleic acid detection (fluorescent quantitative PCR) kit: the diagnostic microbiology department of the martial arts institute developed against the RBD2 gene.
The main instrument is as follows:
high throughput nucleic acid automatic purification instrument: QIAcube HT 9001793 (Qiagen)
Fluorescent quantitative PCR instrument: CFX96 Touch Real-Time PCR Detection System (Bio-rad)
Experiment design:
(1) Amplification and titer determination of SARS-CoV-2 virus (WIV 04, genBank: MN 996528.1): p6 generation SARS-CoV-2 virus inoculation of Vero-E6 cells at 37℃with 5% CO 2 Culturing in incubator for 48 hr, collecting cultured virus supernatant, centrifuging, packaging, and freezing at-80deg.C. Viral titers were determined in Vero-E6 cells using classical plaque assay.
(2) Preliminary evaluation of antibodies: vero-E6 cells were spread evenly on 24-well plates (10) 5 And/well), after 16 hours, the medium containing different antibodies (final concentration of antibody 10. Mu.g/mL) was added, infection was performed with SARS-CoV-2 at 0.005MOI, and after 24 hours and 72 hours, cell culture supernatants were collected for virus RNA extraction and detection.
(3) Effective antibody concentration gradient verification: concentration gradient experiments (10. Mu.g/mL, 2. Mu.g/mL, 0.4. Mu.g/mL) were performed on antibodies effective in the step 2 screen, and the protocol was the same.
(4) Plaque reduction neutralization assay (Plaque reduction neutralization test, PRNT)
Adding an antibody to be detected into a DMEM culture medium containing 2% FBS, carrying out 3-time gradient dilution, and adding the diluted antibody into a 96-well plate, wherein each well is 100 mu L; diluting the virus to 2000PFU/mL, and respectively taking 100 mu L of the virus to be mixed with antibodies with different concentrations; (final antibody concentrations were 30. Mu.g/mL, 10. Mu.g/mL, 3.33. Mu.g/mL, 1.11. Mu.g/mL, 0.37. Mu.g/mL, 0.12. Mu.g/mL, 0.04. Mu.g/mL, 0.014. Mu.g/mL, 0.005. Mu.g/mL, respectively), incubated at 37℃for 30min, and the antibody/virus mixture was added to the cell culture, and the PRNT50 concentration was calculated by a classical plaque assay after 96h of virus infection.
Data processing and statistical analysis
PRNT50 is calculated using GraphPad prism software.
Experimental results
Preliminary evaluation of candidate antibody Single concentration
After each candidate antibody (10. Mu.g/mL) was added to the cell culture medium, 0.005MOI virus was added, and after 24 hours, the supernatant viral RNA content was examined, and the viral RNA content was reduced by about 2 orders of magnitude in the groups of Ab2001.08, ab2001.09, ab2001.10, ab2001.12, ab2001.13, ab2001.16 and Ab2001.08 combined with Ab2001.10 (5. Mu.g each), ab2001.11 combined with Ab2001.12 (5. Mu.g each), as shown in FIG. 8A; the detection of the supernatant viral RNA copies for 72h, the combination of ab2001.10, ab2001.08 and ab2001.10 decreased by more than 2 orders of magnitude, ab2001.08 decreased by more than 1 order of magnitude, as shown in fig. 8B; the cell states of three groups of cells, ab2001.08, ab2001.10, combination and the like, are observed in 120 hours, and the lesions of the other groups are obvious.
Candidate antibody multi-concentration gradient validation
Ab2001.08, ab2001.10 and combination of the two were subjected to multi-concentration gradient assay, wherein 10 mug/mL, 2 mug/mL and 0.4 mug/mL of antibody are added to a cell culture medium, then 0.005MOI virus is added, and after 24 hours, the RNA content of the supernatant virus is detected to be reduced by about 2 orders of magnitude, as shown in FIG. 9A; after 72h the supernatant was assayed for viral RNA content, only the ab2001.08, combination group was reduced by about 2 orders of magnitude, as shown in fig. 9B.
Determination of half-maximal reduction of plaque neutralization concentration of candidate antibodies
The determination of half-maximal reduction of plaque neutralization concentrations was performed according to the protocol of the experimental design for Ab2001.08, ab2001.10 and combinations thereof, and the results are shown in FIG. 10. As can be seen from fig. 10, the candidate antibodies ab2001.08, ab2001.10 and both have the activity of neutralizing SARS-CoV-2, and the PRNT50 concentrations (plaque reduction half-neutralization concentrations) are respectively: 0.012 mug/mL, 0.062 mug/mL and 0.022 mug/mL, is expected to become a specific medicine for preventing and treating new crown epidemic situation.
Example 6 binding and blocking Capacity with different novel crown mutant strains
6.1 rate of dissociation of neutralizing antibodies to SARS-CoV-2S1RBD and mutants thereof
The binding dissociation rates of neutralizing antibodies Ab2001.08, ab2001.10 and SARS-CoV-2S1RBD protein and mutant S1RBD proteins (doi: https:// doi.org/10.1101/2020.03.15.991844) circulating in different regions were determined.
The neutralizing antibodies were immobilized on an AHC sensor (Fortebio, cat# 18-0015) and their binding dissociation rates with SARS-CoV-2S1RBD and its mutants (ACRO, cat# SPD-S52H4, SPD-S52H5, SPD-S52H3, SPD-S52H 8) were determined. Each cycle comprises the steps of: 1) Regenerating a sensor; 2) Immersing in buffer (PBST, 10. Mu.L Tween 20 in 50mL PBS) for 60 seconds; 3) 10 μg/mL of the fully human antibody was immobilized on an AHC sensor for 15 seconds; 4) The sensor is immersed in the buffer for 180 seconds; 5) 100nM SARS-CoV-2S1RBD bound to antibody for 180 seconds; 6) Dissociation of antigen-antibody takes 5 minutes. The affinity results were carried out by Octet Data Analysis Software (fortebio) 1:1 fit analysis, the results are shown in fig. 11.
As can be seen from the results of FIG. 11, although each mutant of SARS-CoV-2S1RBD has a higher affinity for human ACE2 receptor than the wild type, the neutralizing antibodies Ab2001.08 and Ab2001.10 have a higher affinity for the RBD mutant and a higher affinity for the RBD protein than for human ACE2 receptor, indicating that the neutralizing antibodies retain good efficacy even when the novel corona strain is mutated.
6.2 neutralizing antibodies blocking SARS-CoV-2S1RBD and its mutants binding to hACE2-Fc
It was further demonstrated by blocking experiments whether neutralizing antibodies could block the binding of SARS-CoV-2S1RBD and its mutants to the human receptor ACE2 very well.
Experimental procedure was performed as in example 3.2, with final concentration of SARS-CoV-2S1RBD and its mutants of 0.1. Mu.g/mL, initial final concentration of antibody and hACE2-Fc of 100nM, 5-fold gradient dilution (7 concentration points+1 concentration point of 0. Mu.g/mL). The results are shown in FIGS. 12A-12E, wherein FIGS. 12A-12E show the case where the neutralizing antibody blocks the binding of SARS-CoV-2S1RBD, SARS-CoV-2S1RBD V357F mutant, SARS-CoV-2S1RBD N356D and D304Y mutant, and SARS-CoV-2S1RBD R408I mutant, respectively, to hACE 2-Fc. From the results of FIGS. 12A-12E, it was found that the candidate antibodies can block the binding between SARS-CoV-2S1RBD mutant and human receptor ACE2 well, and are more potent than hACE 2-Fc.
The embodiments of the present application have been described in detail above, but the present application is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solutions of the present application within the scope of the technical concept of the present application, and all the simple modifications belong to the protection scope of the present application. In addition, the specific features described in the foregoing embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described in detail. Moreover, any combination of the various embodiments of the present application may be made without departing from the spirit of the present application, which should also be considered as disclosed herein.
Sequence listing
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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 Glu Ile Lys
100 105
<210> 22
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR1
<400> 22
Ser Tyr Ala Ile Ser
1 5
<210> 23
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 23
Arg Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln
1 5 10 15
Gly
<210> 24
<211> 14
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 24
Leu Ala Ser Tyr Ser Ser Gly Trp Glu Asp Val Phe Asp Ile
1 5 10
<210> 25
<211> 123
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> VH
<400> 25
Gln 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 Ser Tyr
20 25 30
Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu 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 Ser Leu Ala Ser Tyr Ser Ser Gly Trp Glu Asp Val Phe Asp Ile
100 105 110
Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 26
<211> 12
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 26
Arg Ala Ser Gln Ser Val Ser Gly Ser Tyr Leu Ala
1 5 10
<210> 27
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR2
<400> 27
Gly Ala Ser Thr Arg Ala Thr
1 5
<210> 28
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR3
<400> 28
Gln Gln Tyr Asp Ser Ser Pro Val Thr
1 5
<210> 29
<211> 108
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> VL
<400> 29
Glu Ile Val 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 Val Ser Gly 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 Gln Tyr Asp Ser Ser Pro
85 90 95
Val Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
100 105
<210> 30
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR1
<400> 30
Ser Phe Asp Ile Asn
1 5
<210> 31
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 31
Trp Met His Ala Ser Ser Gly Asn Ala Gly Tyr Ala Gln Lys Phe Gln
1 5 10 15
Gly
<210> 32
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 32
Tyr Val Pro His Asp Ala Phe Gln Ile
1 5
<210> 33
<211> 118
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> VH
<400> 33
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 Ser Ser Phe
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 His Ala Ser Ser Gly Asn Ala Gly 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 Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Asn Tyr Val Pro His Asp Ala Phe Gln Ile Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser
115
<210> 34
<211> 12
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 34
Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala
1 5 10
<210> 35
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR2
<400> 35
Gly Ala Ser Asn Arg Ala Thr
1 5
<210> 36
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR3
<400> 36
Gln Gln Tyr Asp Ser Ser Pro Ile Thr
1 5
<210> 37
<211> 108
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> VL
<400> 37
Glu Ile Val 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 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 Asn 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 Asp Ser Ser Pro
85 90 95
Ile Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 38
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 38
Trp Met His Ala Gly Ser Gly Asn Ala Gly Tyr Ala Gln Lys Phe Gln
1 5 10 15
Gly
<210> 39
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 39
Tyr Val Pro His Gly Ala Phe Gln Ile
1 5
<210> 40
<211> 118
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> VH
<400> 40
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 Ser Ser Phe
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 His Ala Gly Ser Gly Asn Ala Gly 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 Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Asn Tyr Val Pro His Gly Ala Phe Gln Ile Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser
115
<210> 41
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 41
Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn
1 5 10
<210> 42
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR2
<400> 42
Ala Ala Ser Ser Leu Gln Ser
1 5
<210> 43
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR3
<400> 43
Gln Gln Ser Tyr Ser Thr Pro Pro Ile Thr
1 5 10
<210> 44
<211> 108
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> VL
<400> 44
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 Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro
85 90 95
Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
100 105
<210> 45
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR1
<400> 45
Asn Ser Asp Met His
1 5
<210> 46
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 46
Ala Ile Gly Asp Ser Gly Asp Thr Tyr Tyr Ser Asp Ser Val Lys Gly
1 5 10 15
<210> 47
<211> 12
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 47
Ala Ala Asn Trp Gly Trp Asp Trp Tyr Phe Asp Leu
1 5 10
<210> 48
<211> 120
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> VH
<400> 48
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 Ser Phe Ser Asn Ser
20 25 30
Asp Met His Trp Val Arg Gln Ala Thr Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Gly Asp Ser Gly Asp Thr Tyr Tyr Ser Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Ser Ser 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 Ala Ala Asn Trp Gly Trp Asp Trp Tyr Phe Asp Leu Trp Gly Arg
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 49
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 49
Asp Ala Gly Pro Tyr Gly Met Asp Val
1 5
<210> 50
<211> 117
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> VH
<400> 50
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 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 Ala Gly Pro Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr
100 105 110
Val Thr Val Ser Ser
115
<210> 51
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR1
<400> 51
Ser Asn Tyr Met Thr
1 5
<210> 52
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 52
Val Ile Tyr Pro Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly
1 5 10 15
<210> 53
<211> 12
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 53
Asp Leu Pro Val Gly Gly Val Asp Ala Phe Asp Ile
1 5 10
<210> 54
<211> 120
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> VH
<400> 54
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 Ser Ile 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 Pro 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 Pro Val Gly Gly Val Asp Ala Phe Asp Ile Trp Gly Gln
100 105 110
Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 55
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR1
<400> 55
Asn Tyr Asp Met Phe
1 5
<210> 56
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 56
Ala Ile Gly Pro Ala Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys Gly
1 5 10 15
<210> 57
<211> 13
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 57
Gly Gly Asp Ala Pro Met Val Gly Trp His Phe Asp Val
1 5 10
<210> 58
<211> 121
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> VH
<400> 58
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 Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Gly Pro Ala Gly Asp 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 Thr
85 90 95
Arg Gly Gly Asp Ala Pro Met Val Gly Trp His Phe Asp Val Trp Gly
100 105 110
Arg Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 59
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 59
Gln Ala Ser Gln Asp Ile Asn Asn Tyr Leu Asn
1 5 10
<210> 60
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR2
<400> 60
Ala Ala Ser Asn Leu Glu Thr
1 5
<210> 61
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR3
<400> 61
Gln Gln Tyr Asp Asn Leu Pro Ile Thr
1 5
<210> 62
<211> 107
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> VL
<400> 62
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 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
Tyr Ala 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 Ile
85 90 95
Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
100 105
<210> 63
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR1
<400> 63
Ser Ser Gly Met Thr
1 5
<210> 64
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 64
Tyr Ile Ser Gly Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val Arg
1 5 10 15
Gly
<210> 65
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 65
Gly Gly Gly Phe Leu Trp Phe Gly Glu Phe
1 5 10
<210> 66
<211> 119
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> VH
<400> 66
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 Ser Ser
20 25 30
Gly Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Tyr Ile Ser Gly Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Arg 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 Thr Gly Gly Gly Phe Leu Trp Phe Gly Glu Phe Trp Gly Gln Gly
100 105 110
Thr Thr Val Thr Val Ser Ser
115
<210> 67
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 67
Ile Ile Tyr Pro Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys Gly
1 5 10 15
<210> 68
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 68
Glu Thr Pro Asn Ala Gly Ser Leu Asp Tyr
1 5 10
<210> 69
<211> 118
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> VH
<400> 69
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 Ile 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 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
Arg Glu Thr Pro Asn Ala Gly Ser Leu Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 70
<211> 13
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 70
Gly Gly Val Ala Pro Met Ala Gly Trp His Phe Asp Leu
1 5 10
<210> 71
<211> 121
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> VH
<400> 71
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 Phe Trp Val Arg Gln Ala Thr Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Gly Pro Ala Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr
85 90 95
Arg Gly Gly Val Ala Pro Met Ala Gly Trp His Phe Asp Leu Trp Gly
100 105 110
Arg Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 72
<211> 13
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 72
Gly Gly Ile Ala Ala Met Val Gly Trp His Phe Asp Leu
1 5 10
<210> 73
<211> 121
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> VH
<400> 73
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 Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Gly Pro Ala Gly Asp 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 Gly Gly Ile Ala Ala Met Val Gly Trp His Phe Asp Leu Trp Gly
100 105 110
Arg Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 74
<211> 23
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LFR1
<400> 74
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
20
<210> 75
<211> 23
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LFR1
<400> 75
Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys
20
<210> 76
<211> 15
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LFR2
<400> 76
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu Tyr
1 5 10 15
<210> 77
<211> 15
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LFR2
<400> 77
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr
1 5 10 15
<210> 78
<211> 15
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LFR2
<400> 78
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
1 5 10 15
<210> 79
<211> 32
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LFR3
<400> 79
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr
1 5 10 15
Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
20 25 30
<210> 80
<211> 32
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LFR3
<400> 80
Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
1 5 10 15
Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys
20 25 30
<210> 81
<211> 32
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LFR3
<400> 81
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
1 5 10 15
Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys
20 25 30
<210> 82
<211> 32
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LFR3
<400> 82
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
1 5 10 15
Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
20 25 30
<210> 83
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LFR4
<400> 83
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
1 5 10
<210> 84
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LFR4
<400> 84
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
1 5 10
<210> 85
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LFR4
<400> 85
Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
1 5 10
<210> 86
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LFR4
<400> 86
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
1 5 10
<210> 87
<211> 30
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HFR1
<400> 87
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 Val Ser
20 25 30
<210> 88
<211> 30
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HFR1
<400> 88
Gln 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
20 25 30
<210> 89
<211> 30
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HFR1
<400> 89
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 Ser
20 25 30
<210> 90
<211> 30
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HFR1
<400> 90
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 Ser Phe Ser
20 25 30
<210> 91
<211> 30
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HFR1
<400> 91
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
20 25 30
<210> 92
<211> 30
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HFR1
<400> 92
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 Ser Ile Ser
20 25 30
<210> 93
<211> 30
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HFR1
<400> 93
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
20 25 30
<210> 94
<211> 30
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HFR1
<400> 94
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 Ile Thr Val Ser
20 25 30
<210> 95
<211> 14
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HFR2
<400> 95
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser
1 5 10
<210> 96
<211> 14
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HFR2
<400> 96
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
1 5 10
<210> 97
<211> 14
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HFR2
<400> 97
Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met Gly
1 5 10
<210> 98
<211> 14
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HFR2
<400> 98
Trp Val Arg Gln Ala Thr Gly Lys Gly Leu Glu Trp Val Ser
1 5 10
<210> 99
<211> 32
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HFR3
<400> 99
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln
1 5 10 15
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
20 25 30
<210> 100
<211> 32
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HFR3
<400> 100
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln
1 5 10 15
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg
20 25 30
<210> 101
<211> 32
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HFR3
<400> 101
Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met Glu
1 5 10 15
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser
20 25 30
<210> 102
<211> 32
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HFR3
<400> 102
Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met Glu
1 5 10 15
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Asn
20 25 30
<210> 103
<211> 32
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HFR3
<400> 103
Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Ser Ser Leu Tyr Leu Gln
1 5 10 15
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
20 25 30
<210> 104
<211> 32
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HFR3
<400> 104
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln
1 5 10 15
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg
20 25 30
<210> 105
<211> 32
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HFR3
<400> 105
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln
1 5 10 15
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Thr
20 25 30
<210> 106
<211> 32
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HFR3
<400> 106
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln
1 5 10 15
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg
20 25 30
<210> 107
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HFR4
<400> 107
Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
1 5 10
<210> 108
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HFR4
<400> 108
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
1 5 10
<210> 109
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HFR4
<400> 109
Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser
1 5 10
<210> 110
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HFR4
<400> 110
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
1 5 10
<210> 111
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 111
Arg Ala Ser Arg Pro Ile Ser Asn Tyr Leu Asn
1 5 10
<210> 112
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR2
<400> 112
Ser Val Ser Arg Leu Gln Ser
1 5
<210> 113
<211> 107
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> VL
<400> 113
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 Arg Pro 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
His Ser Val Ser Arg 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 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 Glu Ile Lys
100 105
<210> 114
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR1
<400> 114
Asp Tyr Ala Met Ser
1 5
<210> 115
<211> 19
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 115
Ser Ile Arg Ser Lys Ala Tyr Gly Gly Thr Thr Glu Tyr Ala Ala Ser
1 5 10 15
Val Lys Gly
<210> 116
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 116
His Arg Tyr Asp Ser Ser Gly Tyr Tyr Pro Gly Pro Gly Phe Asp Tyr
1 5 10 15
<210> 117
<211> 127
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> VH
<400> 117
Glu 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 Thr Ala Ser Gly Phe Thr Phe Gly Asp Tyr
20 25 30
Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Ser Ile Arg Ser Lys Ala Tyr Gly Gly Thr Thr Glu Tyr Ala Ala
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile
65 70 75 80
Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Thr Glu His Arg Tyr Asp Ser Ser Gly Tyr Tyr Pro Gly Pro
100 105 110
Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 118
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Ab2001.02LCDR1; Ab2001.03LCDR1; Ab2001.11LCDR1; Ab2001.12LCDR1; Ab2001.13LCDR1; Ab2001.14LCDR1; Ab2001.23LCDR1; Ab2001.24LCDR1; Ab2001.25LCDR1; Ab2001.16LCDR1
<220>
<221> misc_feature
<222> (5)..(5)
<223> Xaa=Gly or Ser
<220>
<221> misc_feature
<222> (8)..(8)
<223> Xaa=Asn or Ser
<220>
<221> misc_feature
<222> (9)..(9)
<223> Xaa=Ser or Tyr
<220>
<221> misc_feature
<222> (11)..(11)
<223> Xaa=Ala or Asn
<400> 118
Arg Ala Ser Gln Xaa Ile Ser Xaa Xaa Leu Xaa
1 5 10
<210> 119
<211> 12
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Ab2001.04LCDR1; Ab2001.08LCDR1; Ab2001.09LCDR1; Ab2001.10LCDR1; Ab2001.19LCDR1
<220>
<221> misc_feature
<222> (1)..(1)
<223> Xaa=Gln or Arg
<220>
<221> misc_feature
<222> (5)..(5)
<223> Xaa=Asp, gly or Ser
<220>
<221> misc_feature
<222> (6)..(6)
<223> Xaa=Ile or Val
<220>
<221> misc_feature
<222> (7)..(7)
<223> Xaa=Asn or Ser
<220>
<221> misc_feature
<222> (8)..(8)
<223> Xaa=Gly, asn or Ser
<220>
<221> misc_feature
<222> (9)..(9)
<223> Xaa=Ser, trp or Tyr
<220>
<221> misc_feature
<222> (10)..(10)
<223> xaa=leu or Tyr
<220>
<221> misc_feature
<222> (11)..(11)
<223> Xaa=Ala, leu or Asn
<220>
<221> misc_feature
<222> (12)..(12)
<223> Xaa=Ala or absence of
<400> 119
Xaa Ala Ser Gln Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10
<210> 120
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Ab2001.02LCDR2; Ab2001.03LCDR2; Ab2001.11LCDR2; Ab2001.12LCDR2; Ab2001.13LCDR2; Ab2001.14LCDR2; Ab2001.23LCDR2; Ab2001.24LCDR2; Ab2001.25LCDR2; Ab2001.16LCDR2
<220>
<221> misc_feature
<222> (4)..(4)
<223> Xaa=Arg or Ser
<220>
<221> misc_feature
<222> (6)..(6)
<223> Xaa=Glu or Gln
<400> 120
Ala Ala Ser Xaa Leu Xaa Ser
1 5
<210> 121
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Ab2001.04LCDR2; Ab2001.08LCDR2; Ab2001.09LCDR2; Ab2001.10LCDR2; Ab2001.19LCDR2
<220>
<221> misc_feature
<222> (1)..(1)
<223> Xaa=Ala, asp or Gly
<220>
<221> misc_feature
<222> (4)..(4)
<223> Xaa=Asn, ser or Thr
<220>
<221> misc_feature
<222> (5)..(5)
<223> Xaa=Leu or Arg
<220>
<221> misc_feature
<222> (6)..(6)
<223> Xaa=Ala or Glu
<400> 121
Xaa Ala Ser Xaa Xaa Xaa Thr
1 5
<210> 122
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Ab2001.02LCDR3; Ab2001.03LCDR3; Ab2001.11LCDR3; Ab2001.12LCDR3; Ab2001.13LCDR3; Ab2001.14LCDR3; Ab2001.23LCDR3; Ab2001.24LCDR3; Ab2001.25LCDR3; Ab2001.16LCDR3
<220>
<221> misc_feature
<222> (8)..(8)
<223> Xaa=Pro or Ser
<220>
<221> misc_feature
<222> (9)..(9)
<223> Xaa=Ile or Thr
<220>
<221> misc_feature
<222> (10)..(10)
<223> Xaa=Thr or absence of
<400> 122
Gln Gln Ser Tyr Ser Thr Pro Xaa Xaa Xaa
1 5 10
<210> 123
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Ab2001.04LCDR3; Ab2001.08LCDR3; Ab2001.07LCDR3; Ab2001.09LCDR3; Ab2001.10LCDR3; Ab2001.19LCDR3
<220>
<221> misc_feature
<222> (4)..(4)
<223> Xaa=Asp or Gly
<220>
<221> misc_feature
<222> (5)..(5)
<223> Xaa=Asn or Ser
<220>
<221> misc_feature
<222> (6)..(6)
<223> xaa=leu or Ser
<220>
<221> misc_feature
<222> (8)..(8)
<223> Xaa= Ile, leu, gln or Val
<400> 123
Gln Gln Tyr Xaa Xaa Xaa Pro Xaa Thr
1 5
<210> 124
<211> 108
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Ab2001.02VL; Ab2001.03VL; Ab2001.11VL; Ab2001.12VL; Ab2001.13VL; Ab2001.14VL; Ab2001.23VL; Ab2001.24VL; Ab2001.25VL; Ab2001.16VL
<220>
<221> misc_feature
<222> (28)..(28)
<223> Xaa=Gly or Ser
<220>
<221> misc_feature
<222> (31)..(31)
<223> Xaa=Asn or Ser
<220>
<221> misc_feature
<222> (32)..(32)
<223> Xaa=Ser or Tyr
<220>
<221> misc_feature
<222> (34)..(34)
<223> Xaa=Ala or Asn
<220>
<221> misc_feature
<222> (48)..(48)
<223> Xaa=lie or Leu
<220>
<221> misc_feature
<222> (53)..(53)
<223> Xaa=Arg or Ser
<220>
<221> misc_feature
<222> (55)..(55)
<223> Xaa=Glu or Gln
<220>
<221> misc_feature
<222> (71)..(71)
<223> Xaa=Phe or Tyr
<220>
<221> misc_feature
<222> (96)..(96)
<223> Xaa=Pro or Ser
<220>
<221> misc_feature
<222> (97)..(97)
<223> Xaa=lie or absence of
<220>
<221> misc_feature
<222> (104)..(104)
<223> Xaa=Lys or Arg
<220>
<221> misc_feature
<222> (105)..(105)
<223> Xaa=Leu or Val
<400> 124
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 Xaa Ile Ser Xaa Xaa
20 25 30
Leu Xaa Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Xaa
35 40 45
Tyr Ala Ala Ser Xaa Leu Xaa Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Xaa 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 Thr Pro Xaa
85 90 95
Xaa Thr Phe Gly Gln Gly Thr Xaa Xaa Glu Ile Lys
100 105
<210> 125
<211> 108
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Ab2001.04VL; Ab2001.08VL; Ab2001.09VL; Ab2001.10VL; Ab2001.19VL
<220>
<221> misc_feature
<222> (1)..(1)
<223> Xaa=Asp or Glu
<220>
<221> misc_feature
<222> (3)..(3)
<223> Xaa=Gln or Val
<220>
<221> misc_feature
<222> (4)..(4)
<223> xaa=leu or Met
<220>
<221> misc_feature
<222> (9)..(9)
<223> Xaa=Gly or Ser
<220>
<221> misc_feature
<222> (10)..(10)
<223> Xaa=Ser or Thr
<220>
<221> misc_feature
<222> (13)..(13)
<223> Xaa=Ala or Leu
<220>
<221> misc_feature
<222> (15)..(15)
<223> Xaa=Pro or Val
<220>
<221> misc_feature
<222> (17)..(17)
<223> Xaa=Asp or Glu
<220>
<221> misc_feature
<222> (19)..(19)
<223> Xaa=Ala or Val
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa=lie or Leu
<220>
<221> misc_feature
<222> (22)..(22)
<223> Xaa=Ser or Thr
<220>
<221> misc_feature
<222> (24)..(24)
<223> Xaa=Gln or Arg
<220>
<221> misc_feature
<222> (28)..(28)
<223> Xaa=Asp, gly or Ser
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa =
<220>
<221> misc_feature
<222> (30)..(30)
<223> Xaa=Asn or Ser
<220>
<221> misc_feature
<222> (31)..(31)
<223> Xaa=Gly, asn or Ser
<220>
<221> misc_feature
<222> (32)..(32)
<223> Xaa=Ser or absence of
<220>
<221> misc_feature
<222> (33)..(33)
<223> Xaa=Trp or Tyr
<220>
<221> misc_feature
<222> (35)..(35)
<223> Xaa=Ala or Asn
<220>
<221> misc_feature
<222> (43)..(43)
<223> Xaa=Lys or Gln
<220>
<221> misc_feature
<222> (46)..(46)
<223> Xaa=Lys or Arg
<220>
<221> misc_feature
<222> (51)..(51)
<223> Xaa=Ala, asp or Gly
<220>
<221> misc_feature
<222> (54)..(54)
<223> Xaa=Asn, ser or Thr
<220>
<221> misc_feature
<222> (55)..(55)
<223> Xaa=Leu or Arg
<220>
<221> misc_feature
<222> (56)..(56)
<223> Xaa=Ala or Glu
<220>
<221> misc_feature
<222> (59)..(59)
<223> Xaa=Ile or Val
<220>
<221> misc_feature
<222> (61)..(61)
<223> Xaa=Asp or Ser
<220>
<221> misc_feature
<222> (74)..(74)
<223> Xaa=Phe or Leu
<220>
<221> misc_feature
<222> (78)..(78)
<223> Xaa=Arg or Ser
<220>
<221> misc_feature
<222> (80)..(80)
<223> Xaa=Glu or Gln
<220>
<221> misc_feature
<222> (84).(84)
<223> Xaa=Phe or Ile
<220>
<221> misc_feature
<222> (86)..(86)
<223> Xaa=Thr or Val
<220>
<221> misc_feature
<222> (93)..(93)
<223> Xaa=Asp or Gly
<220>
<221> misc_feature
<222> (94)..(94)
<223> Xaa=Asn or Ser
<220>
<221> misc_feature
<222> (95)..(95)
<223> xaa=leu or Ser
<220>
<221> misc_feature
<222> (97)..(97)
<223> Xaa= Ile, leu, gln or Val
<220>
<221> misc_feature
<222> (101)..(101)
<223> Xaa=Gly or Gln
<220>
<221> misc_feature
<222> (104)..(104)
<223> Xaa=Lys or Arg
<220>
<221> misc_feature
<222> (105)..(105)
<223> Xaa=Leu or Val
<400> 125
Xaa Ile Xaa Xaa Thr Gln Ser Pro Xaa Xaa Leu Ser Xaa Ser Xaa Gly
1 5 10 15
Xaa Arg Xaa Thr Xaa Xaa Cys Xaa Ala Ser Gln Xaa Xaa Xaa Xaa Xaa
20 25 30
Xaa Leu Xaa Trp Tyr Gln Gln Lys Pro Gly Xaa Ala Pro Xaa Leu Leu
35 40 45
Ile Tyr Xaa Ala Ser Xaa Xaa Xaa Thr Gly Xaa Pro Xaa Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Xaa Thr Ile Ser Xaa Leu Xaa
65 70 75 80
Pro Glu Asp Xaa Ala Xaa Tyr Tyr Cys Gln Gln Tyr Xaa Xaa Xaa Pro
85 90 95
Xaa Thr Phe Gly Xaa Gly Thr Xaa Xaa Glu Ile Lys
100 105

Claims (30)

1. An isolated antigen binding protein comprising an LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 selected from the group consisting of:
(i) LCDR1 is composed of the amino acid sequence shown in SEQ ID NO. 1, LCDR2 is composed of the amino acid sequence shown in SEQ ID NO. 2, LCDR3 is composed of the amino acid sequence shown in SEQ ID NO. 3, HCDR1 is composed of the amino acid sequence shown in SEQ ID NO. 5, HCDR2 is composed of the amino acid sequence shown in SEQ ID NO. 6, and HCDR3 is composed of the amino acid sequence shown in SEQ ID NO. 7;
(ii) LCDR1 is composed of the amino acid sequence shown in SEQ ID NO. 1, LCDR2 is composed of the amino acid sequence shown in SEQ ID NO. 2, LCDR3 is composed of the amino acid sequence shown in SEQ ID NO. 3, HCDR1 is composed of the amino acid sequence shown in SEQ ID NO. 5, HCDR2 is composed of the amino acid sequence shown in SEQ ID NO. 9, and HCDR3 is composed of the amino acid sequence shown in SEQ ID NO. 7;
(iii) LCDR1 is composed of the amino acid sequence shown in SEQ ID NO. 18, and LCDR2 is composed of SEQ ID
The amino acid sequence shown in SEQ ID NO. 19, LCDR3 consists of the amino acid sequence shown in SEQ ID NO. 20, HCDR1 consists of the amino acid sequence shown in SEQ ID NO. 22, HCDR2 consists of the amino acid sequence shown in SEQ ID NO. 23, and HCDR3 consists of the amino acid sequence shown in SEQ ID NO. 24;
has one or more of the following properties:
1) Blocking the binding of RBD of S protein of SARS-CoV-2 or mutant thereof to human ACE 2;
2) Blocking the binding of RBD of S protein of SARS-CoV to human ACE 2;
3) In the Octet assay, at 5.0 x 10 -8 K below M D Value RBD that specifically binds to S protein of SARS-CoV-2;
4) In the Octet assay, 6.0×10 -10 K below M D Mutants of RBD that specifically bind to the S protein of SARS-CoV-2;
5) The affinity of RBD with S protein of SARS-CoV-2 is stronger than that of human ACE2-Fc;
6) RBD that specifically binds to S protein of SARS-CoV;
7) Hydrophilic;
8) The main peak of the charge heterogeneity analysis is 45% -85%;
9) In a Thermal shift assay, the Tm is at least 75 ℃;
10 Has activity in neutralizing SARS-CoV-2.
2. The isolated antigen binding protein of claim 1, comprising an antibody or antigen binding fragment thereof.
3. The isolated antigen binding protein of claim 2, wherein the antigen binding fragment comprises a Fab, fab ', F (ab) 2, fv fragment, F (ab') 2, scFv, and/or di-scFv.
4. The isolated antigen binding protein of claim 2, wherein the antibody is a fully human antibody.
5. The isolated antigen binding protein of any one of claims 1-4, comprising a light chain variable region comprising framework regions L-FR1, L-FR2, L-FR3, and L-FR4.
6. The isolated antigen binding protein of claim 5, wherein the C-terminus of the L-FR1 is directly or indirectly linked to the N-terminus of the LCDR1 and the L-FR1 consists of the amino acid sequence shown in SEQ ID No. 74.
7. The isolated antigen binding protein of any one of claims 5, wherein the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 consists of an amino acid sequence of any one of SEQ ID NOs 76 and 78.
8. The isolated antigen binding protein of claim 5, wherein the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 consists of an amino acid sequence of any one of SEQ ID NOs 79, 81.
9. The isolated antigen binding protein of claim 5, wherein the N-terminus of L-FR4 is directly or indirectly linked to the C-terminus of LCDR3 and the L-FR4 consists of the amino acid sequence of any one of SEQ ID No. 83, SEQ ID No. 84.
10. The isolated antigen binding protein of any one of claims 1-4, comprising a light chain variable region consisting of the amino acid sequence set forth in any one of SEQ ID NOs 4, 21.
11. The isolated antigen binding protein of any one of claims 1-4, comprising an antibody light chain constant region, and the antibody light chain constant region comprises a human igκ constant region or a human igλ constant region.
12. The isolated antigen binding protein of any one of claims 1-4, comprising a heavy chain variable region comprising framework regions H-FR1, H-FR2, H-FR3, and H-FR4.
13. The isolated antigen binding protein of claim 12, wherein the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1 and the H-FR1 consists of the amino acid sequence of any one of SEQ ID NOs 87, 88.
14. The isolated antigen binding protein of claim 12, wherein the H-FR2 is located at the
Between HCDR1 and said HCDR2, and said H-FR2 consists of an amino acid sequence set forth in any one of SEQ ID NO:95, SEQ ID NO: 96.
15. The isolated antigen binding protein of claim 12, wherein the H-FR3 is located at the
Between HCDR2 and said HCDR3, and said H-FR3 consists of an amino acid sequence as set forth in any one of SEQ ID NO 99, SEQ ID NO 101.
16. The isolated antigen binding protein of claim 12, wherein the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the HCDR3 and the H-FR4 consists of the amino acid sequence of SEQ ID No. 107.
17. The isolated antigen binding protein of any one of claims 1-4, comprising a heavy chain variable region consisting of an amino acid sequence set forth in any one of SEQ ID NOs 8, 10, 25.
18. The isolated antigen binding protein of claim 10, comprising a heavy chain variable region consisting of an amino acid sequence set forth in any one of SEQ ID NOs 8, 10, 25.
19. The isolated antigen binding protein of any one of claims 1-4, comprising an antibody heavy chain constant region, and the antibody heavy chain constant region comprises a human IgG constant region.
20. The isolated antigen binding protein of claim 19, wherein the human IgG constant region is a human IgG1 constant region.
21. The isolated antigen binding protein of any one of claims 1-4, comprising a light chain variable region and a heavy chain variable region, wherein
i) The light chain variable region consists of an amino acid sequence shown in SEQ ID NO. 4, and the heavy chain variable region consists of an amino acid sequence shown in SEQ ID NO. 8;
ii) the light chain variable region consists of the amino acid sequence shown in SEQ ID NO. 4 and the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO. 10;
iii) The light chain variable region consists of the amino acid sequence shown in SEQ ID NO. 21, and the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO. 25.
22. An isolated one or more nucleic acid molecules encoding the isolated antigen binding protein of any one of claims 1-21.
23. A vector comprising the nucleic acid molecule of claim 22.
24. A cell comprising the nucleic acid molecule of claim 22 or the vector of claim 23.
25. A method of making the isolated antigen binding protein of any one of claims 1-21, the method comprising culturing the cell of claim 24 under conditions such that the isolated antigen binding protein of any one of claims 1-21 is expressed.
26. A pharmaceutical composition comprising the isolated antigen binding protein of any one of claims 1-21, the nucleic acid molecule of claim 22, the vector of claim 23 and/or the cell of claim 24, and optionally a pharmaceutically acceptable adjuvant.
27. Use of the isolated antigen binding protein of any one of claims 1-21, the nucleic acid molecule of claim 22, the vector of claim 23, the cell of claim 24 and/or the pharmaceutical composition of claim 26 in the manufacture of a medicament for preventing, alleviating and/or treating an infection by a coronavirus.
28. The use of claim 27, wherein the infection by coronavirus comprises covd-19.
29. A non-diagnostic and therapeutic method of blocking the binding of RBD of S protein of SARS-CoV-2 or a mutant thereof to human ACE2 comprising the step of administering the isolated antigen binding protein of any one of claims 1-21, the nucleic acid molecule of claim 22, the vector of claim 23, the cell of claim 24 and/or the pharmaceutical composition of claim 26.
30. A non-diagnostic and therapeutic method of blocking the binding of RBD of S protein of SARS-CoV to human ACE2 comprising the step of administering the isolated antigen binding protein of any one of claims 1-21, the nucleic acid molecule of claim 22, the vector of claim 23, the cell of claim 24 and/or the pharmaceutical composition of claim 26.
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