WO2021190500A1

WO2021190500A1 - Preparation and application of cross-neutralizing antibody of sars-cov-2 and sars-cov

Info

Publication number: WO2021190500A1
Application number: PCT/CN2021/082374
Authority: WO
Inventors: 谢良志; 孙春昀; 孔德生
Original assignee: 神州细胞工程有限公司
Priority date: 2020-03-25
Filing date: 2021-03-23
Publication date: 2021-09-30
Also published as: CN115279785A

Abstract

Involved is the preparation of a plurality of cross-neutralizing humanized monoclonal antibodies. The antibodies can cross-block the binding of SARS-CoV-2 and SARS-CoV spike proteins (S proteins) to an ACE2 receptor, and efficiently neutralize cells infected by SARS-CoV-2 and SARS-CoV viruses. Provided are a nucleic acid sequence (comprising a heavy chain/light chain variable region) encoding the antibodies, a carrier comprising the nucleic acid sequence, a pharmaceutical composition, and a reagent kit. The prepared cross-neutralizing humanized antibodies can be used as a specific antibody medication for simultaneously preventing and treating an acute respiratory infectious disease caused by the SARS-CoV-2 and SARS-CoV viruses. The cross-neutralizing antibodies also have the function of diagnosis, etc.

Description

Preparation and application of SARS-CoV-2 and SARS-CoV cross-neutralizing antibodies

Cross-references to related applications

This application claims the rights and interests of the Chinese patent application 202010219867.1 filed on March 25, 2020, the content of which is incorporated herein by reference.

Technical field

The present invention relates to the technical field of cellular immunity, and provides multiple cross-blocking SARS-CoV-2 and SARS-CoV spike protein (S protein) binding to ACE2 receptors, and efficient neutralization of SARS-CoV-2 and SARS-CoV Humanized antibodies that infect cells with viruses. It can be used to treat infectious diseases caused by SARS-CoV-2 and SARS-CoV. The present invention also provides a nucleic acid sequence encoding the antibody, a vector and a cell containing the nucleic acid sequence.

Background technique

In December 2019, the new coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2) infection broke out in Wuhan, and the number of patients with new coronavirus pneumonia (Corona Virus Disease 2019, COVID-19) caused by the virus infection was in a short period of time. The rapid increase within the country has triggered the spread of the epidemic [1]. The spread of SARS-CoV-2 from person to person is similar to SARS-CoV (Severe Acute Respiratory Syndrome Coronavirus) and MERS-CoV (Middle East Respiratory Syndrome). It is mainly spread by respiratory droplets and can also be spread through contact. The main source of infection of SARS-CoV-2 is COVID-19 patients, and those with asymptomatic infection may also become the source of infection. Relevant studies have shown that the basic infection number (R0 value) of SARS-CoV-2 is between 2.24 and 3.58, suggesting that it has a strong transmission capacity [2]. The population is generally susceptible to SARS-CoV-2, with an incubation period of 1-14 days, mostly 3-7 days. The main clinical symptoms are fever, dry cough, and fatigue. Mild patients only manifested as low-grade fever, mild fatigue, etc., without pneumonia. Severely ill patients often manifest as dyspnea and/or hypoxemia, acute respiratory distress syndrome, septic shock, difficult to correct metabolic acidosis, coagulation dysfunction, and multiple organ failure [3,4]. As an acute respiratory infectious disease, COVID-19 has been included in the Class B infectious disease stipulated in the "Law of the People's Republic of China on the Prevention and Control of Infectious Diseases" and is managed as Class A.

SARS-CoV-2 and SARS-CoV share a common host cell receptor protein, angiotensin converting enzyme 2 (ACE2) [5]. The trimeric spike protein (S protein) of the virus binds to the ACE2 receptor and is cleaved by the host protease into the S1 polypeptide containing the receptor binding domain (RBD) and the S2 polypeptide responsible for mediating the fusion of the virus with the cell membrane. Then invade the body [6]. Therefore, finding and preparing effective antibodies to prevent the SARS-CoV-2 RBD protein from binding to the ACE2 receptor, thereby inhibiting the virus from infecting cells, has become one of the methods to prevent and treat SARS-CoV-2 virus infection. At present, clinical exploration of patients' recovery period plasma therapy has been used, and positive therapeutic effects have been achieved. However, the source of plasma in the recovery period is limited, the operation is troublesome, the cost is high, and there are problems such as safety. By preparing anti-SARS-CoV-2 RBD protein monoclonal antibodies, screening neutralizing antibodies that can specifically bind to them, and further humanizing them has become an effective means of preparing preventive or therapeutic antibody drugs. The structure of the S protein between SARS-CoV-2 and SARS-CoV is similar and the RBD amino acid sequence homology is relatively high, about 75% and 73.7%, respectively. In view of the common receptors and high sequence homology between the two, screening antibodies that can effectively neutralize SARS-CoV-2 and SARS-CoV at the same time may become a specific anti-coronavirus drug.

As there is no therapeutic drug for SARS-CoV-2 infection, the current treatment options for COVID-19 are mostly to relieve symptoms, prevent secondary infections, reduce complications, and provide organ function support. Therefore, there is an urgent need in the art to develop high-affinity coronavirus neutralizing antibodies, especially monoclonal antibodies, with good virus neutralizing effects. The humanized monoclonal antibody of the invention can effectively neutralize SARS-CoV-2 and SARS-CoV viruses, and can be used as a specific antibody drug to prevent and treat acute SARS-CoV-2 and SARS-CoV viruses at the same time. Infectious respiratory diseases.

Summary of the invention

The first aspect of the present invention provides an isolated, blocking SARS-CoV-2 spike protein and/or SARS-CoV spike protein and ACE2 receptor binding antibody or antigen-binding fragment thereof, which comprises a)-d ), where

a) i) Heavy chain variable region, whose heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 domains are respectively SEQ ID NOs: 13, 14 and 15 or at least 85%, 88%, 90%, 95% , 98% or 99% sequence identity,

ii) The light chain variable region, whose light chain CDR1, light chain CDR2 and light chain CDR3 domains are respectively SEQ ID NOs: 10, 11 and 12 or have at least 75%, 78%, 80%, 85%, 90 %, 91%, 95%, 98% or 99% sequence identity;

b) i) The heavy chain variable region, whose heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 domains are respectively SEQ ID NO: 13, 14 and 15 with at least 85%, 88%, 90%, 95%, 98% or 99% sequence identity,

ii) The light chain variable region, whose light chain CDR1, light chain CDR2 and light chain CDR3 domains are respectively SEQ ID NO: 45, 11 and 46 or at least 75%, 78%, 80%, 85%, 90 %, 91%, 95%, 98% or 99% sequence identity;

c) i) The heavy chain variable region, whose heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 domains are respectively SEQ ID NOs: 67, 68 and 69 with at least 85%, 88%, 90%, 95%, 98% or 99% sequence identity,

ii) The light chain variable region, whose light chain CDR1, light chain CDR2 and light chain CDR3 domains are respectively SEQ ID NOs: 10, 11 and 12 or have at least 75%, 78%, 80%, 85%, 90 %, 91%, 95%, 98%, or 99% sequence identity; and

d) i) The heavy chain variable region, whose heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 domains are respectively SEQ ID NO: 67, 68, and 69 with at least 85%, 88%, 90%, 95%, 98% or 99% sequence identity,

ii) The light chain variable region, whose light chain CDR1, light chain CDR2 and light chain CDR3 domains are respectively SEQ ID NO: 45, 11 and 12 or have at least 75%, 78%, 80%, 85%, 90 %, 91%, 95%, 98%, or 99% sequence identity.

In a specific embodiment, the antibody or antigen-binding fragment thereof comprises any one of a) to d), wherein:

a) i) Heavy chain variable region, whose sequence is SEQ ID NO: 22 or has at least 85%, 88%, 90%, 95%, 98%, or 99% sequence identity with it;

ii) The light chain variable region, whose sequence is SEQ ID NO: 23 or has at least 85%, 88%, 90%, 95%, 98%, or 99% sequence identity with it;

b) i) Heavy chain variable region, whose sequence is SEQ ID NO: 51 or has at least 85%, 88%, 90%, 95%, 98% or 99% sequence identity with it;

ii) The light chain variable region, whose sequence is SEQ ID NO: 52 or has at least 85%, 88%, 90%, 95%, 98%, or 99% sequence identity with it;

c) i) The heavy chain variable region, whose sequence is SEQ ID NO: 74 or has at least 85%, 88%, 90%, 95%, 98%, or 99% sequence identity with it;

ii) The light chain variable region whose sequence is SEQ ID NO: 75 or has at least 85%, 88%, 90%, 95%, 98%, or 99% sequence identity with it;

d) i) Heavy chain variable region, whose sequence is SEQ ID NO: 94 or has at least 85%, 88%, 90%, 95%, 98% or 99% sequence identity with it;

ii) The light chain variable region whose sequence is SEQ ID NO: 95 or has at least 85%, 88%, 90%, 95%, 98%, or 99% sequence identity with it.

The average KD of its binding affinity to SARS-CoV-2 S1 is 0.9E-11～8.7E-10M, preferably 2.0E-11～3E-10M, more preferably 2.6E-10, 2.9E-10, 2.1E -10 and 2.7E-11M; the average KD of its binding affinity to SARS-CoV S1 is 0.4E-11～6.0E-10M, preferably 1.0E-11～8E-10M, more preferably 1.2E-11, 1.1 E-10, 2.0E-10 and 7.5E-11M.

After being administered to mice by a single intravenous injection, the average in vivo exposure C _max and AUC _last were 136.15 μg/mL and 10930.35 h×μg/mL, respectively, the average half-life t _1/2 was 281.20 h, and the clearance rate Cl was 0.27mL/h/kg.

In one embodiment, the antibody is expressed by Fut8 knockout mammalian cells. Preferably, the cell is expressed by Fut8 knockout HEK-293, which exhibits significantly better binding ability to CD16a than IgG1 subtype, At high concentrations, it has weak binding to CD32a or CD32b protein, and the binding level of CD64, C1q complement protein and FcRn is similar to that of IgG1 subtype antibody; it is significantly better than the ADCC function of IgG1 subtype and its similar ADCP function, and the CDC function has no change .

In one embodiment, the antibody further comprises:

The heavy chain constant region, preferably, its sequence is SEQ ID NO: 106 or has at least 90%, 92%, 95%, 98% or 99% sequence identity with it;

The light chain constant region, preferably, its sequence is SEQ ID NO: 25 or has at least 90%, 92%, 95%, 98% or 99% sequence identity with it. It has the following characteristics: no binding to CD32a, CD32b, CD16a and C1q complement proteins, low binding to CD64 under high concentration conditions, and binding to FcRn similar to IgG1 subtype antibodies under pH 6.0; no significant ADCC, CDC and ADCP functions; after a single intravenous injection to mice, the average exposure C _max and AUC _last in vivo were 144.66μg/mL and 11940.01h×μg/mL, respectively, and the average half-life t _1/2 was 290.08h. The clearance rate Cl is 0.26mL/h/kg.

In one embodiment, the antibody further comprises:

i) The heavy chain constant region, preferably, its sequence is SEQ ID NO: 108 or has at least 90%, 92%, 95%, 98% or 99% sequence identity with it;

ii) The light chain constant region, preferably, its sequence is SEQ ID NO: 25 or has at least 90%, 92%, 95%, 98% or 99% sequence identity with it. It has the following characteristics: no binding to CD32a, CD32b, CD16a, CD64 and C1q complement proteins, and a very weak binding level to FcRn under pH 6.0 and high concentration conditions; basically no ADCC, CDC and ADCP functions. After a single intravenous injection to mice, the average in vivo exposure C _max and AUC _last were 125.11μg/mL and 1202.18h×μg/mL, respectively, the average half-life t _{1/2 was} only 11.72h, and the clearance rate Cl was 4.13mL/h/kg.

In one embodiment, it is a monoclonal antibody.

In one embodiment, it is Fv, Fab, Fab', Fab'-SH, F(ab')2, Fd fragment, Fd' fragment, single-chain antibody molecule or single-domain antibody; wherein the single-chain antibody molecule is preferably scFv, di-scFv, tri-scFv, diabody or scFab.

In one embodiment, the epitope is the structural region of SARS-CoV-2 and SARS-CoV virus spike proteins containing S375, K378, D405 and R408.

The second aspect of the present invention relates to an antibody-drug conjugate comprising the aforementioned antibody or antigen-binding fragment thereof and another therapeutic agent, preferably the antibody or antigen-binding fragment thereof and the other therapeutic agent are connected by a linker .

The third aspect of the present invention relates to a nucleic acid, which encodes the aforementioned antibody or antigen-binding fragment thereof. It can be DNA and/mRNA.

In one embodiment, it comprises

a) The nucleotide sequence of the heavy chain variable region shown in SEQ ID NO: 30, 55, 78 and 98 and/or the light chain variable region shown in SEQ ID NO: 31, 56, 79 and 99, respectively Nucleotide sequence; and optionally

b) The nucleotide sequence of the heavy chain constant region shown in SEQ ID NO: 6, 105 and 107 and/or the nucleotide sequence of the light chain constant region shown in SEQ ID NO: 7 respectively; or a) and b) Variants.

The third aspect of the present invention relates to an expression vector comprising the aforementioned nucleic acid.

The fourth aspect of the present invention relates to a host cell comprising the aforementioned nucleic acid or the aforementioned expression vector.

The fifth aspect of the present invention relates to a method for producing the aforementioned antibody or antigen-binding fragment thereof, which comprises culturing the aforementioned host cell under conditions suitable for antibody expression, and recovering the expressed antibody from the culture medium.

The sixth aspect of the present invention relates to a pharmaceutical composition comprising the aforementioned antibody or antigen-binding fragment thereof

Or the aforementioned antibody-drug conjugate or the aforementioned nucleic acid or the aforementioned expression vector, and a pharmaceutically acceptable carrier,

Optionally one or more other therapeutic agents.

The seventh aspect of the present invention relates to the aforementioned antibody or its antigen-binding fragment or the aforementioned antibody-drug conjugate or the aforementioned pharmaceutical composition, which is used to prevent and treat SARS-CoV-2 and/or SARS-CoV infections. disease.

The eighth aspect of the present invention relates to the use of the aforementioned antibody or its antigen-binding fragment or the aforementioned antibody-drug conjugate for the preparation of a medicine for the prevention and treatment of diseases caused by SARS-CoV-2 and/or SARS-CoV infection application.

The ninth aspect of the present invention relates to a pharmaceutical combination comprising the aforementioned antibody or antigen-binding fragment thereof or the aforementioned antibody-drug conjugate or the aforementioned pharmaceutical composition and one or more additional therapeutic agents.

The tenth aspect of the present invention relates to a kit comprising the aforementioned antibody or antigen-binding fragment thereof or the aforementioned antibody-drug conjugate or the aforementioned pharmaceutical composition, preferably, further comprising a device for administration.

The eleventh aspect of the present invention relates to a method for preventing and treating diseases caused by SARS-CoV-2 and/or SARS-CoV infection, which comprises administering to a subject the aforementioned antibody or antigen-binding fragment thereof or the aforementioned antibody- The drug conjugate or the foregoing drug composition, the foregoing drug combination, or the foregoing kit.

The twelfth aspect of the present invention relates to an isolated, blocking SARS-CoV-2 spike protein/SARS-CoV spike protein and ACE2 receptor binding antibody or antigen-binding fragment thereof, and its binding epitope contains S375 , K378, D405 and R408 structure area.

The thirteenth aspect of the present invention relates to a SARS-CoV-2 spike protein/SARS-CoV spike protein binding epitope, which is comprised of SARS-CoV-2 spike protein/SARS-CoV spike protein The structure area of S375, K378, D405 and R408.

Description of the drawings

Figure 1: Screening of monoclonal phage cross-binding SARS-CoV-2 and SARS-CoV proteins.

Figure 2: The cross-binding ability of murine antibodies with SARS-CoV-2 and SARS-CoV S1 proteins.

Figure 3: Flow cytometric detection of the binding of mouse antibodies to SARS-CoV-2 S1 protein.

Figure 4: The murine antibody cross-competes the binding of ACE2 to SARS-CoV-2 or SARS-CoV and RBD protein.

Figure 5: Murine antibody cross-neutralizes SARS-CoV-2 and SARS-CoV pseudoviruses.

Figure 6: Humanized antibody binding ability to SARS-CoV-2 and SARS-CoV RBD.

Figure 7: Humanized antibodies compete for the binding of ACE2 protein to SARS-CoV-2 and SARS-CoV and RBD proteins.

Figure 8: The affinity detection of humanized antibody with SARS-CoV-2 S1 protein (A) and SARS-CoV S1 protein (B).

Figure 9: Humanized antibodies cross-neutralize SARS-CoV-2 and SARS-CoV pseudoviruses.

Figure 10: Epitope diagram of SARS-2-H014 epitope analysis (A) and ELISA test results (B). SARS-CoV-2 RBD in A is represented by a white surface model, and all designed mutation sites are represented by light gray. The identified SARS-2-H014 highly significant epitope and significant epitope are represented by black and dark gray respectively, and ACE2 is represented by The gray tube band model indicates.

Figure 11: Binding of SARS-2-H014 antibody with different Fc functional forms to CD16a.

Figure 12: Binding of SARS-2-H014 antibody with different Fc functional forms to CD32.

Figure 13: Binding of SARS-2-H014 antibody with different Fc functional forms to CD64.

Figure 14: Binding of SARS-2-H014 antibody with different Fc functional forms to C1q.

Figure 15: Binding of SARS-2-H014 antibody to FcRn in different Fc functional forms.

Figure 16: Different Fc functional forms of SARS-2-H014 antibody on ADCC mediated by target cells expressing SARS-CoV-2 S protein (A) or SARS-CoV S protein (B).

Figure 17: Different effector cells against different Fc functional forms SARS-2-H014 antibody on ADCP mediated by target cells expressing SARS-CoV-2 S protein (A) or SARS-CoV S protein (B).

Figure 18: Different Fc functional forms of SARS-2-H014 antibody on CDC mediated by target cells expressing SARS-CoV-2 S protein (A) or SARS-CoV S protein (B).

Figure 19: The mean blood concentration-time curve of mice after a single intravenous injection of SARS-2-H014 (n=4).

Figure 20: The mean blood concentration-time curve of mice after a single intravenous injection of SARS-2-H014-Fd11-IgG4 (n=6).

Figure 21: The mean blood concentration-time curve of mice after a single intravenous injection of SARS-2-H014-Fd19-IgG4 (n=4).

Detailed ways

definition

Unless otherwise specified, all technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the technical field to which the present invention belongs. For the purpose of the present invention, the following terms are further defined.

When used herein and in the appended claims, the singular forms "a", "an", "another" and "the" include plural referents unless the context clearly dictates otherwise.

The term "antibody" means an immunoglobulin molecule, and refers to any form of antibody that exhibits the desired biological activity. Including but not limited to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and multispecific antibodies (such as bispecific antibodies), and even antibody fragments. Typically, the full-length antibody structure preferably comprises 4 polypeptide chains, usually 2 heavy (H) chains and 2 light (L) chains connected to each other by disulfide bonds. Each heavy chain contains a heavy chain variable region and a heavy chain constant region. Each light chain contains a light chain variable region and a light chain constant region. In addition to this typical full-length antibody structure, its structure also includes other derivative forms.

The term "variable region" refers to the domain in the heavy or light chain of an antibody that is involved in the binding of the antibody to the antigen. The variable regions of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures and can be further subdivided into hypervariable regions interspersed in more conservative regions (called framework regions (FR)) (Called the complementarity determining region (CDR)).

The term "complementarity determining region" (CDR, such as CDR1, CDR2, and CDR3) refers to the amino acid residues of the variable region of an antibody, the presence of which is necessary for antigen binding. Each variable region usually has 3 CDR regions identified as CDR1, CDR2, and CDR3. Each complementarity determining region may contain amino acid residues from the “complementarity determining region” defined by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD). .1991)) and/or those residues from the "hypervariable loop" (Chothia and Lesk; J Mol Biol 196:901-917 (1987)).

The term "framework" or "FR" residues are those variable region residues other than the CDR residues as defined herein.

Each heavy chain variable region and light chain variable region usually contains 3 CDRs and up to 4 FRs, and the CDRs and FRs are arranged in the following order, for example, from the amino terminal to the carboxy terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The complementarity determining region (CDR) and framework region (FR) of a given antibody can be identified by the Kabat system (Kabat et al.: Sequences of Proteins of Immunological Interest, 5th edition, US Department of Health and Human Services, PHS, NIH, NIH publication No. 91-3242, 1991).

The term "constant region" refers to such amino acid sequences on the light chain and heavy chain of an antibody that do not directly participate in the binding of the antibody to the antigen, but exhibit a variety of effector functions, such as antibody-dependent cytotoxicity.

According to the amino acid sequence of the constant region of the heavy chain, complete antibodies can be classified into five types of antibodies: IgA, IgD, IgE, IgG, and IgM, among which IgG and IgA can be further divided into subclasses (isotypes), such as IgG1, IgG2 , IgG3, IgG4, IgA1 and IgA2. Correspondingly, the heavy chains of the five types of antibodies are classified into α, δ, ε, γ, and μ chains, respectively. According to the amino acid sequence of the constant region of its light chain, the light chain of an antibody can be classified into κ and λ. .

An "antigen-binding fragment of an antibody" includes a portion of a complete antibody molecule that retains at least some of the binding specificity of the parent antibody, and usually includes at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody. Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, Fd fragment, Fd' fragment, single-chain antibody molecules (e.g., scFv, di-scFv, or tri-scFv , Diabody or scFab), single domain antibody.

An "antibody fragment" is a non-complete antibody molecule that retains at least some biological properties of the parent antibody, and examples thereof include, but are not limited to, Fc fragments in addition to those mentioned in the above-mentioned "antigen-binding fragments".

The term "modified drug molecule" refers to an antibody or fragment thereof, such as an antigen-binding fragment that forms a covalent or non-covalent link with another molecule or forms a recombinant multi-target fusion drug, and the other molecule is selected from a small molecule compound or a biological Macromolecule.

The term "chimeric" antibody refers to an antibody in which a part of the heavy chain and/or light chain is derived from a specific source or species, and the remaining part is derived from a different source or species. "Humanized antibodies" are a subset of "chimeric antibodies."

The term "humanized antibody" or "humanized antigen-binding fragment" is defined herein as an antibody or antibody fragment: (i) derived from a non-human source (for example, a transgenic mouse carrying a heterologous immune system) And antibodies based on human germline sequences; or (ii) chimeric antibodies in which the variable region is of non-human origin and the constant region is of human origin; or (iii) CDR-grafted, in which the CDR of the variable region is derived from a non-human source, and One or more framework regions of the variable region are of human origin, and the constant region (if any) is of human origin. The purpose of "humanization" is to eliminate the immunogenicity of non-human source antibodies in the human body, while retaining the greatest possible affinity. It is advantageous to select a human framework sequence that is most similar to the framework sequence of a non-human source antibody as a template for humanization. In some cases, it may be necessary to replace one or more amino acids in the human framework sequence with corresponding residues in the non-human framework to avoid loss of affinity.

A "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, the population comprising a single antibody is identical except for possible mutations (such as natural mutations) that may be present in very small amounts. Therefore, the term "monoclonal" indicates the nature of the antibody, that is, it is not a mixture of unrelated antibodies. In contrast to polyclonal antibody preparations, which usually include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibody preparations is that they are generally not contaminated by other antibodies. The term "monoclonal" should not be understood as requiring the production of the antibody by any specific method. The term monoclonal antibody specifically includes chimeric antibodies, humanized antibodies and human antibodies.

The antibody "specifically binds" to an antigen of interest, such as a virus-associated antigen protein (herein, spike protein S), that is, binds to the antigen with sufficient affinity so that the antibody can be used as a therapeutic agent, and targets those expressing the antigen. Viruses or cells, and have no significant cross-reactivity with other proteins or with proteins other than homologs and variants (such as mutant forms, splice variants, or proteolytically truncated forms) of the antigen target mentioned above No significant cross-reaction.

The term "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule and its binding partner. Unless otherwise specified, "binding affinity" as used herein refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). "KD", "association rate constant k _on "and "dissociation rate constant k _off " are usually used to describe the affinity between a molecule (such as an antibody) and its binding partner (such as an antigen), that is, how tightly a ligand binds to a specific protein. degree. Binding affinity is affected by interactions between non-covalent molecules, such as hydrogen bonds, electrostatic interactions, hydrophobicity between two molecules and van der Waals forces. In addition, the binding affinity between the ligand and its target molecule may be affected by the presence of other molecules. Affinity can be analyzed by conventional methods known in the art, including the ELISA described herein.

The term "epitope" includes any protein determinant capable of specifically binding to an antibody or T cell receptor. Epitope determinants usually consist of chemically active surface groups of molecules (such as amino acids or sugar side chains, or combinations thereof), and usually have specific three-dimensional structural characteristics and specific charge characteristics.

"Isolated" antibodies are antibodies that have been identified and isolated from cells that naturally express the antibody. Isolated antibodies include in situ antibodies in recombinant cells as well as antibodies that are usually prepared through at least one purification step.

The "sequence identity" between two polypeptide or nucleic acid sequences means the percentage of the number of residues that are identical between the sequences to the total number of residues. When calculating the percent identity, the sequences being compared are aligned in a way that produces the largest match between the sequences, and the gaps in the alignment (if any) are resolved by a specific algorithm. Preferred computer program methods for determining the identity between two sequences include, but are not limited to, the GCG program package, including GAP, BLASTP, BLASTN and FASTA (Altschul et al., 1990, J. Mol. Biol. 215: 403-410) . The above procedures are publicly available from the International Center for Biotechnology Information (NCBI) and other sources. The well-known Smith Waterman algorithm can also be used to determine identity.

The term "receptor" is a biochemical concept that refers to a class of molecules that can transmit extracellular signals and produce specific effects in cells. The effect may only last for a short period of time, such as changing cell metabolism or cell movement. It may also be a long-term effect, such as up-regulating or down-regulating the expression of a certain gene or genes.

The term "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. Natural sequence human FcR is preferred, and receptors (γ receptors) that bind to IgG antibodies are preferred, which include FcγRI, FcγRII and FcγRIII subtypes, and variants of these receptors. Other FcRs are included in the term "FcR". The term also includes the neonatal receptor (FcRn), which is responsible for the transport of maternal IgG to the fetus (Guyer et al., Journal of Immunology 117:587 (1976) and Kim et al., Journal of Immunology 24:249 (1994)).

The term "neonatal Fc receptor", abbreviated as "FcRn", binds to the Fc region of an IgG antibody. The neonatal Fc receptor (FcRn) plays an important role in the metabolic fate of IgG antibodies in the body. FcRn functions to rescue IgG from the lysosomal degradation pathway, thereby reducing its clearance in serum and increasing its half-life. Therefore, the in vitro FcRn binding properties/characteristics of IgG indicate its in vivo pharmacokinetic properties in the blood circulation.

The term "effector function" refers to those biological activities attributable to the Fc region of an antibody, which vary with antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, "antibody-dependent cell-mediated cytotoxicity" (ADCC), antibody-dependent cellular phagocytosis (ADCP), Cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, down-regulation of cell surface receptors (such as B cell receptors), and B cell activation.

The term "effector cells" refers to leukocytes that express one or more FcRs and perform effector functions. In one aspect, the effector cell at least expresses FcyRIII and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from natural sources, for example, blood. Effector cells are usually lymphocytes associated with the effector stage and function to produce cytokines (helper T cells), kill cells infected by pathogens (cytotoxic T cells) or secrete antibodies (differentiated B cells) .

"Immune cells" include cells that have hematopoietic origin and play a role in immune responses. Immune cells include: lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which it binds to certain cytotoxic cells (such as NK cells, neutrophils, and macrophages). The secretion of Ig on the Fcγ receptor enables these cytotoxic effector cells to specifically bind to the target cell carrying the antigen, and then kill the target cell using, for example, a cytotoxin. In order to evaluate the ADCC activity of the antibody of interest, an in vitro ADCC assay can be performed, such as the in vitro ADCC assay described in U.S. Patent No. 5,500,362 or 5,821,337 or U.S. Patent No. 6,737,056 (Presta), and the method described in the Examples of this application . Useful effector cells for such assays include PBMC and NK cells.

"Complement dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. The activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass), wherein the antibody binds to its corresponding antigen. In order to assess complement activation, CDC assays can be performed, such as the CDC assays described in Gazzano-Santoro et al., J. Immunol Methods 202:163 (1996), such as the methods described in the examples of this application, such as The method described in U.S. Patent No. 6,194,551B1 and WO1999/51642, in which polypeptide variants with altered Fc region amino acid sequence (polypeptides with variant Fc region) and polypeptide variants with enhanced or reduced C1q binding are described .

"Antibody-dependent cellular phagocytosis" (ADCP) refers to a cell-mediated response in which non-specific cytotoxic cells expressing FcγR recognize bound antibodies on target cells and subsequently cause phagocytosis of target cells.

The amino acid sequence and nucleotide sequence of the antibody of the present invention, as well as Fc functional modification

The present invention firstly uses recombinant SARS-CoV RBD protein to immunize mice, and then obtains four scFv antibody clones that bind to SARS-CoV-2 and SARS-CoV RBD protein through phage antibody library screening. After that, the nucleotide sequences encoding the heavy chain and light chain variable regions of the scFv antibody were spliced with the nucleotide sequences encoding the mouse IgG1 heavy chain constant region and the mouse kappa light chain constant region, respectively, and inserted into the transient Transform the expression vector for culture expression. The protein A purification column was used for purification to obtain high-purity mouse antibodies.

The classical CDR transplantation method is used to humanize mouse antibodies [10,11]. Respectively, the similarity with the murine light chain and heavy chain variable regions is more than 50%, and the framework regions of the light chain and heavy chain variable regions are similar to the amino acid sequences of the framework regions of the light chain and heavy chain variable regions of the antibody to be modified Antibodies with sex above 50% are used as humanized templates. By comparing the IMGT human antibody heavy and light chain variable region germline gene database, the heavy and light chain variable region germline genes with high homology were selected as humanization templates, and the four mouse antibody light chain and The 3 CDR sequences of the heavy chain were respectively transplanted into the corresponding human template. Since the key points of the mouse-derived framework region are essential to support the activity of the CDR, the key points were backmutated to the sequence of the murine antibody. The light chain/heavy chain signal peptide sequence, the back-mutated humanized antibody light chain/heavy chain variable region sequence, and the human IgG4 heavy chain constant region/human kappa light chain constant region sequence were spliced in sequence to obtain the humanization The amino acid and nucleotide sequences of antibodies SARS-2-H014, SARS-2-H157, SARS-2-H202 and SARS-2-H697.

The present invention further modified the Fc function of SARS-2-H014. They are: 1) Defucosylated IgG1 subtype expressed in mammalian cells knocked out with Fut8 gene; 2) Humanized antibody SARS-2-H014-Fd11-IgG4 of IgG4 subtype with reduced Fc function and 3) The FcRn binding IgG4 subtype humanized antibody SARS-2-H014-Fd19-IgG4 was removed.

Nucleic acid of the present invention

The invention also relates to nucleic acid molecules encoding the antibodies of the invention or parts thereof. Some example sequences of these nucleic acid molecules are shown in the sequence listing.

The nucleic acid molecule of the present invention is not limited to the sequence disclosed herein, but also includes variants and other nucleic acid forms corresponding thereto, such as mRNA, cDNA and variants thereof. The variants of the present invention can be described with reference to their physical characteristics in hybridization. Those skilled in the art will recognize that using nucleic acid hybridization techniques, nucleic acids can be used to identify their complements and their equivalents or homologs. It will also be recognized that hybridization can occur with less than 100% complementarity. However, considering the appropriate selection of conditions, hybridization techniques can be used to distinguish DNA sequences based on their structural correlation with specific probes. For guidance on such conditions, see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 and Ausubel, FM, Brent, R., Kingston, RE, Moore, DD, Sedman, JG, Smith, JA, & Struhl, K. eds. (1995). Current Protocols in Molecular Biology. New York: John Wiley and Sons.

Recombinant vector and expression

The invention also provides a recombinant construct comprising one or more nucleotide sequences of the invention. The recombinant construct of the present invention can be used with a vector, such as a plasmid, phagemid, phage or viral vector, into which the nucleic acid molecule encoding the antibody of the present invention is inserted.

The antibodies provided herein can be prepared by recombinantly expressing nucleotide sequences encoding light and heavy chains or parts thereof in a host cell. In order to express the antibody in a recombinant method, one or more recombinant expression vectors carrying the nucleotide sequence encoding the light chain and/or the heavy chain or part thereof can be used to transfect the host cell so that the light chain and the heavy chain are in the Expressed in host cells. Standard recombinant DNA methodology is used to prepare and/or obtain nucleic acids encoding heavy and light chains, incorporate these nucleic acids into recombinant expression vectors and introduce the vectors into host cells, such as Sambrook, Fritsch and Maniatis (eds. ), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, (1989), Ausubel, FMet al. (eds.) Current Protocols in Molecular Biology, Greene Publishing, (1989) and Boss et al. Those described in US Patent No. 4,816,397.

In addition, the nucleotide sequence encoding the variable region of the heavy chain and/or light chain can be converted into, for example, a nucleotide sequence encoding a full-length antibody chain, Fab fragment or scFv: for example, the variable region encoding the light chain can be The DNA fragment of the region or heavy chain variable region is operably linked (so that the amino acid sequences encoded by the two DNA fragments are in frame) to another DNA fragment encoding, for example, an antibody constant region or a flexible linker. The sequences of human heavy and light chain constant regions are known in the art (see, for example, Kabat, EA, el. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242), DNA fragments including these regions can be obtained by standard PCR amplification.

To express the antibody, standard recombinant DNA expression methods can be used (see, for example, Goeddel; Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). For example, the nucleotide sequence encoding the desired antibody can be inserted into an expression vector, and then the expression vector can be transfected into a suitable host cell. Suitable host cells are prokaryotic cells and eukaryotic cells. Examples of prokaryotic host cells are bacteria, and examples of eukaryotic host cells are yeast, insect or mammalian cells. It should be understood that the design of the expression vector including the selection regulatory sequence is affected by many factors, such as the choice of host cell, the expression level of the desired protein, and whether the expression is constitutive or inducible.

The antibody of the present invention can be recovered and purified from recombinant cell culture by known methods, including but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, protein A affinity chromatography, protein G affinity chromatography, anion Or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification. See, for example, Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001), such as

Chapters

1, 4, 6, 8, 9, and 10, each by reference The full text is included in this article.

The antibodies of the present invention include natural purified products, products of chemical synthesis methods, and products produced by recombinant technology from prokaryotic and eukaryotic hosts. The eukaryotic hosts include, for example, yeast, higher plants, insects and mammalian cells. The antibodies of the invention can be glycosylated or non-glycosylated. Such methods are described in many standard laboratory manuals, such as Sambrook above, sections 17.37-17.42; Ausubel above,

chapters

10, 12, 13, 16, 18 and 20.

Therefore, an embodiment of the present invention is also a host cell comprising the vector or nucleic acid molecule, wherein the host cell may be a higher eukaryotic host cell such as mammalian and insect cells, a lower eukaryotic host cell such as yeast cell, and It may be a prokaryotic cell such as a bacterial cell.

Characteristics and functions of the antibody of the present invention

The ELISA test showed that the obtained four mouse antibodies SARS-2-mh014, SARS-2-mh157, SARS-2-mh202 and SARS-2-mh697 can block SARS-CoV-2 and SARS-CoV RBD and ACE combines and cross-neutralizes SARS-CoV-2 and SARS-CoV pseudoviruses.

Humanized antibodies SARS-2-H014, SARS-2-H157, SARS-2-H202 and SARS-2-H697 have good cross-binding and cross-competition with SARS-CoV-2 and SARS-CoV RBD proteins. The receptor has high affinity with SARS-CoV-2 and SARS-CoV RBD protein, and with SARS-CoV-2 S1 protein. Cross-neutralize SARS-CoV-2 and SARS-CoV pseudoviruses. The humanized SARS-2-H014 antibody can effectively neutralize the SARS-CoV-2 new coronavirus at the cellular level. After a single intravenous injection to mice, the average exposure C _max and AUC _last in the body were 136.15. μg/mL and 10930.35h×μg/mL, the average half-life t _1/2 was 281.20h, and the clearance rate Cl was 0.27mL/h/kg.

The humanized SARS-2-H014 antibody expressed by mammalian cells knocked out of the Fut8 gene showed significantly better binding capacity to CD16a than IgG1 subtype, and weakly binds to CD32a or CD32b protein at high concentrations, and The binding level of CD64, C1q complement protein and FcRn is similar to that of IgG1 subtype antibody; the ADCC function of IgG1 subtype and the similar ADCP function are significantly better than that of IgG1 subtype, and the CDC function has no change.

SARS-2-H014-Fd11-IgG4 antibody does not bind to CD32a, CD32b, CD16a and C1q complement proteins, and has a weak binding level to CD64 under high concentration conditions, and FcRn, which is similar to IgG1 subtype antibodies under pH 6.0 conditions Binding; no obvious ADCC, CDC and ADCP functions; after a single intravenous injection to mice, the average in vivo exposure C _max and AUC _last were 144.66μg/mL and 11940.01h×μg/mL, respectively, and the average half-life t _{1 /2} is 290.08h, and the clearance rate Cl is 0.26mL/h/kg.

SARS-2-H014-Fd19-IgG4 antibody has the following characteristics: it does not bind to CD32a, CD32b, CD16a, CD64 and C1q complement proteins, and has a very weak binding level to FcRn under pH 6.0 and high concentration conditions; basically no ADCC, CDC and ADCP functions. After a single intravenous injection to mice, the average in vivo exposure C _max and AUC _last were 125.11μg/mL and 1202.18h×μg/mL, respectively, the average half-life t _{1/2 was} only 11.72h, and the clearance rate Cl was 4.13mL/h/kg.

use

The antibody of the present invention can be used to treat, prevent or detect diseases caused by SARS-CoV-2 and SARS-CoV viruses, such as acute respiratory infections caused by SARS-CoV-2 and SARS-CoV viruses.

Pharmaceutical composition

One or more of the antibody, antigen-binding fragment, antibody-drug conjugate, nucleic acid, carrier of the present invention and at least one other chemical agent can be prepared into a pharmaceutical composition, which includes the above-mentioned active ingredients and one or more A pharmaceutically acceptable carrier, diluent or excipient; optionally, one or more other therapeutic agents may also be included.

Reagent test kit

The invention also relates to a pharmaceutical package and a kit comprising one or more containers containing the above-mentioned pharmaceutical composition of the invention. Related to this type of container may be a reminder in the form prescribed by a government agency that regulates the production, use, or sale of drugs or biological products, which reflects that the product is approved for human administration by the agency that produces, uses, or sells the product.

Preparation and storage

The pharmaceutical composition of the present invention can be prepared in a manner known in the art, for example, by conventional mixing, dissolving, granulating, grinding, emulsifying, wrapping, embedding or freeze-drying methods.

After the pharmaceutical composition containing the compound of the present invention formulated in an acceptable carrier has been prepared, they can be placed in a suitable container and labeled for the treatment of the indicated condition. Such labels would include the amount, frequency, and method of administration.

Drug combination

The above-mentioned pharmaceutical composition comprising the antibody of the present invention is also combined with one or more other therapeutic agents, wherein the resulting combination does not cause unacceptable adverse effects.

The following examples are used to exemplarily illustrate the present invention, but not to limit the present invention.

Example

Example 1: Screening of murine antibodies that cross-bind SARS-CoV-2 and SARS-CoV using phage antibody display library

1.1 Mouse Immunization

50 μg of recombinant SARS-CoV RBD protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd., Cat.40150-V08B2, SEQ ID NO:1) was mixed with Freund's adjuvant and injected subcutaneously to immunize mice. For specific immunization methods, refer to the literature [7].

1.2 Construction of phage antibody library

TriPure Isolation Reagent kit (source: Roche, Cat. No. 11 667 165 001) was used to extract RNA from mouse spleen tissue, and the reverse transcription kit Reverse transcription Kit (source: Beijing Yiqiao Shenzhou Technology Co., Ltd., Cat. No. .SRT) cDNA is obtained after reverse transcription. After PCR amplification of the mouse antibody light chain and heavy chain variable region nucleotide sequence, the overlap extension splicing PCR method was used to splice the nucleotide sequence encoding the mouse antibody light chain and heavy chain variable region sequence into a scFv encoding Nucleotide sequence, light and heavy chain variable regions through linker:

Connect [8], and then connect to the phage vector pComb3x (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) by restriction endonuclease Sfi I (source: Fermentas). Mouse phage display scFv antibody library.

1.3 Screening of cross-binding SARS-CoV-2 and SARS-CoV RBD phage

The solid-phase screening method was used to screen phages that bind to both SARS-CoV-2 and SARS-CoV RBD proteins. Solution 1: Coat the SARS-CoV S1 protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd., Cat.40150-V08B1) at a concentration of 10μg/mL on a 96-well plate, with 100μL per well, and coat overnight at 4°C. Perform the first round of screening. The plate was washed the next day and blocked at room temperature for 1 hour, then added the phage antibody library and incubated for 2 hours at 37°C, washed the plate to remove unbound phages, added 800 μL Elution buffer (source: Shenzhou Cell Engineering Co., Ltd.) and incubated for 8 minutes, and added 10 μL 2M Tris buffer to each well. Source: Shenzhou Cell Engineering Co., Ltd.) Zhonghe. The eluted phage was soaked into X-BLUE strain (source: Biomed) and added with helper phage for expression, and the expressed phage antibody library was collected the next day. In the second round of screening, 10 μg/mL SARS-CoV-2 RBD protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd., Cat.40592-V05H) was coated on a 96-well plate, and the screening method was the same as above. Scheme 2: Coat the SARS-CoV-2 RBD protein at a concentration of 5μg/mL on a 96-well plate, and the specific screening method is the same as above.

Monoclonal phages were picked from the enriched library for expression, and their binding to SARS-CoV-2 and SARS-CoV S protein was detected by ELISA. SARS-CoV-2 S1, SARS-CoV S1 (source: Beijing Yiqiao Shenzhou Technology Co., Ltd., Cat.40591-V05H), SARS-CoV-2 RBD, SARS-CoV RBD and negative control at a concentration of 5μg/mL CD155(D1)-mFc (source: Shenzhou Cell Engineering Co., Ltd.) protein was coated on a 96-well plate, 100 μL per well, and coated overnight at 4°C. The plate was washed the next day and blocked at room temperature for 2 hours. The plate was washed to remove unbound proteins, then 10 times diluted phage monoclonal was added to incubate, the plate was washed to remove unbound phage, and X-BLUE was added to incubate and the plate was washed repeatedly. Add the substrate color solution for color development, and read the OD ₄₅₀ by the microplate reader after termination. Take the detected protein as the abscissa and OD ₄₅₀ as the ordinate, analyze and plot with GraphPadPrism software.

The results are shown in Figure 1. From the enriched library, four scFv clones that specifically cross-bind with SARS-CoV-2 and SARS-CoV S1 and RBD proteins were obtained from the enriched library, namely SARS-2-m014, SARS-2 -m157, SARS-2-m202 and SARS-2-m697, the nucleotide sequence of scFv antibody (SEQ ID NO:3/40/62/85) was obtained by sequencing.

1.4 Production of mouse-derived antibodies that cross-conjugate SARS-CoV-2 and SARS-CoV and RBD

The nucleotide sequences of the heavy chain variable region of SARS-2-m014, SARS-2-m157, SARS-2-m202 and SARS-2-m697scFv antibodies were amplified by PCR, and inserted into the signal with heavy chain by In-fusion method The human-mouse chimeric antibody SARS was obtained from the pSE vector (source: self-prepared) digested with ScaI+NheI (source: Fermentas) of peptide (SEQ ID NO: 28) and human IgG1 constant region (SEQ ID NO: 6) -2-mh014 heavy chain (SEQ ID NO: 36), SARS-2-mh157 heavy chain (SEQ ID NO: 58), SARS-2-mh202 heavy chain (SEQ ID NO: 81) and SARS-2-mh697 heavy chain The expression vector of the chain (SEQ ID NO: 101). The nucleotide sequences of the light chain variable region of SARS-2-m014, SARS-2-m157, SARS-2-m202 and SARS-2-m697scFv antibodies were amplified by PCR, and inserted into the light chain signal by In-fusion method Peptide (SEQ ID NO: 29) and human kappa constant region (SEQ ID NO: 7) were digested with Sca I+BsiWI (source: Fermentas) pSE vector to obtain the human-mouse chimeric SARS-2-mh014 light chain ( SEQ ID NO: 37), SARS-2-mh157 light chain (SEQ ID NO: 59), SARS-2-mh202 light chain (SEQ ID NO: 82) and SARS-2-mh697 light chain (SEQ ID NO: 102) ) Expression vector.

Amplification variable region primers:

Passage 293E cells to 200 mL/bottle with SCD4-4-TC2 medium (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.), initial seeding density of 0.3～0.4×10 ⁶ _{cell/mL, CO 2} at 37°C and rotating speed of 175 rpm Cell culture is performed in a shaker. After the cell density reaches 1.5～3×10 ⁶ cells/mL, add a total of 100μg of light and heavy chain plasmid DNA mixed 1:1 and 800μL of TF2 transfection reagent (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.), and shake Continue to grow in the bed until the 7th day of material collection. The culture solution was centrifuged at 4000 rpm for 25 minutes, the supernatant was collected, and 1/5 of the supernatant volume was added to the stockbuffer (source: Shenzhou Cell Engineering Co., Ltd.). Equilibrate the protein A chromatography column (source: Shenzhou Cell Engineering Co., Ltd.) with PBS for 5-10 times the column volume, add the filtered culture supernatant to the chromatography column, and equilibrate again for 5-10 times the column volume, then use sodium acetate Buffer (source: Shenzhou Cell Engineering Co., Ltd.) to elute the sample. After the sample is eluted, it is neutralized to neutral with Tris buffer for later use.

Example 2: Functional detection of cross-binding SARS-CoV-2 and SARS-CoV murine antibodies

2.1 Cross-binding of mouse antibody with SARS-CoV-2 and SARS-CoV S1 protein

The 0.3μg/mL, 0.1μg/mL, 0.03μg/mL and 0.01μg/mL SARS-CoV-2 or SARS-CoV S1 protein were coated on 96-well plates with 100μL per well and coated overnight at 4°C. Wash the plate the next day, block at room temperature for 1 hour, add 100 μL of 1 μg/mL mouse antibody and incubate for 1 hour, then wash the plate to remove unbound antibody, add 0.25 μg/mL Goat anti-human IgG Fc/HRP (source: KPL) and incubate After that, the plate was washed repeatedly, and the substrate color developing solution was added for color development. After termination, the OD ₄₅₀ was detected.

The results are shown in Figure 2. The SARS-2-mh014, SARS-2-mh157, SARS-2-mh202, and SARS-2-mh697 antibodies have good results with SARS-CoV-2 S1 protein and SARS-CoV S1 protein. Cross-binding and concentration-dependent.

This example further verified the binding ability of the murine antibody to WH(2019)nCoV-SPIKE-8 D3 cells transiently expressing SARS-CoV-2 S1 protein by flow cytometry. Take WH(2019)-Ncov-SPIKE-8 D3 cells (source: Shenzhou Cell Engineering Co., Ltd.) in the logarithmic growth phase and place them in a flow tube, 5×10 ⁵ cells/tube. Add 4 murine antibodies at 16.67μg/mL, mix with H7N9-R1 negative control antibody (source: Shenzhou Cell Engineering Co., Ltd.), mix and incubate at 4°C for 20 minutes, wash with PBS washing solution, and centrifuge to remove unbound antibodies. Add FITC-labeled Goat anti-Human IgG Fc secondary antibody (source: KPL company), mix and incubate at 4°C for 20 minutes, and then repeat washing with PBS. Cells were resuspended in 200μL PBS, filtered with 400 mesh, and tested by flow cytometer. The results are shown in Figure 3. The four mouse-derived antibodies all have good binding to WH(2019)-nCoV-SPIKE-8 D3 cells, and SARS-2-mh014 has a slightly higher binding capacity, and the negative control has no binding.

2.2 Mouse-derived antibodies cross-compete the binding of ACE2 receptor with SARS-CoV-2 and SARS-CoV and RBD proteins

Coat the SARS-CoV-2 or SARS-CoV RBD protein at a concentration of 1μg/mL on a 96-well plate, with 100μL per well, and coat overnight at 4°C. The plate was washed the next day and blocked at room temperature for 1 hour, then 100μL 0.08μg/mL ACE2 protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) was added, and 1μg/mL mouse antibody and negative control antibody H7N9-R1 were added at the same time to incubate. . Wash the plate to remove the unbound antibody, add 0.5μg/mL C-his-R023/HRP (source: Shenzhou Cell Engineering Co., Ltd.), incubate the plate repeatedly, add the substrate color development solution for color development, and detect OD _{450 after termination} . Inhibition rate PI% = (OD _blank- OD _sample ) / OD _blank × 100, where OD _blank represents the OD value of the normal coating group with only ACE2 and no antibody, and OD _sample represents the detection group with normal coating and ACE2 and antibody at the same time OD value.

The results are shown in Figure 4. The ACE2 protein can bind to the coated SARS-CoV-2 and SARS-CoV RBD proteins, and all four murine antibodies can effectively cross-compete the ACE2 protein with SARS-CoV-2 and SARS-CoV RBD. For protein binding, the negative control antibody has no competitive effect.

2.3 Murine antibody cross-neutralizes SARS-CoV-2 and SARS-CoV pseudovirus

Lennti-X 293 (source: Clontech) was used to package pseudoviruses expressing SARS-CoV-2 or SARS-CoV S full-length protein. Mix 62μg of PSD, pWPXL-Luc and pCMV3-SARS-CoV-2-S or pCMV3-SARS-CoV-S plasmid (source: Shenzhou Cell Engineering Co., Ltd.) according to the ratio of 3:4:2, and add 72μL of Sinofection TF02 (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) transfection reagent. After mixing, let it stand at room temperature for 10 minutes, and then add it to Lenti-X 293 cells. Place the cell plate in a 37°C, 5% CO ₂ incubator for 6 hours and then change the medium. After culturing for 48 hours, the supernatant was collected, and the cell debris was removed by filtration with a 0.45 μm filter membrane to obtain a pseudovirus solution, which was stored at -80°C.

Use the limiting dilution method to perform 10-fold gradient dilution of the virus, set a total of 10 virus concentrations, each with 6 replicate wells. A 96-well plate was seeded with a suspension of VERO-E6 cells (source: Basic Medical Cell Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences) ^{with a density of 5×10 5 cell/mL, 100 μL/well.} Add 50 μL of the virus in gradient dilution to each well, use the cell culture medium as a negative control, mix well, and place it in a 37°C, 5% CO ₂ incubator for 24 hours. After incubation, add 5×passive lysis buffer (source: Promega), 30μL/well, and mix well to lyse the cells. Take 10μL/well and transfer it to 96-well white bottom plate to detect the fluorescence signal. Reed-Muench method calculates the half of the tissue cell infection dose (TCID ₅₀ ) value.

Add different concentrations (80.0μg/mL, 26.7μg/mL, 8.9μg/mL, 3.0μg/mL, 0.99μg/mL, 0.33μg/mL, 0.11μg/mL, 0.037μg/mL and 0.012μg/mL) antibody, 50μL/well. _{Add 300TCID 50} pseudovirus to each well, 50μL/well. The virus and antibody-free group was used as a positive control, and the virus-free and antibody-free group was used as a negative control. After mixing, incubate in a 37°C, 5% CO ₂ incubator for 1 hour. After the incubation was completed, 100 μL/well was inserted into ^{the VERO-E6 cell suspension with a density of 5×10 5} cells/mL, mixed and placed in a 37°C, 5% CO ₂ incubator for static culture for 24 hours. After incubation, add 5×passive lysis buffer (source: Promega), 30μL/well, and mix well to lyse the cells. Take 10μL/well and transfer it to 96-well white bottom plate fluorescence signal value (RLU), and calculate the neutralization rate. Neutralization rate %=(positive control RLUs-sample RLUs)/(positive control RLUs-negative control RLUs)×100%. The results are shown in Figure 5. The four mouse-derived antibodies can effectively neutralize SARS-CoV-2 and SARS-CoV pseudoviruses in a concentration-dependent manner.

Example 3: Humanized modification and production of murine antibodies

3.1 Determination of the CDR of the mouse antibody light chain and heavy chain

According to the nucleotide sequence determined in Example 1.3, the amino acid sequence of the variable region of the heavy chain and light chain of 4 strains of murine cross-neutralizing antibody was deduced: the amino acid sequence of the variable region of the heavy chain of SARS-2-mh014 antibody (SEQ ID NO: 8) and light chain variable region amino acid sequence (SEQ ID NO: 9); SARS-2-mh157 antibody heavy chain variable region amino acid sequence (SEQ ID NO: 43) and light chain variable region amino acid sequence ( SEQ ID NO: 44); SARS-2-mh202 antibody heavy chain variable region amino acid sequence (SEQ ID NO: 65) and light chain variable region amino acid sequence (SEQ ID NO: 66); SARS-2-mh697 antibody The amino acid sequence of the heavy chain variable region (SEQ ID NO: 88) and the amino acid sequence of the light chain variable region (SEQ ID NO: 89).

Refer to Kabat[9] and IMGT numbering to determine the amino acid sequence of each of the 3 CDRs of the light chain and the heavy chain of the 4 strains of murine neutralizing antibodies. The light chain and heavy chain CDRs of the above four antibody strains were directly transplanted into the final humanized antibodies obtained in the subsequent CDR transplantation humanization step. The CDR sequences and homology analysis of the light chain and heavy chain of the 4 strains of murine neutralizing antibodies are shown in Table 1 and Table 2.

Table 1 Mouse-derived neutralizing antibody light chain CDR sequence and homology analysis

Table 2 Murine neutralizing antibody heavy chain CDR sequence and homology analysis

3.2 Mouse antibody humanized CDR transplantation

The classical CDR transplantation method is used to humanize mouse antibodies [10,11]. Respectively, the similarity with the murine light chain and heavy chain variable regions is more than 50%, and the framework regions of the light chain and heavy chain variable regions are similar to the amino acid sequences of the framework regions of the light chain and heavy chain variable regions of the antibody to be modified Antibodies with sex above 50% are used as humanized templates. By comparing the IMGT human antibody heavy and light chain variable region germline gene database, the heavy and light weights with high homology to SARS-2-mh014, SARS-2-mh157, SARS-2-mh202 and SARS-2-mh697 were selected respectively The germline gene of the chain variable region was used as a template for humanization, and the three CDR sequences of the light chain and heavy chain of the four murine antibody strains were transplanted into the corresponding human template. In this example, the selection of the humanized templates of the four mouse-derived cross-neutralizing antibodies and the homology with the mouse-derived corresponding antibodies are shown in Table 3.

Table 3 Selection of humanized templates for the framework region of SARS-CoV-2 neutralizing antibody

3.3 Back mutation of the framework region of the humanized variable region sequence

Since the key points of the mouse-derived framework region play a vital role in maintaining the stability of the CDR spatial structure, the key points need to be backmutated to the corresponding amino acids of the mouse antibody. In this example, the four strains of CDR grafted humanized SARS-CoV-2 neutralizing antibody humanized template framework region back mutation design are shown in Table 4.

After CDR humanization transplantation and framework region back mutation, 4 humanized antibodies were obtained: SARS-2-H014, SARS-2-H157, SARS-2-H202 and SARS-2-H697. The 4 humanized antibodies The amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 22/51/74/94, and the amino acid sequence of the variable region of the light chain is SEQ ID NO: 23/52/75/95. The 4 humanized antibodies contain signals The heavy chain amino acid sequences of the peptides are respectively SEQ ID NO: 18/49/72/92, which respectively include the heavy chain signal peptide amino acid sequence (SEQ ID NO: 20), and the heavy chain variable region amino acid sequence (SEQ ID NO :22/51/74/94) and heavy chain constant region amino acid sequence (SEQ ID NO: 24). The light chain amino acid sequences of the four humanized antibodies containing signal peptides are respectively SEQ ID NO: 19/50/73/93, which respectively contain the light chain signal peptide amino acid sequence (SEQ ID NO: 21) connected in sequence, and the heavy chain can The amino acid sequence of the variable region (SEQ ID NO: 23/52/75/95) and the amino acid sequence of the light chain constant region (SEQ ID NO: 25). The CDR sequences and homology analysis of the light chain and heavy chain of the 4 humanized antibodies are shown in Table 5 and Table 6.

Table 4 CDR transplantation humanized SARS-CoV-2 neutralizing antibody back mutation design

Note: For example, V71I means that the 71 position V is mutated back to I according to the Kabat numbering system.

Table 5 Humanized antibody light chain CDR sequence and homology analysis

Table 6 Humanized antibody heavy chain CDR sequence and homology analysis

3.4 Production of humanized antibodies

The SARS-2-H014 heavy chain variable region (SEQ ID NO: 30), the SARS-2-H157 heavy chain variable region (SEQ ID NO: 55), and the SARS-2-H202 heavy chain variable region (SEQ ID NO: 55) were obtained through the method of full gene synthesis. Chain variable region (SEQ ID NO: 78) and SARS-2-H697 heavy chain variable region (SEQ ID NO: 98) nucleotide sequence. Inserted into the pSE vector digested with Sca I+Nhe I (source: Fermentas) with heavy chain signal peptide (SEQ ID NO: 28) and heavy chain IgG1 constant region (SEQ ID NO: 32) by In-fusion method Obtained SARS-2-H014 heavy chain (SEQ ID NO: 26) expression vector, SARS-2-H157 heavy chain (SEQ ID NO: 53) expression vector, SARS-2-H202 heavy chain (SEQ ID NO: 76) expression Vector and SARS-2-H697 heavy chain (SEQ ID NO: 96) expression vector.

The SARS-2-H014 light chain variable region (SEQ ID NO: 31) and SARS-2-H202 light chain variable region (SEQ ID NO: 79) were obtained through the method of full gene synthesis, and inserted through the In-fusion method Into the pSE vector digested with Sca I+BsiW I (source: Fermentas) with the light chain signal peptide (SEQ ID NO: 29) and the light chain kappa constant region nucleotide sequence (SEQ ID NO: 33) SARS-2-H014 light chain (SEQ ID NO: 27) expression vector, SARS-2-H202 light chain (SEQ ID NO: 77) expression vector.

The nucleotide sequences of SARS-2-H157 light chain (SEQ ID NO: 54) and SARS-2-H697 light chain (SEQ ID NO: 97) were obtained by splicing PCR, and inserted by HindIII+Xba I by In-fusion method. (Source: Fermentas) The SARS-2-H157 light chain (SEQ ID NO: 54) expression vector and the SARS-2-H697 light chain (SEQ ID NO: 97) expression vector were obtained from the digested pSE vector.

After the plasmid was extracted, it was transfected into HEK-293 cells (source: Invitrogen) for culture and expression for 7 days, and purified by a protein A purification column to obtain high-purity antibodies.

Full-gene synthesis of SARS-2-H014 heavy chain variable region primers:

Full-gene synthesis of SARS-2-H014 light chain variable region primers:

Full-gene synthesis of SARS-2-H202 heavy chain variable region primers:

Full-gene synthesis of SARS-2-H202 light chain variable region primers:

Full-gene synthesis of SARS-2-H697 heavy chain variable region primers:

Full-gene synthesis of SARS-2-H157 heavy chain variable region primers:

Splicing the SARS-2-H697 light chain primer:

Splicing the SARS-2-H157 light chain primer:

Example 4: Detection of antigen binding and neutralization ability of humanized antibodies

4.1 Cross-binding of humanized antibodies with SARS-CoV-2 and SARS-CoV RBD proteins

Different concentrations (1000ng/mL, 333.3ng/mL, 111.1ng/mL, 37.0ng/mL, 12.3ng/mL, 1.37ng/mL and 0.46ng/mL) SARS-CoV-2 or SARS-CoV RBD protein Coated on a 96-well plate, 100μL per well, coated overnight at 4°C. Wash the plate the next day, block at room temperature for 1 hour, add 100 μL of 1 μg/mL humanized antibody and incubate for 1 hour, then wash the plate to remove unbound antibody, add 0.25 μg/mL Goat anti-human IgG Fc/HRP (source: KPL) After incubation, the plate was washed repeatedly, and the substrate color developing solution was added for color development. After termination, the OD ₄₅₀ was detected.

The results are shown in Figure 6. The humanized antibodies SARS-2-H014, SARS-2-H157, SARS-2-H202, and SARS-2-H697 have better RBD proteins than SARS-CoV-2 and SARS-CoV. The cross-combination of, and the binding ability is similar, the combination shows an "S"-shaped curve growth.

4.2 Humanized antibodies cross-compete the binding of ACE2 receptor to SARS-CoV-2 and SARS-CoV and RBD proteins

Refer to Example 2.2 to detect the ability of humanized antibodies to cross-compete ACE2 receptors for binding to SARS-CoV-2 and SARS-CoV and RBD proteins. The results are shown in Figure 7. The humanized antibodies SARS-2-H014, SARS-2-H157, SARS-2-H202 and SARS-2-H697 can effectively inhibit the ACE2 protein and SARS-CoV-2 and SARS- CoV RBD protein binding, and its inhibitory ability is similar.

4.3 Detection of affinity between humanized antibody and SARS-CoV-2 and SARS-CoV S1 protein

A biomolecular interaction analysis system (model: OctetRED96e, manufacturer: Fortebio) was used to determine the binding of humanized antibodies to biotinylated SARS-CoV-2 and SARS-CoV S1 proteins (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) Affinity. Select SA Sensor, add 2μg/mL of biotinylated SARS-CoV-2 or SARS-CoV S1 protein after 60s equilibration, and then equilibrate again for 100s to wash out unbound proteins. Different concentrations of humanized antibodies (4.0μg/mL, 2.0μg/mL, 1.0μg/mL, 0.5μg/mL, 0.25μg/mL, 0.13μg/mL, 0.06μg/mL) were added for 300s and dissociated for 300s. Use the Data Analysis Octet software to process the data and calculate the antibody affinity (KD), binding constant (kon), and dissociation constant (kdis).

The results are shown in Table 7. The four humanized antibodies have high affinity with SARS-CoV-2 and SARS-CoV S1 proteins. The affinity between SARS-2-H014 and SARS-CoV-2 S1 protein is 2.6E-10M, the binding constant is 4.5E+05 1/Ms, and the dissociation constant is 1.2E-04 1/s; it is compatible with SARS-CoV S1 protein The affinity is 1.2E-11M, the binding constant is 2.7E+05 1/Ms, and the dissociation constant is 3.3E-06 1/s. The affinity between SARS-2-H157 and SARS-CoV-2 S1 protein is 2.9E-10M, the binding constant is 6.9E+05 1/Ms, and the dissociation constant is 2.0E-04 1/s; and SARS-CoV S1 protein The affinity is 1.1E-10M, the binding constant is 4.8E+05 1/Ms, and the dissociation constant is 5.3E-05 1/s. The affinity between SARS-2-H202 and SARS-CoV-2 S1 protein is 2.1E-10M, the binding constant is 5.8E+05 1/Ms, and the dissociation constant is 1.2E-04 1/s; and SARS-CoV S1 protein The affinity is 2.0E-10M, the binding constant is 4.2E+05 1/Ms, and the dissociation constant is 8.6E-05 1/s. The affinity of SARS-2-H697 and SARS-CoV-2 S1 protein is 2.7E-11 M, the binding constant is 9.1E+05 1/Ms, and the dissociation constant is 2.5E-05 1/s; and SARS-CoV S1 The affinity of the protein is 7.5E-11M, the binding constant is 4.6E+05 1/Ms, and the dissociation constant is 3.4E-05 1/s. The above results indicate that the four humanized antibodies have similar binding capacity to SARS-CoV-2 and SARS-CoV S1 proteins. The specific kinetic characteristic parameter curves are shown in Figure 8.

Table 7 Detection of affinity between humanized antibodies and SARS-CoV-2 S1 protein

4.4 Humanized antibodies cross-neutralize SARS-CoV-2 and SARS-CoV pseudoviruses

Refer to Example 2.3 to evaluate the ability of humanized antibodies to cross-neutralize SARS-CoV-2 and SARS-CoV pseudoviruses. The results are shown in Figure 9. The four humanized antibodies SARS-2-H014, SARS-2-H157, SARS-2-H202 and SARS-2-H697 can effectively neutralize SARS-CoV-2 and SARS-CoV The pseudovirus is concentration-dependent. The humanized transformation did not change the cross-neutralization ability of the antibody, and the neutralization ability of the four humanized antibodies against SARS-CoV-2 and SARS-CoV pseudoviruses was similar.

Example 5: Analysis of drug quality and drug stability of humanized antibody SARS-2-H014

5.1 The purity and particle size analysis of SARS-2-H014

Application of sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS polyacrylamide gel electrophoresis, SDS-PAGE), molecular sieve chromatography (size-exclusion high performance liquid chromatograph, SEC-HPLC) analysis of SARS-2-H014 purity. SDS-PAGE specific operation steps: (1) SDS-PAGE gel preparation: 3.9% concentrated gel, 7.5% separation gel (non-reducing electrophoresis), 13% separation gel (reducing electrophoresis); (2) Samples are boiled at 100°C for 2 minutes and centrifuged After loading the sample 8μg; (3) electrophoresis at 100V voltage for 1h; (4) Coomassie brilliant blue staining and decolorization, using BandScan software to calculate the purity of the sample band. SEC-HPLC operation steps are: (1) Instrument: liquid chromatography system (Agilent company, model: Agilent1260), hydrophilic silica gel high-performance molecular exclusion chromatography column (Tosoh company, model: TSK-GEL G3000SW _XL (7.8×300mm, 5μm)); (2) Mobile phase: 200mM NaH ₂ PO ₄ , 100mM Arginine, pH 6.5; (2) Sample amount is 80μg; (3) Detection wavelength is 280nm, analysis time is 30min, flow rate is 0.5mL/min , The column temperature is 25℃; (4) Calculate the ratio of each peak according to the area normalization method.

The purity of reduced SDS-PAGE and non-reduced SDS-PAGE of SARS-2-H014 were 99.4% and 92.7%, respectively; the main peak ratio of SEC-HPLC was 99.4%, and the aggregate ratio was 0.6% (Table X). Both test results indicate that the purity of SARS-2-H014 is relatively high, with no other ingredients except for a very small amount of aggregates.

Use dynamic light scattering (DLS) to detect the particle size and uniformity of SARS-2-H014. Specific steps: (1) Instrument: Dynamic Light Scattering (Wyatt Technology, Model: DynaPro NanoStar); (2) ) The sample volume is 50μL; (3) After collecting the data, use the Dynamics 7.1.8 software to analyze the data.

The radius of SARS-2-H014 is 5.7nm and the percentage dispersion (%Pd) is 12.4%, indicating that the SARS-2-H014 particles are small, the size of normal IgG1 antibody particles, and the uniformity is good (Table 8).

Table 8 SEC and DLS test results of SARS-2-H014 fusion protein

5.2 Thermal stability analysis of SARS-2-H014

Differential scanning fluorescence (DSF) was used to detect the thermal stability of SARS-2-H014. Specific operation steps: (1) Instrument: Uncle system (Unchained Labs, Model: UNCLE-0330); (2) The sample volume is 9μL; (3) The experimental detection parameters are set: the temperature range is 25℃～95℃, heating Speed 0.3℃/min; (4) Use UNcle Analysis software to analyze the data, take the midpoint value of the internal fluorescence curve under UV266 as Tm, and take the polymerization start temperature of the aggregate curve formed by the static light scattering signal under UV266/Blue473 They are Tagg266 and Tagg473.

The thermal stability test results of SARS-2-H014 in Histidine buffer (40mM Histidine, 120mM NaCl, 0.02% Tween80, pH 6.0) are shown in Table 9, showing good thermal stability.

Table 9 Tm test results of SARS-2-H014 fusion protein

5.3 Analysis of acid-base isomers of SARS-2-H014

IgG1 antibody has asparagine (Asn) deamidation, lysine (Lys) glycation, methionine (Met) oxidation, etc., so there will be uneven charge, showing acidic and basic isomers . Cation exchange high performance liquid chromatograph (CEX-HPLC) and capillary isoelectric focusing (cIEF) were used to analyze the acid and base isomer levels of SARS-2-H014. CEX-HPLC operation steps: (1) Instrument: liquid chromatography system (Agilent company, model: Agilent1260), cation exchange chromatography column (Thermo company, model: ProPac ^TM WCX-10 (4×250mm, 5μm)); (2) ) Mobile phase A: buffer A, pH 5.6 (Thermo company, Cat: 083273); (3) Mobile phase B: buffer B, pH 10.2 (Thermo company, Cat: 083275); (4) The sample amount is 80 μg; ( 5) The detection wavelength is 280nm, the analysis time is 50min, the flow rate is 0.5mL/min, and the column temperature is 25°C; (6) The ratio of each peak is calculated by the area normalization method. cIEF operation steps: (1) Instrument: imaging capillary electrophoresis instrument (Proteinsimple, model: iCE3); (2) Take 10μL of 5mg/ml sample and 8μL Pharmalyte 3-10, 70μL 1% methyl cellulose (MC), 2μL PI marker And ddH ₂ O was prepared into an analysis solution with a total volume of 200 μL; (3) The sample was placed in an iCE3 instrument, 1500V was pre-focused for 1 minute, and then 3000V was collected for 6 minutes; (4) Chrom Perfect software was used to analyze the data.

The ratio of acidic peaks of SARS-2-H014 detected by CEX-HPLC is 7.9% and the ratio of basic peaks is 4%; the ratio of acidic peaks detected by cIEF is 16.0%, and the ratio of basic peaks is 2.2% (Table 10) . Both test results show that the acidic and basic isomers of SARS-2-H014 are at a relatively low level.

Table 10 CEX-HPLC and cIEF test results of SARS-2-H014 fusion protein

5.4 Thermal acceleration stability analysis of SARS-2-H014

After the SARS-2-H014 sample was stored at 45°C for 1 week, the purity of the sample was analyzed by SDS-PAGE and SEC-HPLC, and the change in particle size of the sample was analyzed by DLS. The specific operation steps refer to Example 5.1.

After SARS-2-H014 was stored at 45°C for 1 week, the purity of reduced SDS-PAGE decreased by 0.9%, but the purity of non-reduced electrophoresis did not decrease; the purity of SEC decreased by 2%, aggregates increased slightly, and a few fragments appeared, indicating that SARS- 2-H014 has a smaller tendency of aggregation and fragmentation after thermal acceleration, but its purity is still higher after thermal acceleration. The DLS test results showed that although the radius of SARS-2-H014 increased by 0.4nm after thermal acceleration, it was still the normal particle size; %Pd increased, and 40nm particle components appeared to reduce Intensity, again indicating that SARS-2-H014 has a better Small aggregation tendency (Table 11). In short, although SARS-2-H014 has a smaller tendency to aggregate, it still shows good thermal acceleration stability.

Table 11 Thermal acceleration stability test results of SARS-2-H014

5.5 The freeze-thaw stability analysis of SARS-2-H014

The SARS-2-H014 sample was stored at -80°C for 3 hours and then transferred to 45°C to thaw for 1 hour. The freeze-thaw cycle was repeated five times. SDS-PAGE and SEC-HPLC were used to analyze the purity of the sample, and DLS was used to analyze the change in particle size of the sample. Refer to Example 5.1 for the specific operation steps.

After five repeated freezing and thawing of SARS-2-H014, the purity of SDS-PAGE and SEC did not change significantly, and the level of aggregates and fragments did not increase significantly; the size of DLS particles did not increase significantly (Table 12). It shows that SARS-2-H014 has good freeze-thaw stability.

Table 12 Freeze-thaw stability test results of SARS-2-H014

5.6 Analysis of the oscillation stability of SARS-2-H014

The SARS-2-H014 sample is placed in a deep well plate and placed on a vortex shaker at 800 rpm for 24 hours. SDS-PAGE and SEC-HPLC are used to analyze the purity of the sample, and DLS is used to analyze the change in particle size of the sample. Refer to the specific operation steps Example 5.1.

After SARS-2-H014 was shaken for 24 hours, the purity of SDS-PAGE and SEC did not change significantly, and the level of aggregates and fragments did not increase significantly; the size of DLS particles did not increase significantly (Table 13). It shows that SARS-2-H014 has good shock stability.

Table 13 Oscillation and stability test results of SARS-2-H014

5.7 High concentration stability analysis of SARS-2-H014

The 10.3 mg/mL SARS-2-H014 sample was concentrated to 25.6 mg/mL, 50.9 mg/mL, 81.0 mg/mL, and 95.1 mg/mL using a 50kDa ultrafiltration tube. The samples were analyzed by SDS-PAGE and SEC-HPLC. For purity, use DLS to analyze the change of sample particle size, and the specific operation steps refer to Example 5.1.

When the concentration of SARS-2-H014 gradually increased, the purity of SDS-PAGE and SEC did not change significantly, and the level of aggregates and fragments did not increase significantly; but the radius of DLS particles increased slowly with the increase of concentration, and the maximum radius was 9.8nm ( Table 14). It shows that SARS-2-H014 has good high concentration stability.

Table 14 High-concentration stability test results of SARS-2-H014

Example 6: Epitope analysis of humanized antibody SARS-2-H014

The results of Example 4 show that SARS-2-H014 can cross-bind the RBD proteins of SARS-CoV-2 and SARS-CoV, and can cross-compete the binding of ACE2 receptor to SARS-CoV-2 and SARS-CoV and RBD proteins. Crystallization by cryo-electron microscopy showed that residues 437-508 are the key amino acid residues required for the binding of SARS-CoV-2 RBD to ACE2 [12]. In addition, by comparing the sequences of SARS-CoV-2 and SARS-CoV, it is found that the ACE2 binding region of the two in RBD has low similarity, while the non-ACE2 binding region of RBD has higher similarity [13]. Based on the above information, it is speculated that the epitope of SARS-2-H014 may be located in a relatively similar region in the structure of SARS-CoV-2 and SARS-CoV RBD, and after SARS-2-H014 is combined with RBD, it will form a spatial position with the structural conformation of ACE2 Prevent conflict. Therefore, in this example, 14 sites that are the same in SARS-CoV-2 and SARS-CoV RBD or residue sites located in the ACE2 binding region and its vicinity were selected, and they were mutated to be different from the original residue types. The larger other residue types produced 13 mutants, namely V367F, K378D, T385Y, T415Y, N439R, N440Y, Y489R, T500Y, Y505E, A372Y, S375Y, D405R/R408D and V503Y.

In this example, SARS-CoV-2 RBD-His was used as a template (sequence source: https://www.gisaid.org/), PCR was used for site-directed mutation, and sequencing verification was performed. The mutant and wild-type (WT) SARS-CoV-2 RBD proteins were transiently transfected, and the binding ability of SARS-2-H014 antibody to the mutant protein was detected by ELISA. At the same time, a non-ACE2 SARS-CoV neutralizing antibody R007 (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) was used as a control.

For the ELISA test results (Figure 10B), the ELISA reading of WT SARS-CoV-2 RBD was used as the standard. When the ELISA binding signal of SARS-2-H014 for a specific mutant was reduced to 75% relative to that of WT SARS-CoV-2 RBD In the following, this residue site is defined as a significant binding epitope. Similarly, if the ELISA binding signal of SARS-2-H014 against a specific mutant drops below 50%, it is defined as a highly significant epitope. As shown in Figure 10, S375 and K378 in SARS-CoV-2 RBD are highly significant epitopes of SARS-2-H014, and D405 and R408 are significant epitopes of H014.

Example 7: Construction and production of humanized antibody SARS-2-H014 with different Fc functional forms

7.1 Construction and production of defucosylated IgG1 subtype SARS-2-H014

Extract SARS-2-H014 heavy chain (SEQ ID NO: 26) expression vector, SARS-2-H014 light chain (SEQ ID NO: 27) expression vector plasmids, transfect HEK-293 (Fut8 gene knockout) cells for culture Expressed for 7 days, purified with a protein A purification column to obtain a high-purity defucosylated IgG1 subtype humanized SARS-2-H014 antibody, namely SARS-2-H014-Ae0-IgG1.

7.2 Construction and production of humanized antibody SARS-2-H014 of IgG4 subtype with reduced Fc function

In order to reduce the immune function mediated by the antibody Fc fragment, nucleotide mutations in the constant region of the IgG4 subtype were carried out with reference to the literature [14] to obtain the genetically engineered heavy chain IgG4 constant region nucleotide sequence (Fd11-IgG4, SEQ ID NO:105). The SARS-2-H014-Fd11-IgG4 heavy chain sequence (SEQ ID NO: 109) was obtained by splicing PCR, which contains the heavy chain signal peptide nucleotide sequence (SEQ ID NO: 28), and the SARS-2-H014 heavy chain can be The nucleotide sequence of the variable region (SEQ ID NO: 30) and the nucleotide sequence of Fd11-IgG4 (SEQ ID NO: 105). The SARS-2-H014-Fd11-IgG4 heavy chain (SEQ ID NO: 109) expression vector was inserted into the pSE vector digested with Hind III+Xba I (source: Fermentas) by In-fusion method.

Splicing SARS-2-H014-Fd11-IgG4 heavy chain primers:

The SARS-2-H014-Fd11-IgG4 heavy chain (SEQ ID NO: 109) expression vector and the SARS-2-H014 light chain (SEQ ID NO: 27) expression vector plasmid were extracted, and transfected into HEK-293 cells for culture and expression 7 Today, a protein A purification column was used to obtain a high-purity IgG4 subtype humanized SARS-2-H014 antibody with reduced Fc function, namely SARS-2-H014-Fd11-IgG4.

7.3 Construction and production of humanized antibody SARS-2-H014 that removes FcRn binding to IgG4 subtype

In order to remove the binding of the antibody to FcRn and reduce the immune function mediated by the Fc fragment of the antibody, refer to the literature to carry out nucleotide mutations in the constant region of the IgG4 subtype [15,16] to obtain the genetically engineered Fc heavy chain IgG4 constant region Nucleotide sequence (Fd19-IgG4, SEQ ID NO: 107). The SARS-2-H014-Fd1d-IgG4 heavy chain sequence (SEQ ID NO: 111) was obtained by splicing PCR, which contains the heavy chain signal peptide nucleotide sequence (SEQ ID NO: 28), and the SARS-2-H014 heavy chain can be The nucleotide sequence of the variable region (SEQ ID NO: 30) and the nucleotide sequence of Fd19-IgG4 (SEQ ID NO: 107). The SARS-2-H014-Fd19-IgG4 heavy chain sequence (SEQ ID NO: 111) was obtained by splicing PCR and inserted into the pSE vector digested with HindIII+Xba I (source: Fermentas) by In-fusion method to obtain SARS-2 -H014-Fd19-IgG4 heavy chain (SEQ ID NO: 111) expression vector.

Splicing SARS-2-H014-Fd19-IgG4 heavy chain primers:

The SARS-2-H014-Fd19-IgG4 heavy chain (SEQ ID NO: 111) expression vector and the SARS-2-H014 light chain (SEQ ID NO: 27) expression vector plasmids were extracted and transfected into HEK-293 cells for culture and expression 7 Today, a protein A purification column was used to obtain a high-purity humanized SARS-2-H014 antibody of the IgG4 subtype with FcRn-free binding and reduced Fc function, namely SARS-2-H014-Fd19-IgG4.

Example 8: Fc function of different forms of humanized antibody SARS-2-H014

8.1 CD16a binding function of humanized antibody SARS-2-H014 with different Fc functional forms

Humanized antibodies with different Fc functional forms at different concentrations (30000ng/mL, 10000ng/mL, 3333.3ng/mL, 1111.1ng/mL, 370.4ng/mL, 123.5ng/mL and 41.2ng/mL): SARS- 2-H014-IgG1, SARS-2-H014-Ae0-IgG1, SARS-2-H014-Fd11-IgG4 and SARS-2-H014-Fd19-IgG4 were coated on 96-well plates, 100μL per well, 4℃ Coated overnight. The plate was washed the next day and sealed at room temperature for 1 hour, and then 5 μg/mL CD16a-His (F158V) protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) was added, 100 μL/well, and incubated for 1 hour. Wash the plate to remove unbound proteins, add 0.5μg/mL anti-His-MM02T/HRP (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.), incubate the plate repeatedly, add the substrate color solution for color development, and then detect after termination OD ₄₅₀ .

The results are shown in Figure 11. The SARS-2-H014-Ae0-IgG1 antibody of the defucosylated IgG1 subtype has a significantly better binding capacity to CD16a than the SARS-2-H014 antibody of the IgG1 subtype, while reducing the antibody Fc Fragment-mediated immune function Fd11-IgG4 and Fd19-IgG4 subtype antibodies SARS-2-H014-Fd11-IgG4 and SARS-2-H014-Fd19-IgG4 did not bind to CD16a.

8.2 CD32 binding function of humanized antibody SARS-2-H014 with different Fc functional forms

Humanized antibodies with different Fc functional forms at different concentrations (30μg/mL, 10μg/mL and 3.3μg/mL): SARS-2-H014-IgG1, SARS-2-H014-Ae0-IgG1, SARS-2- H014-Fd11-IgG4 and SARS-2-H014-Fd19-IgG4 were respectively coated on a 96-well plate with 100 μL per well and coated overnight at 4°C. The plate was washed the next day and blocked at room temperature for 1 hour, then 5μg/mL CD32a-His or CD32b-His protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) was added, 100μL/well, and incubated for 1h. Wash the plate to remove unbound proteins, add 0.5μg/mL anti-His-MM02T/HRP (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.), incubate the plate repeatedly, add the substrate color solution for color development, and then detect after termination OD ₄₅₀ .

The results are shown in Figure 12. Among the SARS-2-H014 antibodies with different Fc functional forms, the IgG1 and Ae0-IgG1 forms of antibodies bind to the CD32a or CD32b protein, and bind in a concentration gradient; Fd11-IgG4 and Fd19-IgG4 The form of the antibody does not bind to CD32a or CD32b protein.

8.3 CD64 binding function of humanized antibody SARS-2-H014 with different Fc functional forms

Humanized antibodies with different Fc functional forms at different concentrations (30000ng/mL, 10000ng/mL, 3333.3ng/mL, 1111.1ng/mL, 370.4ng/mL, 123.5ng/mL and 41.2ng/mL): SARS- 2-H014-IgG1, SARS-2-H014-Ae0-IgG1, SARS-2-H014-Fd11-IgG4 and SARS-2-H014-Fd19-IgG4 were coated on 96-well plates, 100μL per well, 4℃ Coated overnight. The plate was washed the next day and sealed at room temperature for 1 hour, then 0.5 μg/mL CD64-his protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) was added, 100 μL/well, and incubated for 1 hour. Wash the plate to remove unbound proteins, add 0.5μg/mL anti-His-MM02T/HRP (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.), incubate the plate repeatedly, add the substrate color solution for color development, and then detect after termination OD ₄₅₀ .

The results are shown in Figure 13. Among the SARS-2-H014 antibodies with different Fc functional forms, the IgG1 and Ae0-IgG1 forms of antibodies and CD64 showed an "S" curve growth, and the binding levels of the two were similar; the Fd11-IgG4 form The antibody binds weakly to CD64 under high concentration conditions, while the Fd19-IgG4 form of antibody does not bind to CD64 protein.

8.4 C1q binding function of humanized antibody SARS-2-H014 with different Fc functional forms

Humanized antibodies with different Fc functional forms at different concentrations (30000ng/mL, 10000ng/mL, 3333.3ng/mL, 1111.1ng/mL, 370.4ng/mL, 123.5ng/mL and 41.2ng/mL): SARS- 2-H014-IgG1, SARS-2-H014-Ae0-IgG1, SARS-2-H014-Fd11-IgG4 and SARS-2-H014-Fd19-IgG4 were coated on 96-well plates, 100μL per well, 4℃ Coated overnight. The plate was washed the next day and blocked for 1 hour at room temperature, then 5μg/mL of C1q complement protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) was added, 100μg/well, and incubated for 1 hour. Wash the plate to remove unbound proteins, add 0.5μg/mL anti-His-MM02T/HRP (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.), incubate the plate repeatedly, add the substrate color solution for color development, and then detect after termination OD ₄₅₀ .

The results are shown in Figure 14. Among the SARS-2-H014 antibodies with different Fc functional forms, the IgG1 and Ae0-IgG1 forms of antibody and C1q complement protein showed an "S" curve growth, and the binding levels of the two were similar; Fd11-IgG4 And Fd19-IgG4 form of antibody does not bind to C1q complement protein.

8.5 FcRn binding function of humanized antibody SARS-2-H014 with different Fc functional forms

10μg/mL NeutrAvidin (source: ThermoFisher) was coated on a 96-well plate, 100μL per well, coated overnight at 4°C. The plate was washed the next day and sealed at room temperature for 1 hour, and 5μg/mL FCGRT&B2M-His-Biotin protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) was added, 100μL/well. In pH 6.0 buffer, add different concentrations (10000ng/mL, 2500ng/mL, 625ng/mL, 156.3ng/mL, 39.1ng/mL, 9.8ng/mL, 2.4ng/mL and 0.61ng/mL) Humanized antibodies with different Fc functional forms: SARS-2-H014-IgG1, SARS-2-H014-Ae0-IgG1, SARS-2-H014-Fd11-IgG4 and SARS-2-H014-Fd19-IgG4, mix well After incubation for 1h. Wash the plate to remove unbound proteins and antibodies, add 0.25μg/mL goat anti-human IgG F(ab)2/HRP (pH6.0) (source: Jackson ImmunoResearch), incubate the plate repeatedly, and add the substrate color development solution. Color development, and detect OD ₄₅₀ after termination.

As shown in Figure 15, among the SARS-2-H014 antibodies with different Fc functional forms, the antibody in the form of IgG1 binds to FCGRT&B2M-His-Biotin protein the strongest, and the antibody in the form of Ae0-IgG1 and Fd11-IgG4 binds to FCGRT&B2M-His-Biotin. The protein binding level is similar, and the Fd19-IgG4 form of antibody does not bind to FCGRT&B2M-His-Biotin protein.

8.6 ADCC function mediated by humanized antibody SARS-2-H014 with different Fc functional forms

HEK293FT cells (HEK293FT-SARS-CoV-2-S or HEK293FT-SARS-CoV-S) stably expressing the full-length SARS-CoV-2 or SARS-CoV S protein were used as target cells to stably transfect CD16AV and NFAT- Luc2P Jurkat cells (Jurkat-NFAT/Luc2P-CD16AV) are effector cells, and the ADCC function of humanized antibodies is detected by the reporter gene method.

In a 96-well plate, ^{target cells with a density of 2×10 6} cells/mL and effector cells with an equal volume and an equal density were inserted at 50 μL/well. After that, 50 μL of humanized antibody and H7N9-R1 negative control antibody of different concentrations (20 μg/mL, 1 μg/mL and 0.05 μg/mL) were added, and the mixture was mixed _{and incubated in a 37°C, 5% CO 2} incubator for 6 hours. Finally, add 5×passive lysis buffer (source: Promega), 30μL/well, and mix well to lyse the cells. Take 10μL/well cell sample to detect RLU value. Use GraphPad Prism software to analyze and draw a dose-response curve, the abscissa is the concentration of the sample, and the ordinate is the RLU value. Bioluminescence intensity induction factor=RLU value of sample group/RLU value of negative control group.

The results are shown in Figure 16. Among the SARS-2-H014 antibodies with different Fc functional forms, the Ae0-IgG1 form antibody can significantly mediate the expression of SARS-CoV-2 (Figure 16A) and SARS-CoV S protein target cells (Figure 16A). 16B) ADCC effect; IgG1 form of antibody can only mediate a weak ADCC effect; Fd11-IgG4 and Fd19-IgG4 form of antibody have no ADCC effect.

8.7 ADCP function mediated by humanized antibody SARS-2-H014 with different Fc functional forms

With HEK293FT-SARS-CoV-2-S or HEK293FT-SARS-CoV-S as target cells, Jurkat cells (Jurkat-NFAT/Luc2P-CD32A, Jurkat-NFAT) stably transfected with CD32A, CD32B or CD64 and NFAT-Luc2P /Luc2P-CD32B or Jurkat-NFAT/Luc2P-CD64) are effector cells, and the reporter gene method is used to detect the ADCP function mediated by humanized antibodies.

In a 96-well plate, ^{target cells with a density of 2×10 6} cells/mL and effector cells with an equal volume and an equal density were inserted at 50 μL/well. Then add 50μL/well of humanized antibody of different concentrations (20μg/mL, 1μg/mL and 0.05μg/mL), and set H7N9-R1 negative antibody control and cell-free control at the same time. After mixing, incubate in a 37°C, 5% CO ₂ incubator for 6 hours. Finally, add 5×passive lysis buffer (source: Promega), 30μL/well, and mix well to lyse the cells. Take 10μL/well cell sample to detect RLU value. Use GraphPad Prism software to analyze and draw a dose-response curve, the abscissa is the concentration of the sample, and the ordinate is the RLU value. Bioluminescence intensity induction factor=RLU value of sample group/RLU value of negative control group.

The results are shown in Figure 17. In the ADCP effect mediated by target cells expressing SARS-CoV-2 Spike protein (Figure 17A), when Jurkat-NFAT/Luc2P-CD32A and Jurkat-NFAT/Luc2P-CD32B are used as effector cells, The SARS-2-H014 antibody in the form of IgG1, Ae0-IgG1 and Fd11-IgG4 can mediate a weak ADCP effect, while the antibody in the form of Fd19-IgG4 has no ADCP effect. When Jurkat-NFAT/Luc2P-CD64 is used as effector cells, SARS-2-H014 antibodies with different Fc functional forms have no ADCP effect. In the ADCP effect on target cells expressing SARS-CoV S protein (Figure 17B), when Jurkat-NFAT/Luc2P-CD32A and Jurkat-NFAT/Luc2P-CD32B are used as effector cells, SARS-2- with different Fc functional forms The H014 antibody has no ADCP effect. When Jurkat-NFAT/Luc2P-CD64 is used as effector cell, SARS-2-H014 antibody in the form of IgG1 and Ae0-IgG1 can mediate a weaker ADCP effect, while antibodies in the form of Fd11-IgG4 and Fd19-IgG4 have no ADCP effect.

8.8 CDC function mediated by humanized antibody SARS-2-H014 with different Fc functional forms

With HEK293FT-SARS-CoV-2-S or HEK293FT-SARS-CoV-S as target cells, WST-8 method was used to detect the CDC function of humanized antibodies.

In a 96-well plate, target cells with a ^{density of 2×10 6 cells/mL were inserted at 50 μL/well.} Add 50μL of rabbit complement (source: One lambda) and different concentrations (100μg/mL, 20μg/mL, 4μg/mL, 0.8μg/mL, 1.16μg/mL, 0.032μg/mL, 0.0064μg/mL and 0.00128μg/ mL) humanized antibody and set up a test blank hole (no cells), a positive control group (only inoculated cells) control, and a H7N9-R1 negative control antibody group. After mixing, incubate in a 37°C, 5% CO ₂ incubator for 2 hours. After incubation, add WST-8 color developing solution, 10μL/well. The 96-well plate was _{incubated in a CO 2} incubator, and after the color was stable, it was placed on a microplate reader to measure the absorbance at 450 nm and 630 nm. Calculate the CDC killing effect of the antibody by using the absorbance value (OD ₄₅₀ -OD ₆₃₀ ) and subtracting the reading value of the blank well. Killing rate %=(positive control OD value-sample OD value)/positive control OD value×100%.

The results are shown in Fig. 18, SARS-2-H014 antibodies of different Fc functional forms have no CDC effect on target cells expressing SARS-CoV-2 S protein (Fig. 18A) or SARS-CoV S protein (Fig. 18B).

Example 9: Evaluation of mouse pharmacokinetics of humanized antibody SARS-2-H014 with different Fc functional forms

9.1 Pharmacokinetic test of IgG1 humanized antibody SARS-2-H014 in mice

C57BL/6 mice (4 mice in total, half male and half male, source: Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were used to give SARS-2-H014 by a single tail vein injection at a dose of 5 mg/kg. The medicine volume is 10mL/kg. Before administration and 5min, 30min, 1h, 3h, 6h, 10h, 24h, 32h, 48h, 72h, 96h, 120h, 168h, 240h, 336h, 504h and 672h after administration, all mice were subjected to orbital blood sampling and centrifugation Take the serum. The blood drug concentration was detected by ELISA, and the non-compartmental model (NCA) in Phoenix-WinNonlin 6.4 software was used to calculate the pharmacokinetic parameters.

During the experiment, all mice were in normal condition. The drug-time curve is shown in Figure 19. The drug concentration in the mice changes continuously with time, and the early decline is faster, but the blood drug concentration is basically stable for a long time after that, with only a very small amount. The rate of decrease, the metabolism is very slow, and there is no obvious gender difference. The pharmacokinetic parameters are shown in Table 15. After a single intravenous injection of SARS-2-H014 in mice, the average in vivo exposure C _max and AUC _last were 136.15μg/mL and 10930.35h×μg/mL, respectively , The average half-life t _1/2 is 281.20h, and the clearance rate Cl is 0.27mL/h/kg.

Table 15 The pharmacokinetic parameters of a single intravenous injection of SARS-2-H014 in mice (0～336h)

9.2 Pharmacokinetic test of SARS-2-H014-Fd11-IgG4 in mice

C57BL/6 mice (a total of 6 mice, half male and half male, source: Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were used to give SARS-2-H014-Fd11-IgG4 antibody by a single tail vein injection. The dose was 5mg/kg, the administration volume is 10mL/kg. Before administration and 5min, 30min, 1h, 3h, 6h, 10h, 24h, 32h, 48h, 72h, 96h, 120h168h, 240h, 336h, 504h and 672h after administration, blood was collected from the orbit of all mice, and serum was collected by centrifugation . The blood drug concentration was detected by ELISA, and the non-compartmental model (NCA) in Phoenix-WinNonlin 6.4 software was used to calculate the pharmacokinetic parameters.

During the experiment, all mice were in normal condition, and the drug-time curve is shown in Figure 20. The drug concentration in mice changes continuously over time, and the early decline is relatively rapid, but the plasma concentration is basically stable for a long time, with only a small decrease, the metabolism is very slow, and there is no obvious gender difference, but No. 978 and 979 The blood concentration of mice in 168～336h decreased significantly. The pharmacokinetic parameters are shown in Table 16. After SARS-2-H014-Fd11-IgG4 was administered to mice by a single intravenous injection, the average in vivo exposure C _max and AUC _last were 144.66 μg/mL and 11940.01 h, respectively. ×μg/mL, the average half-life t _1/2 is 290.08h, and the clearance rate Cl is 0.26mL/h/kg.

Table 16 The pharmacokinetic parameters of a single intravenous injection of SARS-2-H014-Fd11-IgG4 in mice (0～336h)

9.3 Pharmacokinetic test of SARS-2-H014-Fd19-IgG4 in mice

C57BL/6 mice (4 mice in total, half male and half male, source: Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were used to give SARS-2-H014-Fd19-IgG4 by a single tail vein injection at a dose of 5 mg /kg, the administration volume is 10mL/kg. Before administration and 5min, 30min, 1h, 3h, 6h, 10h, 24h, 32h, 48h and 72h after administration, blood was collected from the orbit of all mice, and serum was collected by centrifugation. The blood drug concentration was detected by ELISA, and the non-compartmental model (NCA) in Phoenix-WinNonlin 6.4 software was used to calculate the pharmacokinetic parameters.

During the entire experiment, the mice were in normal clinical observation. The serum drug concentration-time curve is shown in Figure 21. The drug is metabolized rapidly in mice, and the drug concentration drops rapidly over time, with no obvious gender difference. The pharmacokinetic parameters are shown in Table 17. After SARS-2-H014-Fd19-IgG4 was administered to mice by a single intravenous injection, the average in vivo exposure C _max and AUC _last were 125.11 μg/mL and 1202.18 h, respectively. ×μg/mL, the average half-life t _1/2 is only 11.72h, the clearance rate Cl is 4.13mL/h/kg, and its metabolic characteristics are related to the molecular structure modification of the FcRn binding site.

Table 17 The pharmacokinetic parameters of a single intravenous injection of SARS-2-H014-Fd19-IgG4 in mice (0～72h)

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Nucleotide and Amino Acid Sequence Listing

Claims

An isolated, blocking SARS-CoV-2 spike protein and/or SARS-CoV spike protein binding to the ACE2 receptor or its antigen-binding fragment, comprising any one of a) to d), wherein

a) i) Heavy chain variable region, whose heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 domains are respectively SEQ ID NOs: 13, 14 and 15 or at least 85%, 88%, 90%, 95% , 98% or 99% sequence identity, and/or

ii) The light chain variable region, whose light chain CDR1, light chain CDR2 and light chain CDR3 domains are respectively SEQ ID NOs: 10, 11 and 12 or have at least 75%, 78%, 80%, 85%, 90 %, 91%, 95%, 98% or 99% sequence identity;

b) i) The heavy chain variable region, whose heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 domains are respectively SEQ ID NO: 13, 14 and 15 with at least 85%, 88%, 90%, 95%, 98% or 99% sequence identity, and/or

ii) The light chain variable region, whose light chain CDR1, light chain CDR2 and light chain CDR3 domains are respectively SEQ ID NO: 45, 11 and 46 or at least 75%, 78%, 80%, 85%, 90 %, 91%, 95%, 98% or 99% sequence identity;

c) i) The heavy chain variable region, whose heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 domains are respectively SEQ ID NOs: 67, 68 and 69 with at least 85%, 88%, 90%, 95%, 98% or 99% sequence identity, and/or

ii) The light chain variable region, whose light chain CDR1, light chain CDR2 and light chain CDR3 domains are respectively SEQ ID NOs: 10, 11 and 12 or have at least 75%, 78%, 80%, 85%, 90 %, 91%, 95%, 98%, or 99% sequence identity; and

d) i) The heavy chain variable region, whose heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 domains are respectively SEQ ID NO: 67, 68, and 69 with at least 85%, 88%, 90%, 95%, 98% or 99% sequence identity, and/or

ii) The light chain variable region, whose light chain CDR1, light chain CDR2 and light chain CDR3 domains are respectively SEQ ID NO: 45, 11 and 12 or have at least 75%, 78%, 80%, 85%, 90 %, 91%, 95%, 98%, or 99% sequence identity.
The antibody or antigen-binding fragment thereof according to claim 1, which comprises any one of a) to d), wherein:

a) i) the heavy chain variable region whose sequence is SEQ ID NO: 22 or has at least 85%, 88%, 90%, 95%, 98%, or 99% sequence identity with it; and/or

ii) The light chain variable region, whose sequence is SEQ ID NO: 23 or has at least 85%, 88%, 90%, 95%, 98%, or 99% sequence identity with it;

b) i) Heavy chain variable region, whose sequence is SEQ ID NO: 51 or has at least 85%, 88%, 90%, 95%, 98%, or 99% sequence identity with it; and/or

ii) The light chain variable region, whose sequence is SEQ ID NO: 52 or has at least 85%, 88%, 90%, 95%, 98%, or 99% sequence identity with it;

c) i) The heavy chain variable region whose sequence is SEQ ID NO: 74 or has at least 85%, 88%, 90%, 95%, 98%, or 99% sequence identity with it; and/or

ii) The light chain variable region whose sequence is SEQ ID NO: 75 or has at least 85%, 88%, 90%, 95%, 98%, or 99% sequence identity with it;

d) i) the heavy chain variable region whose sequence is SEQ ID NO: 94 or has at least 85%, 88%, 90%, 95%, 98% or 99% sequence identity with it; and/or

ii) The light chain variable region whose sequence is SEQ ID NO: 95 or has at least 85%, 88%, 90%, 95%, 98%, or 99% sequence identity with it.
The antibody or antigen-binding fragment thereof according to any one of claims 1-2, which is a humanized antibody or a chimeric antibody.
The antibody or antigen-binding fragment thereof according to any one of claims 1-2, whose antibody constant region is IgG, IgM, IgA subtype, preferably,

It is an antibody of IgG1, IgG2 or IgG4 subtype; more preferably

It is an IgG1, IgG2, or IgG4 subtype antibody whose binding function to Fc receptor, C1q complement and FcRn receptor is changed due to changes in the amino acid sequence and/or glycosylation form of its Fc region.
The antibody or antigen-binding fragment thereof according to any one of claims 1-2, wherein the antibody further comprises:

a) The heavy chain constant region, preferably its sequence is SEQ ID NO: 24 or has at least 90%, 92%, 95%, 98% or 99% sequence identity with it; and/or

b) The light chain constant region, preferably, its sequence is SEQ ID NO: 25 or has at least 90%, 92%, 95%, 98% or 99% sequence identity with it.
The antibody or antigen-binding fragment thereof according to any one of claims 1-5,

a) The average KD of its binding affinity to SARS-CoV-2 S1 is 0.9E-11 to 8.7E-10M, preferably 2.0E-11 to 3E-10M, more preferably 2.6E-10, 2.9E-10, 2.1E-10 or 2.7E-11M; and/or

b) The average KD of its binding affinity to SARS-CoV S1 is 0.4E-11 to 6.0E-10M, preferably 1.0E-11 to 8E-10M, more preferably 1.2E-11, 1.1E-10, 2.0E -10 or 7.5E-11M.
The antibody or antigen-binding fragment thereof according to claim 5, whose heavy chain variable region and light chain variable region are as described in claim 2 a),

After being administered to mice by a single intravenous injection, the average in vivo exposure C max and AUC last were 136.15 μg/mL and 10930.35 h×μg/mL, respectively, the average half-life t 1/2 was 281.20 h, and the clearance rate Cl was 0.27mL/h/kg.
The antibody or antigen-binding fragment thereof according to claim 2 a) or claim 5, which is expressed by Fut8 gene knockout mammalian cells, preferably, the cells are Fut8 gene knockout CHO and HEK-293 cells .
The antibody or antigen-binding fragment thereof according to claim 8, wherein:

i) It exhibits significantly better binding ability to CD16a than IgG1 subtype, weakly binds to CD32a or CD32b protein at high concentrations, and closes CD64, C1q complement protein and FcRn binding levels to IgG1 subtype antibodies; and/ or

ii) It shows a significantly better ADCC function than the IgG1 subtype and a similar ADCP function, and there is no change in the CDC function.
The antibody or antigen-binding fragment thereof according to claim 2 a), wherein the antibody further comprises:

i) The heavy chain constant region, preferably, its sequence is SEQ ID NO: 106 or has at least 90%, 92%, 95%, 98% or 99% sequence identity with it; and/or

ii) The light chain constant region, preferably, its sequence is SEQ ID NO: 25 or has at least 90%, 92%, 95%, 98% or 99% sequence identity with it.
The antibody or antigen-binding fragment thereof according to claim 10, having the following characteristics:

i) No binding to CD32a, CD32b, CD16a and C1q complement proteins, a weak binding level to CD64 under high concentration conditions, and binding to FcRn similar to IgG1 subtype antibodies under pH 6.0; and/or

ii) No obvious ADCC, CDC and ADCP functions; and/or

iii) After a single intravenous injection to mice, the average in vivo exposure C max and AUC last were 144.66 μg/mL and 11940.01 h×μg/mL, respectively, the average half-life t 1/2 was 290.08 h, and the clearance rate was Cl It is 0.26mL/h/kg.
The antibody or antigen-binding fragment thereof of claim 2 a), wherein the antibody further comprises:

i) The heavy chain constant region, preferably its sequence is SEQ ID NO: 108 or has at least 90%, 92%, 95%, 98% or 99% sequence identity with it; and/or

ii) The light chain constant region, preferably, its sequence is SEQ ID NO: 25 or has at least 90%, 92%, 95%, 98% or 99% sequence identity with it.
The antibody or antigen-binding fragment thereof according to claim 12, having the following characteristics:

i) No binding to CD32a, CD32b, CD16a, CD64 and C1q complement proteins, and a weak binding level of FcRn under pH 6.0 and high concentration conditions; and/or

ii) Basically no ADCC, CDC and ADCP functions; and/or

iii) After a single intravenous injection to mice, the average in vivo exposure C max and AUC last were 125.11μg/mL and 1202.18h×μg/mL, respectively, the average half-life t 1/2 was only 11.72h, and the clearance rate Cl is 4.13mL/h/kg.
The antibody or antigen-binding fragment thereof according to any one of claims 1-13, which is a monoclonal antibody.
The antibody or antigen-binding fragment thereof according to any one of claims 1-14, wherein the antigen-binding fragment is Fv, Fab, Fab', Fab'-SH, F(ab')2, Fd fragment, Fd' Fragment, single chain antibody molecule or single domain antibody; wherein the single chain antibody molecule is preferably scFv, di-scFv, tri-scFv, diabody or scFab.
The antibody or antigen-binding fragment thereof according to any one of claims 1-15, whose epitope is the structural region of SARS-CoV-2 and SARS-CoV viral spike proteins including S375, K378, D405 and R408.
An antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-16 and another therapeutic agent, preferably the antibody or antigen-binding fragment thereof and another therapeutic agent Connect through the connector.
A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-17, which is mRNA and/or DNA.
The nucleic acid of claim 18, which comprises

a) The nucleotide sequence of the heavy chain variable region shown in SEQ ID NO: 30, 55, 78 and 98 and/or the light chain variable region shown in SEQ ID NO: 31, 56, 79 and 99, respectively Nucleotide sequence; and optionally

b) The nucleotide sequence of the heavy chain constant region shown in SEQ ID NO: 6, 105 and 107 and/or the nucleotide sequence of the light chain constant region shown in SEQ ID NO: 7 respectively;

Or variants of a) and b).
An expression vector comprising the nucleic acid according to claim 18 or 19.
A host cell comprising the nucleic acid according to claim 18 or 19 or the expression vector according to claim 20.
A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 1-16, which comprises culturing the host cell according to claim 21 under conditions suitable for antibody expression, and from culturing The expressed antibody is recovered from the base.
A pharmaceutical composition comprising

The antibody or antigen-binding fragment thereof according to any one of claims 1-16 or the antibody-drug conjugate according to claim 17 or the nucleic acid according to any one of claims 18-19 or as claimed 20 the expression vector;

A pharmaceutically acceptable carrier; optionally

One or more other therapeutic agents, preferably, the other therapeutic agents are selected from antiviral drugs or inflammatory factor inhibitors, small molecule chemical drugs of other mechanisms; preferably, the antiviral drugs are selected from including but not limited to type I interferon Drugs, antibodies, protease inhibitors, RNA-dependent RNA polymerase (RdRP) inhibitors, and host-targeted antiviral drugs.
The antibody or antigen-binding fragment thereof according to any one of claims 1-16, the antibody-drug conjugate according to claim 17, the nucleic acid according to any one of claims 18-19, as claimed The expression vector according to 20 and the pharmaceutical composition according to claim 23, which are used to prevent and treat diseases caused by SARS-CoV-2 and/or SARS-CoV infection.
The antibody or antigen-binding fragment thereof according to any one of claims 1-16, the antibody-drug conjugate according to claim 17, the nucleic acid according to any one of claims 18-19, as claimed Use of the expression vector of 20 and the pharmaceutical composition of claim 23 in the preparation of a medicine for preventing and treating diseases caused by SARS-CoV-2 and/or SARS-CoV infection.
A drug combination comprising

The antibody or antigen-binding fragment thereof according to any one of claims 1-16, the antibody-drug conjugate according to claim 17, the nucleic acid according to any one of claims 18-19, as claimed The expression vector of 20, the pharmaceutical composition of claim 23; and

One or more additional therapeutic agents.
A kit containing

The antibody or antigen-binding fragment thereof according to any one of claims 1-16, the antibody-drug conjugate according to claim 17, the nucleic acid according to any one of claims 18-19, as claimed The expression vector according to 20, the pharmaceutical composition according to claim 23; preferably,

It further includes a device for administration.
A method for preventing and treating diseases caused by SARS-CoV-2 and/or SARS-CoV infection, which comprises administering to a subject

The antibody or antigen-binding fragment thereof according to any one of claims 1-16, the antibody-drug conjugate according to claim 17, the nucleic acid according to any one of claims 18-19, as claimed The expression vector according to 20, the pharmaceutical composition according to claim 23, the pharmaceutical combination according to claim 26, or the kit according to claim 27.
An isolated, blocking SARS-CoV-2 spike protein/SARS-CoV spike protein and ACE2 receptor binding antibody or antigen-binding fragment thereof, its binding epitope is a structural region containing S375, K378, D405 and R408 .
A binding epitope of SARS-CoV-2 spike protein/SARS-CoV spike protein, which is the structure of SARS-CoV-2 spike protein/SARS-CoV spike protein containing S375, K378, D405 and R408 area.