WO2023019723A1 - 单克隆抗体32c7及其制备方法和用途 - Google Patents

单克隆抗体32c7及其制备方法和用途 Download PDF

Info

Publication number
WO2023019723A1
WO2023019723A1 PCT/CN2021/125690 CN2021125690W WO2023019723A1 WO 2023019723 A1 WO2023019723 A1 WO 2023019723A1 CN 2021125690 W CN2021125690 W CN 2021125690W WO 2023019723 A1 WO2023019723 A1 WO 2023019723A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
antigen
seq
binding fragment
amino acid
Prior art date
Application number
PCT/CN2021/125690
Other languages
English (en)
French (fr)
Inventor
朱永群
邓凯
Original Assignee
上海浙江大学高等研究院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海浙江大学高等研究院 filed Critical 上海浙江大学高等研究院
Publication of WO2023019723A1 publication Critical patent/WO2023019723A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the field of biomedicine, specifically, the present invention relates to antibodies for preventing and treating novel coronavirus infection and its preparation, especially monoclonal antibody 32C7 and its preparation and use.
  • S The most prominent target for mediating antibody responses on the surface of SARS-CoV-2 virions is the homotrimeric spike (S) protein.
  • the spike protein facilitates viral entry through the interaction of the receptor domain (RBD) with angiotensin-converting enzyme 2 (ACE2). Therefore, antibodies targeting the RBD domain of the spike protein are particularly important in the fight against the current pandemic.
  • RBD receptor domain
  • ACE2 angiotensin-converting enzyme 2
  • Cheolmin Kim et al. screened human monoclonal antibody mAbs against the receptor-binding domain of the viral spike protein through an antibody library constructed from peripheral blood mononuclear cells of recovered patients.
  • the results showed that the CT-P59 mAb effectively neutralized SARS-CoV-2 isolates, including the D614G variant, without antibody-dependent potentiation, which blocked the interaction of the receptor-binding domain with angiotensin-converting enzyme 2 (ACE2) The interaction region of the receptor.
  • ACE2 angiotensin-converting enzyme 2
  • the therapeutic effect of CT-P59 which was able to reduce viral titers, was evaluated in three animal models (ferrets, hamsters, and rhesus monkeys).
  • CN113024640A discloses an epitope peptide antigen detection neutralizing antibody kit based on the screening of the new coronavirus RBD and the ACE2 receptor binding domain, which contains the amino acid sequence shown in SEQ ID NO: 1-4 based on the new coronavirus Epitope peptide antigen for RBD and ACE2 receptor binding domain screening.
  • the purpose of the present invention is to make up for the deficiencies of the prior art, to provide a new neutralizing antibody 32C7 and its preparation method and application, so as to provide a safe and effective antibody for the clinical treatment of COVID-19, the antibody of the present invention or Its antigen-binding fragment has broad application prospects. details as follows.
  • an antibody 32C7 or an antigen-binding fragment thereof which can specifically bind to the spike protein, and which can bind to a protein selected from the group consisting of 345, 346, 347, 348, 349, 351, or
  • the epitope is at least one epitope selected from the group consisting of T345, R346, F347, S349, N440, L441, K444, N450, Y351 and Y451 of the spike protein, to inhibit or block the spike protein Spike protein binding to receptors.
  • the epitope consists of 345, 346, 347, 348, 349, 351, 352, 354, 440, 441, 442, 443 of the spike protein , 444, 445, 448, 450, 451, 452, 466, 499, 509 amino acid residues, or the epitope consists of T345, R346, F347, S349, N440, L441, K444, Composed of N450, Y351 and Y451.
  • an antibody 32C7 or an antigen-binding fragment thereof which comprises a heavy chain and a light chain, and which exhibits a Kd for the receptor domain of the spike protein of less than 120 nM.
  • the third aspect of the present invention provides an antibody 32C7 or an antigen-binding fragment thereof, which contains the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region, and its amino acid sequences are SEQ ID NO: 2, SEQ ID NO : 4, SEQ ID NO: 6; and
  • amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12, respectively.
  • the amino acid sequence of its heavy chain variable region includes the antigen complementarity determinations shown in SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6 region and have at least 80%, preferably at least 90%, preferably at least 95%, and more preferably at least 99% identity with the sequence shown in SEQ ID NO: 14; and the amino acid sequence of its light chain variable region comprises SEQ ID NO 8, SEQ ID NO: 10, the antigen complementarity determining region shown in SEQ ID NO: 12 and has at least 80%, preferably at least 90%, preferably at least 95%, more preferably at least 95% of the sequence shown in SEQ ID NO: 17 99% identity, and at the same time retain at least the function of the pre-mutation sequence.
  • the antibody 32C7 or its antigen-binding fragment preferably, wherein the antibody 32C7 or its antigen-binding fragment is a monoclonal antibody; the antigen-binding fragment includes Fab fragment, Fab', F(ab ') 2 fragment, single chain variable fragment scFv, scFv-Fc fragment or single chain antibody ScAb.
  • the fourth aspect of the present invention provides a nucleic acid molecule encoding the antibody 32C7 or an antigen-binding fragment thereof according to the first, second or third aspect of the present invention.
  • the fifth aspect of the present invention provides a vector comprising the nucleic acid molecule according to the fourth aspect of the present invention.
  • the sixth aspect of the present invention provides a method for preparing antibody 32C7 or an antigen-binding fragment thereof, the method comprising culturing cells under conditions suitable for the expression of the antibody 32C7 or an antigen-binding fragment thereof, thereby producing the monoclonal Cloning an antibody, wherein the cell comprises the vector according to the fifth aspect of the present invention.
  • the seventh aspect of the present invention provides a composition containing the antibody 32C7 or an antigen-binding fragment thereof according to the first, second or third aspect of the present invention.
  • the eighth aspect of the present invention provides the use of the antibody 32C7 or its antigen-binding fragment according to the first aspect, the second aspect or the third aspect of the present invention in the preparation of drugs or reagents for treating or preventing ACE2-related diseases,
  • the disease is selected from coronavirus infection, also preferably, the coronavirus includes but not limited to SARS-CoV-2 (D614 and G614), SARS-CoV or HCoV-NL63.
  • the antibody 32C7 or its antigen-binding fragment can specifically bind to the Spike protein, and it can bind to a protein selected from the group consisting of 345, 346, 347, 348, 349, 351, 352, 354, 440, 441, 442, 443, 444, 445, 448, 450, 451, 452, 466, 499, 509 amino acid residues, or the epitope is selected from the group consisting of thorns T345, R346, F347, S349, N440, L441, K444, N450, Y351 and Y451 of the spike protein to inhibit or block the binding of the spike protein to the receptor.
  • the novel coronavirus includes, but is not limited to, the original strain, the British mutant virus strain B.1.1.7, the South African mutant virus strain B.1.351 and B. 1.617.2 (Indian Delta strain).
  • the ninth aspect of the present invention provides a method for preventing and/or treating coronavirus infection in a subject, comprising administering to a subject in need an effective amount of the antibody or antigen-binding fragment of the present invention, a composition thereof, or Drugs or agents of the present invention.
  • the therapeutically effective amount is about 0.1-200 mg/kg, preferably 20 mg/kg.
  • the tenth aspect of the present invention provides a pharmaceutical composition, which contains the antibody 32C7 or an antigen-binding fragment thereof, a nucleic acid molecule, or a carrier, and a pharmaceutically acceptable carrier and/or excipient according to the present invention.
  • the pharmaceutical composition is a vaccine, preferably a subunit vaccine, and the excipient is an adjuvant.
  • the adjuvant includes aluminum adjuvant, Freund's adjuvant, aluminum phosphate, calcium phosphate, paraffin oil, lanolin, surfactant, calcium alginate, Polynucleotide, muramide, saponin, RIBI adjuvant system, cholera toxin, polymer of acrylic acid or methacrylic acid, water-in-oil emulsion, oil-in-water emulsion, preferably aluminum adjuvant agent.
  • the eleventh aspect of the present invention provides a method for preventing or treating coronavirus infection in a subject, preferably, administering to the subject an effective amount of the tenth coronavirus according to the present invention via oral administration, intramuscular injection or nasal instillation.
  • the pharmaceutical composition described in aspect is described in detail.
  • the antibody 32C7 of the present invention has high affinity with the RBD domain of the new coronavirus, and the dissociation is very slow.
  • the antibody of the present invention can effectively inhibit the infection of SARS-CoV-2 live virus, not only showing a significant inhibitory effect on the original strain, but also having a good neutralizing inhibitory effect on the mutant virus strain, which shows that relatively Compared with the existing representative antibodies, the antibodies of the present invention have excellent effects and application prospects.
  • animal model experiments further prove that the antibody of the present invention can greatly reduce the virus titer of SARS-CoV-2, and the relevant inflammatory factors are significantly reduced, and the inflammatory infiltration is significantly improved.
  • the inventors further characterized the structure of the combination of the antibody and RBD, and surprisingly found that the antibody of the present invention avoids the mutation site of the current mutant strain of the new coronavirus, making it have a good inhibitory effect on a variety of mutant strains .
  • Figure 1 is the in vitro anti-new coronavirus effect of the antibody 32C7 according to Example 2 of the present invention, wherein, part A of Figure 1 shows the gradient-dependent interaction results of the antibody 32C7 and the new coronavirus SPR domain, in which from top to The lower concentrations are 120, 60, 30, 15 and 7.5 in sequence, and the unit is nM; Part B of Figure 1 shows that the antibody 32C7 can dose-dependently inhibit the infection of various subtypes of SARS-CoV-2 mutant strains.
  • Figure 2 is the effect of the antibody 32C7 according to Example 3 of the present invention in the transgenic mouse new coronavirus infection model, wherein, part A of Figure 2 is the titer of new coronavirus in the mouse lung grinding liquid; part B of Figure 2 is the small The expression levels of related inflammatory factors in the mouse lung grinding solution; part C of Figure 2 shows the pathological changes of the mouse lung.
  • Figure 3 is an electron microscope observation result of the binding of antibody 32C7 to the S protein of the new coronavirus according to Example 4 of the present invention, wherein, part A of Figure 3 is a structural diagram of the binding of the antibody 32C7 to the S protein of the new coronavirus; part B of Figure 3 is the antibody The epitope of 32C7 binding to the S protein of the new coronavirus.
  • “about X” includes a numerical range of ⁇ 20%, ⁇ 10%, ⁇ 5%, ⁇ 2%, ⁇ 1%, ⁇ 0.5%, ⁇ 0.2%, or ⁇ 0.1% of X, where X is a value.
  • the term “about” refers to a numerical range of 5% more or less than the specified value.
  • the term “about” refers to a numerical range of 2% more or less than the specified value.
  • the term “about” refers to a numerical range of 1% more or less than the specified value.
  • spike protein refers to a protein with a trimeric structure located on the outermost layer of the new coronavirus, also known as spike glycoprotein or S protein. It is a structural protein derived from coronaviruses including SARS-CoV and 2019-nCoV.
  • the coronavirus S protein e.g., 2019-nCoV S protein, SARS-CoV S protein
  • epitope includes any protein determinant capable of specifically binding to an immunoglobulin or fragment thereof or a T-cell receptor.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules (such as amino acids or sugar side chains) and usually have specific three-dimensional structural characteristics as well as specific charge characteristics.
  • the epitope specifically bound by the antibody of the present invention is preferably selected from the 345th, 346, 347, 348, 349, 351, 352, 354, 440, 441, 442, 443, 444, 445, 448, 450 of the spike protein , 451, 452, 466, 499, 509 amino acid residues.
  • it specifically binds at least one epitope among T345, R346, F347, S349, Y351, N450, Y451, N440, L441 and K444 of the spike protein.
  • the term “antibody” refers to an immunologically active portion of an immunoglobulin (Ig) molecule, ie, a molecule that contains an antigen binding site that specifically binds (immunoreacts with) an antigen. "Specifically binds” or “immunoreacts with” means that an antibody reacts with one or more epitopes of the desired antigen and does not react or bind with much lower affinity (Kd) with other polypeptides.
  • Kd lower affinity
  • the antibodies of the invention exhibit a Kd for the receptor domain of the Spike protein of less than 120 nM.
  • the receptor domain of the spike protein has a Kd for the receptor domain of the spike protein of less than 80nM, such as 70nM, 60nM, 50nM, 40nM, 30nM, 20nM, 10nM, even less than 8nM, such as 7nM, or even less than 5nM.
  • Antibodies include, but are not limited to, monoclonal antibodies, chimeric antibodies, dAbs (domain antibodies), single chain antibodies, Fab, Fab' and F(ab') 2 fragments, scFvs.
  • the antibodies of the invention are monoclonal antibodies or antigen-binding fragments thereof.
  • antibody as used herein also includes humanized antibodies, recombinant antibodies, human antibodies produced from transgenic non-human animals, and antibodies selected from libraries using enrichment techniques available to those skilled in the art.
  • an antibody is a glycoprotein or an antigen-binding portion thereof comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • a heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH1, CH2 and CH3).
  • a light chain comprises a light chain variable region (VL) and a light chain constant region (CL).
  • the variable regions of the heavy and light chains comprise framework regions (FRs) and complementarity determining regions (CDRs). The four FRs are relatively conserved, while the CDR regions (CDR1, CDR2 and CDR3) contain hypervariable regions.
  • Antibody structural units are known to generally comprise tetramers.
  • Each tetramer is composed of two pairs of identical polypeptide chains, each pair having one light chain and one heavy chain.
  • the amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the carboxy-terminal portion of each chain defines a constant region primarily responsible for effector functions.
  • the FRs and CDRs are arranged as follows from the NH2 end to the COOH end: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant regions can mediate the binding of the immunoglobulin to host tissues or factors.
  • Antibody constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cellular cytotoxicity (ADCC) or complement-mediated cytotoxicity (CDC) of the antibody.
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-mediated cytotoxicity
  • antibody molecules involve IgG, IgM, IgA, IgE, and IgD, which differ from each other by the nature of the heavy chains present in the molecule.
  • the complementarity determining regions CDR1, CDR2 and CDR3 of the antibody heavy chain variable region are the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 respectively; the antibody light chain variable region
  • the antigen complementarity determining regions CDR1, CDR2 and CDR3 are the amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12, respectively.
  • antibody fragment includes a portion of an intact antibody, such as the antigen-binding or variable region of an intact antibody.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , Fv fragments, scFv fragments, diabodies, or linear antibodies.
  • Papain digestion of antibodies produces two identical "Fab” fragments, or antigen-binding fragments, each with a single antigen-binding site, and a residual "Fc” fragment (whose name reflects its ability to readily crystallize).
  • Pepsin treatment of the antibody produces an F(ab') 2 fragment, which has two antigen combining sites and which retains its ability to cross-link antigen.
  • Fv denotes the minimal antibody fragment that contains a complete antigen recognition and antigen binding site. This fragment contains a dimer of one heavy chain variable region domain and one light chain variable region domain in tight non-covalent association. Folding of these two domains results in the formation of six hypervariable loops (three loops each from the H and L chains) that facilitate amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, albeit with lower affinity.
  • Single-chain Fv (“sFv” or “scFv”) is an antibody fragment comprising the VH and VL antibody domains linked into a single polypeptide chain.
  • the sFv polypeptide may also comprise a polypeptide linker between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding.
  • Fab fragment as used herein contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of several residues at the carboxy-terminus of the CH1 domain of the heavy chain, including one or more cysteines from the antibody hinge region.
  • monoclonal antibody refers to immunoglobulins obtained from a clone of cells, having identical structural and chemical properties, and specific for a single antigenic determinant .
  • Monoclonal antibodies differ from conventional polyclonal antibody preparations (which typically have different antibodies directed against different determinants) in that each monoclonal antibody is directed against a single determinant on the antigen.
  • monoclonal antibodies have the advantage that they are obtained in hybridoma or recombinantly engineered cell culture and are not contaminated with other immunoglobulins.
  • the modifier "monoclonal” indicates the identity of the antibody obtained from a homogeneous population of antibodies, but this should not be construed as requiring any particular method for producing the antibody.
  • Antibodies of the invention may comprise an Fc region derived from an IgG, such as IgGl, IgG2, IgG3 or IgG4.
  • an antibody or antigen-binding fragment thereof described herein is an isolated antibody or antigen-binding fragment thereof.
  • isolated refers to a nucleic acid or antibody that has been extracted from its natural environment. Nucleic acids, peptides and proteins that have been “isolated” thus include nucleic acids and proteins purified by standard purification methods. The term also includes nucleic acids, peptides and proteins produced by recombinant expression in host cells as well as chemically synthesized nucleic acids and/or polypeptides.
  • receptor binding domain refers to the region where the S protein associates or binds with ACE2, also known as RBD.
  • antigen binding site or "binding portion” refers to the portion of an immunoglobulin molecule that participates in antigen binding.
  • the antigen binding site is formed by the amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light (“L”) chains.
  • V N-terminal variable
  • H heavy
  • L light
  • Three highly divergent segments termed “hypervariable regions” in the V regions of the heavy and light chains are inserted between more conserved flanking segments called “framework regions” or "FRs.”
  • FR refers to the naturally found amino acid sequences between and adjacent hypervariable regions of an immunoglobulin.
  • the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface.
  • the antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity determining regions" or "CDRs".
  • binding and “immunological binding” are used interchangeably and refer to a non-covalent interaction that occurs between an immunoglobulin molecule and an antigen specific for said immunoglobulin.
  • the strength or affinity of an immunological binding interaction can be expressed in terms of a dissociation constant (Kd), where a smaller Kd represents a higher affinity.
  • Kd dissociation constant
  • Affinity refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen).
  • binding affinity refers to intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen).
  • the affinity of a molecule X for its partner Y is usually expressed in terms of a binding-dissociation equilibrium constant (Kd). Affinity can be measured by common methods known in the art, including those known in the art and described herein.
  • the term “specific binding” generally means that an antibody binds to an epitope through its antigen-binding domain, and the binding requires complementarity between the antigen-binding domain and the epitope. Thus, when an antibody binds to that epitope through its antigen-binding domain more readily than it binds to a random, unrelated epitope, it is said to “specifically bind” to that epitope.
  • the term “specificity” is used in the present invention to define the relative affinity with which a certain antibody binds to a certain epitope.
  • antibody “A” can be said to have a higher specificity for a particular epitope than antibody "B”, or antibody “A” can be said to bind epitope “C” with a higher specificity than it binds the related epitope "D”. ".
  • fragment fragment
  • antibody fragment fragment
  • antigen-binding fragment fragment
  • antigen-binding fragment fragment
  • antigen-binding fragment fragment
  • variant antibodies are also included within the scope of the present invention. Therefore, variants of the sequences listed in the present invention are also included in the scope of the present invention. Other variants of antibody sequences with improved affinity can be obtained using methods known in the art and are included within the scope of the present invention.
  • the amino acid sequence of a polypeptide can be modified by those skilled in the art using recombinant methods and/or synthetic chemistry techniques for producing variant polypeptides. For example, amino acid substitutions can be used to obtain antibodies with further improved affinity. Alternatively, codon optimization of the nucleotide sequence can be used to increase translation efficiency in the expression system used to produce the antibody.
  • Such variant antibody sequences have 80% or more (i.e., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the sequences recited in the present invention sex. Said sequence identity is calculated with respect to the sequences recited in the present invention. Or when performing an optimal alignment, such as via the program GAP or BESTFIT using the default gap weights.
  • the percent homology between two amino acid sequences is equal to the percent identity between the two sequences.
  • the comparison of sequences and the determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)). Additionally, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)).
  • the antibodies of the present invention also include antibodies with modified amino acid sequences.
  • the antibodies of the present invention comprise heavy chain variable domains containing CDR1, CDR2 and CDR3 sequences and light chain variable domains containing CDR1, CDR2 and CDR3 sequences,
  • One or more of these CDR sequences comprise specific amino acid sequences or modifications based on the antibodies described herein, and the modified antibodies retain the desired functional properties of the anti-new coronavirus antibody of the present invention.
  • modification means that the amino acid modification does not significantly affect or alter the binding characteristics of an antibody comprising the amino acid sequence. Such modifications include amino acid substitutions, additions and deletions. Preferably, residue positions that are not identical differ by conservative amino acid substitutions.
  • Antibodies of the invention may include glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or non-naturally occurring amino acid modifications, among others.
  • Conservative amino acid substitutions refer to the interchangeability of residues with similar side chains.
  • the groups of amino acids with aliphatic side chains are glycine, alanine, valine, leucine, and isoleucine; the groups of amino acids with aliphatic-hydroxyl side chains are serine and threonine;
  • the amino acid groups with side chains are asparagine and glutamine; the amino acid groups with aromatic side chains are phenylalanine, tyrosine and tryptophan;
  • the amino acid groups with basic side chains are lysine, arginine and histidine; and the groups of amino acids with sulfur-containing side chains are cysteine and methionine.
  • Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-tianmen Paragmate and Asparagine-Glutamine.
  • one or more amino acid residues in a CDR region of an antibody of the invention may be replaced with other amino acid residues from the same side chain family.
  • the invention encompasses minor variations in the amino acid sequence of an antibody or immunoglobulin molecule, provided that the amino acid sequence maintains at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99% Mutations.
  • conservative amino acid substitutions are contemplated. Conservative substitutions are those that occur within a family of amino acids with respect to their side chains.
  • amino acids are usually divided into the following families: (1) acidic amino acids are aspartic acid, glutamic acid; (2) basic amino acids are lysine, arginine, histidine; (3) non-polar amino acids Sexual amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan and (4) uncharged polar amino acids are glycine, natural Paragine, glutamine, cysteine, serine, threonine, tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine.
  • Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine.
  • Other families of amino acids include (i) serine and threonine, which are aliphatic-hydroxyl families; (ii) asparagine and glutamine, which are amide-containing families; (iii) alanine, valine, leucine and isoleucine, which are of the aliphatic family; and (iv) phenylalanine, tryptophan and tyrosine, which are of the aromatic family.
  • a split substitution of isoleucine or valine for leucine, glutamic acid for aspartic acid, serine for threonine, or an amino acid similar to a structurally related amino acid would be reasonably expected to There will be no significant effect on the binding or properties of the resulting molecule, especially if the substitution does not involve an amino acid in a framework position.
  • Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the polypeptide derivative.
  • the assay method is not particularly limited, and it can be assayed by methods known in the art. Fragments or analogs of antibody or immunoglobulin molecules can be readily prepared by those skilled in the art.
  • the amino- and carboxyl-termini of the fragments or analogs occur near functional domain boundaries.
  • Structural and functional domains can be identified by comparison of nucleotide and/or amino acid sequence data to public or private sequence databases.
  • computerized comparison methods are used to identify sequence motifs or predicted protein conformational domains that occur in other proteins of known structure and/or function. Methods for identifying protein sequences that fold into a known three-dimensional structure are known.
  • Preferred amino acid substitutions are those that: (1) decrease susceptibility to proteolysis; (2) decrease susceptibility to oxidation; (3) alter binding affinity for protein complex formation; (4) alter binding affinity and ( 4) Imparting or modifying the physicochemical or functional properties of the analogue.
  • Analogs may include various muteins of sequences other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally occurring sequence (preferably in portions of the polypeptide other than the domains that form intermolecular contacts). Conservative amino acid substitutions should not substantially alter the structural characteristics of the parent sequence (eg, the substituting amino acids should not tend to disrupt helices occurring in the parent sequence, or disrupt other types of secondary structure that characterize the parent sequence).
  • variable region modification is to mutate amino acid residues in the VH and/or VK CDR1, CDR2 and/or CDR3 regions to improve one or more binding properties (e.g., affinity) of the antibody of interest.
  • Mutations can be introduced by site-directed mutagenesis or PCR-mediated mutagenesis. Conservative modifications (as described above) are preferably introduced. Mutations may be amino acid substitutions, additions or deletions, but are preferably substitutions. Furthermore, typically no more than one, two, three, four or five residues are changed in the CDR regions.
  • the amino acid sequence of the heavy chain variable region of the monoclonal antibody 32C7 or its antigen-binding fragment comprises the antigen complementarity determining region shown in SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6 and is identical to SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6.
  • the sequence shown in ID NO: 14 has at least 80% identity; and the amino acid sequence of its light chain variable region includes the antigen complementarity determining region shown in SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12 And have at least 80% identity with the sequence shown in SEQ ID NO:17.
  • the identity variant here can be that the mutation occurs in the CDR region of the variable region, or it can occur in the FR region of the framework region.
  • the function of the pre-mutation sequence, that is, the mutation in the above region will not affect the binding characteristics, binding epitope and spatial conformation of the antibody, and will not affect the neutralizing effect of the antibody of the present invention.
  • the invention provides nucleic acid molecules that encode the antibodies of the invention.
  • Nucleic acids may be present in intact cells, in cell lysates, or in partially purified or substantially pure form. When purified by standard techniques including base/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art to remove other cellular components or other contaminants, such as other cellular nucleic acids or Nucleic acids are "isolated" from proteins.
  • a nucleic acid of the invention may be, for example, DNA or RNA, and may or may not contain intronic sequences. In preferred embodiments, the nucleic acid is a cDNA molecule.
  • the nucleic acid of the present invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NO.: 2, 4, 6, 8, 10, 12, or encoding and being selected from SEQ ID NO.: 2, 4, 6
  • the amino acid sequence shown in any one of , 8, 10, 12 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
  • the coding sequence consists of the sequence shown in SEQ ID NO.: 1, 3, 5, 7, 9, 11.
  • Nucleic acids of the invention can be obtained using standard molecular biology techniques. Once the DNA fragments encoding the VH and VL segments are obtained, these DNA fragments are further manipulated by standard recombinant DNA techniques, eg, to convert variable region genes to full length antibody chain genes, Fab fragment genes or scFv genes. In these manipulations, a VL- or VH-encoding DNA fragment is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker.
  • the term "operably linked” as used herein is intended to mean the joining of two DNA fragments such that the amino acid sequences encoded by the two DNA fragments remain in-frame.
  • the isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding the heavy chain constant regions (CH1, CH2 and CH3).
  • the sequence of the human heavy chain constant region gene is known in the art.
  • the isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operably linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL.
  • the sequences of the human light chain constant region genes are known in the art.
  • the invention also provides polynucleotide variants encoding the peptide sequences of the heavy and light chains of the antibodies of the invention or antigen-binding fragments thereof.
  • These polynucleotide variants may have at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, compared to the polynucleotide sequences of the present invention. %, or at least 97%, or at least 98%, or at least 99% or greater sequence identity.
  • Such contiguous sequences may encode CDR sequences, or may encode entire variable regions.
  • Variable region sequences may be fused to any suitable constant region sequences as known in the art. These values can be adjusted appropriately to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, etc., as understood by those skilled in the art.
  • sequences engineered with host codon preferences on the basis of the antibody coding sequences disclosed in the present invention are applicable to the present invention.
  • the base sequence of the present invention can be modified preferentially according to degenerate codons. Codon bias modification generally does not change the sequence of the product protein or polypeptide.
  • nucleic acid and “polynucleotide” are used interchangeably herein to refer to single- or double-stranded RNA, DNA, or mixed polymers.
  • the nucleic acid encoding it is inserted into a vector for further cloning (amplification of the DNA) or for expression.
  • DNA encoding the antibody of the present invention was isolated according to the methods set forth in the Examples.
  • Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
  • vectors refer to expression vectors, including but not limited to plasmids, retroviruses, YACs, episomal genes derived from EBV, and the like.
  • a suitable vector is one encoding a fully functional human CH or CL immunoglobulin sequence with suitable restriction sites engineered to allow easy insertion and expression of any VH or VL sequence.
  • the resulting chimeric antibodies can bind to any strong promoter, including retroviral LTRs, eg, the SV-40 early promoter, Rous Sarcoma Virus LTR, and Moloney Murine Leukemia Virus LTR.
  • native Ig promoters and the like can be used.
  • Retroviral vectors include Moloney murine leukemia virus.
  • DNA viral vectors are preferred.
  • These vectors include pox vectors such as smallpox or fowl pox vectors, herpes virus vectors such as herpes simplex I virus (HSV) vectors.
  • HSV herpes simplex I virus
  • the choice of a particular vector will depend on the target cells and the conditions being treated. Introduction can be by standard techniques such as infection, transfection, transduction or transformation. Examples of gene transfer modes include, eg, naked DNA, CaPO4 precipitation, DEAE polydextrose, electroporation, protoplast fusion, lipofection, microinjection of cells, and viral vectors.
  • Suitable host cells for cloning or expressing DNA are prokaryotic cells, yeast cells or higher eukaryotic cells.
  • prokaryotic host cells include Escherichia coli, Bacillus subtilis, and the like.
  • eukaryotic host cells include yeast cells, insect cells, mammalian cells, and the like.
  • the host cells transformed with the above-mentioned expression or cloning vectors used for the production of the antibody of the present invention are cultured in an appropriately modified conventional nutrient medium for inducing promoters, selecting transformants or amplifying genes encoding desired sequences.
  • Antibodies produced from the cells can be purified using purification techniques known to those of ordinary skill in the art.
  • subject and patient are used interchangeably herein to refer to any animal that may be in need of the antibody-related formulations or drugs, treatments and vaccines described herein.
  • Subjects and patients thus include, but are not limited to, primate (including humans), canine, feline, murine and other mammalian subjects.
  • the subject is a human.
  • subject and patient means a subject or patient susceptible to infection by a coronavirus and/or a subject or patient infected by a coronavirus.
  • treatment refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow down (reduce) the progression of an undesired physiological change or disorder, such as an autoimmune disease.
  • beneficial or desired clinical outcomes include, but are not limited to, the following, whether detectable or not, including relief of symptoms, reduction in extent of disease, stabilization of disease state (i.e., not worsening), delay or slowing of disease progression, Amelioration or palliation as well as alleviation (whether partial or total) of a disease state.
  • Treatment also means prolonging survival as compared to expected survival if not receiving treatment.
  • Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
  • the invention provides a composition comprising the antibody 32C7 of the invention or an antigen-binding fragment thereof.
  • the composition may be a conjugate comprising the aforementioned monoclonal antibody or antigen-binding portion thereof coupled to other substances including cytotoxins , drugs, radioactive toxins.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to (eg, kills) cells.
  • agents include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, Ruubicin, daunorubicin, dihydroxyanthraxin diketone, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, butyl Caine, lidocaine, propranolol, and puromycin, and their analogs or homologues.
  • the composition may also include, for example, antimetabolites (such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), alkylating agents (such as Dichloroethylamine, thiotepa, chlorambucil, melphalan, carmustine and lomustine, cyclophosphamide, busulfan, dibromomannitol, streptozotocin, silk Mitomycin C, and cis-Dichlorodiamidoplatinum(II) (DDP) (cisplatin), anthracyclines (such as daunorubicin (formerly daunorubicin) and doxorubicin), antibiotics (such as actinomycin D), bleomycin, mithramycin, and anthranimycin (AMC)), and antimitotic agents (such as vincristine and vinblastine), duocarmycin, calicheamic
  • the antibodies of the invention, fragments thereof, can be used as therapeutic drugs.
  • Such drugs will generally be used to diagnose, predict, monitor, treat, alleviate, prevent and/or delay the progression of a disease or pathology associated with the spike protein. In some embodiments, it can treat or prevent respiratory system damage caused by a new coronavirus infection.
  • Antibody preparations preferably antibodies with high specificity and high affinity for their target antigens, are administered to a subject and will generally have an effect due to their binding to the target. Administration of the antibody can reduce, antagonize, neutralize, eliminate or inhibit or interfere with the binding of the target to its naturally associated endogenous ligand.
  • the medicament of the present invention comprises the antibody of the present invention or an antigen-binding portion thereof, and a pharmaceutically acceptable carrier.
  • “Pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the agents of the invention may also be administered in combination therapy, ie in combination with other therapeutic agents and their agents.
  • Such therapeutic agents include any anti-infective active agents, small molecule drugs, including but not limited to remdesivir, ribavirin, oseltamivir, zanamivir, hydroxychloroquine, interferon, analgesics, azithromycin and corticosteroids.
  • an effective amount means the amount of a drug or agent that elicits the biological or pharmaceutical response of a tissue, system, animal or human being sought, for example, by a researcher or clinician.
  • therapeutically effective amount means an amount that causes an improved treatment, cure, prevention, or alleviation of a disease, disorder, or side effect, or reduces the rate of progression of a disease or condition, compared to a corresponding subject not receiving that amount amount.
  • the term also includes within its scope amounts effective to enhance normal physiological function.
  • an effective amount herein will vary depending on factors such as the given drug or compound, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the subject being treated, etc., but can still be Routinely determined by those skilled in the art. Effective amounts of compounds of the present invention can be readily determined by those skilled in the art by routine methods known in the art.
  • the present invention provides the use of the monoclonal antibody 32C7 or its antigen-binding fragment in the preparation of medicines or reagents for treating or preventing human novel coronavirus infection.
  • the new coronavirus includes but is not limited to the original strain, B.1.351, B.1.1.7, P1, B.1.617.2 and other mutant strains. More specifically, the mutant virus strain has a mutation selected from: B.1.1.7(N501Y); B.1.351(K417N, E484K, N501Y); P1(K417T, E484K, N501Y); B.1.617. 2 (L452R, T478K).
  • original strain used herein refers to the SARS-CoV-2 (new coronavirus) virus strain whose sequence was published in January 2020 and has the same sequence as the strain with GISAID number EPI_ISL_403934.
  • mutant strain refers to a new coronavirus strain that has a mutation in sequence compared with the EPI_ISL_403934 strain.
  • This example is the preparation of monoclonal antibody 32C7, as follows.
  • the S5 chip was purchased from GE Company, the filter was purchased from PALL Company, and the DMEM medium and fetal bovine serum (FBS) were purchased from GIBCO Company.
  • FBS fetal bovine serum
  • the present invention obtains the antibody 32C7 against the new coronavirus.
  • the heavy chain gene sequence and light chain gene sequence corresponding to the antibody are obtained from single-cell transcripts of RBD protein-specific B cells through antibody gene-specific PCR amplification and PCR product sequencing, and are obtained through the IMGT website (http:// www.imgt.org) to obtain the variable region sequences corresponding to the heavy chain gene and the light chain gene.
  • Antibody 32C7 has the heavy chain variable region nucleic acid sequence shown in SEQ ID NO: 13, the heavy chain variable region amino acid sequence shown in SEQ ID NO: 14 and the heavy chain constant region amino acid sequence shown in SEQ ID NO: 15; The light chain variable region nucleic acid sequence shown in SEQ ID NO: 16, the light chain variable region amino acid sequence shown in SEQ ID NO: 17 and the light chain constant region amino acid sequence shown in SEQ ID NO: 18.
  • Antibody 32C7 has the heavy chain CDR1 region nucleotide sequence shown in SEQ ID NO: 1, the amino acid sequence shown in SEQ ID NO: 2; the CDR2 region nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: amino acid sequence shown in 4; CDR3 region nucleotide sequence shown in SEQ ID NO: 5, amino acid sequence shown in SEQ ID NO: 6.
  • Antibody 32C7 has the nucleotide sequence of the light chain CDR1 region shown in SEQ ID NO: 7, the amino acid sequence shown in SEQ ID NO: 8; the nucleotide sequence of the CDR2 region shown in SEQ ID NO: 9, SEQ ID NO : the amino acid sequence shown in 10; the nucleotide sequence of the CDR3 region shown in SEQ ID NO: 11, the amino acid sequence shown in SEQ ID NO: 12.
  • This example is the anti-new coronavirus effect of monoclonal antibody 32C7 in vitro, as follows.
  • Monoclonal antibody 32C7 was diluted two-fold from 120nM with buffer, filtered through a 0.2 filter and centrifuged to remove air bubbles.
  • the monoclonal antibody 32C7 can efficiently and rapidly bind to the RBD domain of the new coronavirus, with an affinity constant of 1.00 ⁇ 10 5 . Its dissociation constant is 1.09 ⁇ 10 -8 , and the equilibrium dissociation constant between the two is 1.09 ⁇ 10 -8 through analysis and calculation. It shows that the monoclonal antibody 32C7 has a high affinity with the RBD domain of the new coronavirus, and the dissociation is very slow.
  • the monoclonal neutralizing antibody 32C7 can effectively inhibit the infection of SARS-CoV-2 live virus, and has a good neutralizing and inhibitory effect on the original strain and the SARS-CoV-2D614G mutant strain (ie, the B.1.1.7 strain) , and their IC 50 are 4.588ng/mL and 127.6ng/mL, respectively.
  • the neutralizing antibody 32C7 still had a certain neutralizing effect, with an IC 50 of 1.42 ⁇ g/mL.
  • the IC 50 of the currently published representative antibodies CC12.1 and REGN10987 against the D614G mutant strain are 22ng/mL and 19.4ng/mL, respectively, and their neutralizing effect on the South African mutant strain is very weak.
  • the IC50 of CV07-270 was 82.3 ng/mL.
  • This example is the antiviral effect of antibody 32C7 on a humanized mouse model, as follows.
  • mice 8-week-old human ACE2 transgenic (hACE) mice were randomly divided into drug evaluation group, 4 mice. 3 mice in the negative control group. Before the formal experiment, the mice were acclimated to the environment for 2-3 days.
  • mice were lightly anesthetized with 1% pentobarbital sodium (about 0.1ml of anesthetic per gram of body weight), and then infected with 4 ⁇ 10 4 PFU of SARS-CoV- 2 virus liquid.
  • mice were sacrificed on the fifth day after infection, and the lungs were removed and ground.
  • the virus titer and the expression of related inflammatory factors in the lung grinding liquid were detected by qRT-PCR.
  • the lungs of the mice were removed for histopathological examination.
  • the antibody 32C7 can effectively reduce the virus titer of SARS-CoV-2 in the transgenic mouse model, and the virus titer in the lungs of the antibody treatment group compared with the virus control group after five days of virus infection The speed is reduced by 90%.
  • the expression of related inflammatory factors in the lungs was also significantly reduced after antibody 32C7 treatment.
  • C validated infiltration in the lungs was also improved.
  • the antibody 32C7 still has an excellent protective effect on the SARS-CoV-2 transgenic mouse model, and it has obvious antiviral ability.
  • This example is the structural analysis of the binding of antibody 32C7 and S protein, as follows.
  • HEK293F cells were cultured in SMM 293T-I medium containing 8% CO2 at 37 °C. When the cell density reached 2 ⁇ 10 6 cells/mL, the S-2P and S-6P were mixed with 25-kDa linear polyethylenimine at a PEI:DNA mass ratio of 3:1 and 1 mg DNA per liter. The plasmid was transiently transfected into HEK293F cells. The cell culture supernatant was collected and centrifuged at 10,000 ⁇ g for 30 minutes.
  • Secreted S-2P and S-6P proteins were purified using HisPurTM cobalt resin and StrepTactin resin. Purification was performed on a Superose 6 10/300 column in a buffer containing 20 mM HEPES pH 7.2, 150 mM NaCl and 10% trehalose.
  • the Fab region of the antibody 32C7 was obtained by papain digestion in a buffer containing 20mM HEPES pH 7.2, 150mM NaCl, 5mM EDTA and 5mM l-cysteine at 37°C for 40min.
  • the resulting Fab was purified for l-cysteine with a desalting column and then further purified with a HiTrap Q column. Purified Fabs were collected and concentrated to 0.6 mg/mL.
  • 32C7 has 21 interacting amino acid residues with the RBD domain of the new coronavirus, and all of them avoid the mutation sites of the current mutant strains of the new coronavirus, making it have a good inhibitory effect on a variety of mutant strains.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Virology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Genetics & Genomics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

提供了一种单克隆抗体32C7及其制备方法和用途。所述单克隆抗体可以有效地与新冠病毒的S蛋白的RBD结构域特异性结合，能够抑制病毒在体内的复制并降低炎症因子的产生和肺部炎症浸润。所述单克隆抗体抑制新冠病毒进入宿主细胞，达到新冠病毒中和抗体的治疗作用，可用于治疗或者预防新冠病毒感染引起的呼吸系统损伤。

Description

单克隆抗体32C7及其制备方法和用途 技术领域
本发明涉及生物医药领域，具体地，本发明涉及预防和治疗新型冠状病毒感染抗体及其制备，尤其涉及单克隆抗体32C7及其制备和用途。
背景技术
SARS-CoV-2病毒粒子表面介导抗体反应的最显著靶点是同源三聚体刺突(S)蛋白。刺突蛋白通过受体结构域(RBD)与血管紧张素转换酶2(ACE2)的相互作用促进病毒进入细胞。因此，以刺突蛋白RBD结构域为靶点的抗体在对抗当前的大流行中尤为重要。
例如，Cheolmin Kim等人通过从康复患者的外周血单个核细胞构建的抗体库筛选针出对病毒刺突蛋白的受体结合域的人单克隆抗体mAb。结果表明，CT-P59单抗能有效中和SARS-CoV-2分离株，包括D614G变异体，而无抗体依赖性增强效应，其阻断受体结合域与血管紧张素转换酶2(ACE2)受体的相互作用区域。此外，在三种动物模型(雪貂、仓鼠和恒河猴)中评估了CT-P59的治疗效果，其能够降低病毒滴度。
再例如，CN113024640A公开了一种基于新冠病毒RBD与ACE2受体结合结构域筛选的表位肽抗原检测中和抗体试剂盒，其含有氨基酸序列如SEQ ID NO:1-4所示的基于新冠病毒RBD与ACE2受体结合结构域筛选的表位肽抗原。
目前，仍亟需以刺突蛋白RBD结构域为靶点的抗体、其制备以及在治疗性药物或其制剂中的应用。
发明内容
本发明的目的在于弥补现有技术的不足，提供一种新的中和抗体32C7及其制备方法和用途，从而为临床上COVID-19的治疗提供一种安全有效的抗体，本 发明的抗体或其抗原结合片段具有广泛的应用前景。具体如下。
本发明的第一方面，提供一种抗体32C7或其抗原结合片段，其能够特异性结合刺突蛋白，并且其能够结合选自刺突蛋白的第345、346、347、348、349、351、352、354、440、441、442、443、444、445、448、450、451、452、466、499、509位的氨基酸残基的表位所组成的组中的至少一种表位，或者所述表位选自刺突蛋白的第T345、R346、F347、S349、N440、L441、K444、N450、Y351和Y451所组成的组中的至少一种表位，以抑制或阻断所述刺突蛋白与受体的结合。
根据本发明所述的抗体32C7或其抗原结合片段，优选地，所述表位由刺突蛋白的第345、346、347、348、349、351、352、354、440、441、442、443、444、445、448、450、451、452、466、499、509位的氨基酸残基组成，或者所述表位由刺突蛋白的第T345、R346、F347、S349、N440、L441、K444、N450、Y351和Y451组成。
本发明的第二方面，提供一种抗体32C7或其抗原结合片段，其包含重链和轻链，并且其表现出对刺突蛋白的受体结构域小于120nM的Kd。
本发明的第三方面，提供一种抗体32C7或其抗原结合片段，其含有重链可变区的抗原互补决定区CDR1、CDR2和CDR3，其氨基酸序列分别为SEQ ID NO：2、SEQ ID NO：4、SEQ ID NO：6；和
轻链可变区的抗原互补决定区CDR1、CDR2和CDR3，其氨基酸序列分别为SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12。
根据本发明第三方面所述的抗体32C7或其抗原结合片段，其重链可变区的氨基酸序列包含SEQ ID NO：2、SEQ ID NO：4、SEQ ID NO：6所示的抗原互补决定区且与SEQ ID NO：14所示序列具有至少80％，优选至少90％，还优选至少95％，进一步优选至少99％的同一性；和其轻链可变区的氨基酸序列包含SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12所示的抗原互补决定区且与SEQ ID NO：17所示序列具有至少80％，优选至少90％，还优选至少95％，进 一步优选至少99％的同一性，并且同时至少保留突变前序列的功能。
根据本发明的一个方面所述的抗体32C7或其抗原结合片段，优选地，其中所述抗体32C7或其抗原结合片段是单克隆抗体；所述抗原结合片段包括Fab片段、Fab’、F(ab’) 2片段、单链可变片段scFv、scFv-Fc片段或单链抗体ScAb。
本发明的第四方面，提供一种核酸分子，其编码根据本发明第一方面，第二方面或第三方面的所述的抗体32C7或其抗原结合片段。
本发明的第五方面，提供一种载体，其包含根据本发明第四方面所述的核酸分子。
本发明的第六方面，提供一种抗体32C7或其抗原结合片段的制备方法，所述方法包括通过在适于所述抗体32C7或其抗原结合片段表达的条件下培养细胞，从而产生所述单克隆抗体，其中所述细胞包含根据本发明第五方面所述的载体。
本发明的第七方面，提供一种组合物，其含有根据本发明第一方面，第二方面或第三方面所述的抗体32C7或其抗原结合片段。
本发明的第八方面，提供根据本发明第一方面，第二方面或第三方面所述的抗体32C7或其抗原结合片段在制备用于治疗或者预防ACE2相关疾病的药物或试剂中的用途，优选地，所述疾病选自冠状病毒感染，还优选地，所述冠状病毒包括但不限于SARS-CoV-2(D614和G614)、SARS-CoV或HCoV-NL63。
根据本发明第八方面所述的用途，优选地，所述抗体32C7或其抗原结合片段能够特异性结合刺突蛋白，并且其能够结合选自刺突蛋白的第345、346、347、348、349、351、352、354、440、441、442、443、444、445、448、450、451、452、466、499、509位的氨基酸残基的表位，或者所述表位选自刺突蛋白的第T345、R346、F347、S349、N440、L441、K444、N450、Y351和Y451，以抑制或阻断所述刺突蛋白与受体的结合。
根据本发明第八方面所述的用途，优选地，所述的新型冠状病毒包括但不限于原始株Original病毒株，英国突变病毒株B.1.1.7，南非突变病毒株B.1.351和 B.1.617.2(印度Delta德尔塔毒株)。
本发明的第九方面，提供在受试者中预防和/或治疗冠状病毒感染的方法，包括向有需要的受试者施用有效量的本发明的抗体或抗原结合片段、其组合物、或本发明的药物或试剂。优选地，其中治疗有效量约为0.1-200mg/kg，优选20mg/kg。
本发明的第十方面，提供一种药物组合物，其含有根据本发明所述的抗体32C7或其抗原结合片段，核酸分子，或载体，和药学可接受载体和/或赋形剂。
根据本发明第十方面所述的药物组合物，优选地，所述药物组合物为疫苗，优选为亚单位疫苗，所述赋形剂为佐剂。
根据本发明第十方面所述的药物组合物，优选地，所述佐剂包括铝佐剂、弗氏佐剂、磷酸铝、磷酸钙、石蜡油、羊毛脂、表面活性剂、藻酸钙、多聚核苷酸、胞壁肽、皂苷、RIBI佐剂系统、霍乱毒素、丙烯酸或甲基丙烯酸的聚合物、油包水乳剂、水包油乳剂中的一种或几种，优选为铝佐剂。
本发明的第十一方面，提供一种预防或治疗受试者中冠状病毒感染的方法，优选地，经口服、肌肉注射或鼻腔滴注给予所述受试者有效量的根据本发明第十方面所述的药物组合物。
本发明的抗体32C7和新冠病毒的RBD结构域之间具有高亲和力，并且解离的十分缓慢。实验证明，本发明的抗体能够有效抑制SARS-CoV-2活毒的感染，不仅对原始株表现出显著的抑制效果，而且对突变病毒株同样具有很好地中和抑制效果，这表明，相对于现有的代表性抗体来说，本发明的抗体具有优异的效果和应用前景。此外，动物模型实验进一步证明，本发明的抗体能够大大降低SARS-CoV-2的病毒滴度，而且相关炎症因子明显减少，炎性浸润情况明显得到改善。本发明人进一步对抗体和RBD结合的结构进行了表征，惊奇地发现，本发明的抗体避开了目前新冠病毒突变株的突变位点，使得其对于多种突变株都有很好的抑制效果。
附图说明
图1是根据本发明实施例2的抗体32C7的体外抗新冠病毒效果，其中，图1的A部分显示的是抗体32C7能够梯度依赖的和新冠病毒SPR结构域的互作结果，其中从上到下浓度依次为120、60、30、15和7.5，单位为nM；图1的B部分显示了抗体32C7可以剂量依赖的抑制多种亚型的SARS-CoV-2突变株的感染。
图2是根据本发明实施例3的抗体32C7在转基因小鼠新冠病毒感染模型中的效果，其中，图2的A部分为小鼠肺研磨液中新冠病毒滴度；图2的B部分为小鼠肺研磨液中相关炎症因子的表达量；图2的C部分是小鼠肺部病理变化。
图3是根据本发明实施例4的抗体32C7与新冠病毒S蛋白结合的电镜观察结果，其中，图3的A部分为抗体32C7与新冠病毒S蛋白结合的结构图；图3的B部分为抗体32C7与新冠病毒S蛋白结合的抗原表位。
具体实施方式
下面将结合具体实施例详细描述本发明的实施例，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围，而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
除非另有说明，否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料，但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入，用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时，以本说明书的内容为准。
另外，对于本发明中的数值范围，应理解为具体公开了该范围的上限和下限 以及它们之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。当与数值相连使用时，术语“约”、“大约地”或“大约”旨在包括数值的集合或范围。例如，“约X”包括为X的±20％、±10％、±5％、±2％、±1％、±0.5％、±0.2％或±0.1％的数值范围，其中X为数值。在一个实施方案中，术语“约”指的是比特定值多或少5％的数值范围。在另一个实施方案中，术语“约”指的是比特定值多或少2％的数值范围。在另一个实施方案中，术语“约”指的是比特定值多或少1％的数值范围。
本发明中，术语“刺突蛋白”是指位于新冠病毒最外层的三聚体结构的蛋白，也称为刺突糖蛋白或S蛋白。其是来源于包括SARS-CoV和2019-nCoV在内的冠状病毒的结构蛋白。因此，术语“针对冠状病毒S蛋白的抗体”、“抗冠状病毒S蛋白的抗体”、“抗S蛋白抗体”、“冠状病毒S蛋白抗体”、“S蛋白抗体”或“结合S蛋白的抗体”在本文中可互换地使用，是指本文的抗体能够以足够的亲和力结合冠状病毒S蛋白(例如，2019-nCoV S蛋白、SARS-CoV S蛋白)，由此所述抗体可以用作靶向冠状病毒S蛋白的诊断剂、预防剂和/或治疗剂。
如本文所用，术语“表位”包括能够特异性结合至免疫球蛋白或其片段或T-细胞受体的任何蛋白质决定簇。表位决定簇通常由分子(例如氨基酸或糖侧链)的化学活性表面组群组成且通常具有特定的三维结构特征以及特定的电荷特征。本发明的抗体特异性结合的表位优选为选自刺突蛋白的第345、346、347、348、349、351、352、354、440、441、442、443、444、445、448、450、451、452、466、499、509位的氨基酸残基。优选地，其特异性结合刺突蛋白的T345、R346、F347、S349、Y351、N450、Y451、N440、L441和K444中的至少一种表位。
如本文所用，术语“抗体”指的是免疫球蛋白分子(Ig)分子的免疫活性部分，即含有特异性结合抗原(与之免疫反应)的抗原结合位点的分子。“特异性结合”或“与之免疫反应”意指抗体与期望的抗原的一个或多个抗原决定簇反应且不与其他多肽反应或以低得多的亲和力(Kd)结合。本发明的抗体表现出对刺突蛋白的受 体结构域小于120nM的Kd。优选地，其具有对刺突蛋白的受体结构域小于80nM的Kd，例如70nM、60nM、50nM、40nM、30nM、20nM、10nM，甚至小于8nM，例如7nM，甚至小于5nM的Kd。抗体包括但不限于单克隆抗体、嵌合抗体、dAb(结构域抗体)、单链抗体、Fab、Fab’和F(ab’) 2片段、scFvs。优选地，本发明的抗体为单克隆抗体或其抗原结合片段。
本文所用术语“抗体”还包括人源化抗体、重组抗体、从转基因的非人动物产生的人抗体和使用本领域技术人员可得到的富集技术选自文库的抗体。
如本领域所理解，抗体是包含通过二硫键互联的至少两个重(H)链和两个轻(L)链的糖蛋白或其抗原结合部分。重链包含重链可变区(VH)和重链恒定区(CH1、CH2和CH3)。轻链包含轻链可变区(VL)和轻链恒定区(CL)。重链和轻链的可变区包含框架区(FR)和互补决定区(CDR)。四个FR是相对保守的，而CDR区域(CDR1、CDR2和CDR3)包含高变区。抗体结构单元已知一般包含四聚体。每个四聚体由两个相同多肽链对构成，每对具有一条轻链和一条重链。每条链的氨基端部分包括主要负责抗原识别的约100个至110个或更多个氨基酸的可变区。每条链羧基端部分定义了主要负责效应子功能的恒定区。FR和CDR从NH 2端至COOH端如下排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。此外，所述恒定区可以介导免疫球蛋白与宿主组织或因子的结合。抗体恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性(ADCC)或补体介导毒性(CDC)。通常，抗体分子涉及IgG、IgM、IgA、IgE及IgD，其彼此间因存在于分子中的重链的性质而不同。
在本发明中，抗体重链可变区的抗原互补决定区CDR1、CDR2和CDR3分别为SEQ ID NO：2、SEQ ID NO：4及SEQ ID NO：6的氨基酸序列；抗体轻链可变区的抗原互补决定区CDR1、CDR2和CDR3分别为SEQ ID NO：8、SEQ ID NO：10及SEQ ID NO：12的氨基酸序列。
本文所用术语“抗体片段”包含完整抗体的一部分，诸如完整抗体的抗原结合 区或可变区。抗体片段的实例包括但不限于Fab、Fab’、F(ab’) 2、Fv片段、scFV片段、双抗体、或线性抗体。抗体的木瓜蛋白酶消化会产生两个相同的“Fab”片段或抗原结合片段(每个具有单个抗原结合位点)和一个残余的“Fc”片段(其名称反映了它的容易结晶的能力)。抗体的胃蛋白酶处理会产生F(ab’) 2片段，其具有两个抗原结合位点且其保留它的交联抗原的能力。
本文所用术语“Fv”表示含有完全抗原识别和抗原结合位点的最小抗体片段。该片段含有紧密地非共价结合的一个重链可变区结构域和一个轻链可变区结构域的二聚体。这两个结构域的折叠导致六个高变环(三个环各自来自H和L链)的形成，它们促进用于抗原结合的氨基酸残基并给所述抗体赋予抗原结合特异性。但是，即使单个可变区(或仅包含三个对抗原特异性的CDR的Fv的一半)具有识别和结合抗原的能力，尽管以较低亲和力。“单链Fv”(“sFv”或“scFv”)是包含连接成单个多肽链的VH和VL抗体结构域的抗体片段。sFv多肽还可以包含在VH和VL结构域之间的多肽接头，其使sFv能够形成期望的结构用于抗原结合。
本文所用术语“Fab”片段含有轻链的恒定结构域和重链的第一恒定结构域(CH1)。Fab’片段与Fab片段的差别在于几个残基在重链CH1结构域的羧基端处的添加，包括来自抗体铰链区的一个或多个半胱氨酸。
本文所用术语“单克隆抗体”，有时也称为“单抗”或mAb，其是指从一纯系细胞得到的免疫球蛋白，具有相同的结构和化学特性，对单一抗原决定簇有特异性。单克隆抗体与常规多克隆抗体制剂(通常是具有针对不同决定簇的不同抗体)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性外，单克隆抗体的好处还在于它们是通过杂交瘤或重组工程细胞培养获得，不会混杂有其它免疫球蛋白。修饰语“单克隆”表示了抗体的特性，是从均一的抗体群中获得的，但这不应被解释成需要用任何特殊方法来生产抗体。
本发明的抗体可包含Fc区，所述Fc区来自IgG，例如IgG1、IgG2、IgG3或IgG4。
除非另有说明，否则本文所述抗体或其抗原结合片段为分离的抗体或其抗原 结合片段。其中所用术语“分离的”是指已经从它的天然环境提取出的核酸或抗体。已经“分离的”核酸、肽和蛋白因而包括通过标准纯化方法纯化的核酸和蛋白。该术语也包括通过在宿主细胞中的重组表达而制备的核酸、肽和蛋白以及化学合成的核酸和/或多肽。
本发明中，术语“受体结合结构域”是指S蛋白与ACE2相联结或结合的区域，也称为RBD。
术语“抗原结合位点”或“结合部分”指的是参与抗原结合的免疫球蛋白分子的部分。抗原结合位点由重(“H”)链及轻(“L”)链的N端可变(“V”)区的氨基酸残基形成。重链及轻链的V区中三个高度趋异段(被称为“高变区”)被插入称为“框架区”或“FR”的更保守的侧翼段之间。因此，术语“FR”指的是在免疫球蛋白高变区之间和相邻的天然发现的氨基酸序列。在抗体分子中，轻链的三个高变区与重链的三个高变区在三维空间中相对于彼此配置而形成抗原结合表面。抗原结合表面与结合的抗原的三维表面互补，且重链及轻链各自的三个高变区被称为“互补决定区”或“CDR”。
本发明中，术语“结合”和“免疫结合”可互换使用，是指发生在免疫球蛋白分子与对所述免疫球蛋白特异的抗原之间的非共价相互作用。免疫结合相互作用的强度或亲和力可以解离常数(Kd)表达，其中较小的Kd代表较高的亲和力。“亲和力”是指分子(例如抗体)的单一结合位点与其结合配偶体(例如抗原)之间全部非共价相互作用总和的强度。除非另有说明，在用于本文时，“结合亲和力”指反映结合对的成员(例如抗体与抗原)之间1∶1相互作用的内在结合亲和力。分子X对其配偶体Y的亲和力通常可用结合解离平衡常数(Kd)来表述。亲和力可通过本领域知道的常用方法来测量，包括现有技术已知以及本文中所描述的那些。
在本发明中，术语“特异性结合”通常是指抗体通过其抗原结合结构域与表位结合，并且该结合需要抗原结合结构域和表位之间具有互补性。因此，当抗体通过其抗原结合结构域与该表位结合时，比它结合到随机的、不相关的表位更容易，其被称为“特异性结合”该表位。术语“特异性”在本发明中用于限定某种抗体与某 个表位结合的相对亲和力。例如，可以认为抗体“A”比抗体“B”对特定表位具有更高的特异性，或者可以认为抗体“A”以比结合相关表位“D”更高的特异性结合表位“C”。
除非另有说明，术语“片段”、“抗体片段”、“抗原-结合片段”及“抗原结合片段”可互换地使用。
变体抗体也都被包括在本发明的范围内。因此，在本发明中列举的序列的变体也都包括在本发明的范围内。具有提高的亲和力的抗体序列的其它变体可以使用本领域已知的方法得到，并都包括在本发明的范围内。本领域技术人员利用用于生产变体多肽的重组方法和/或合成化学技术可以修改多肽的氨基酸序列。例如，可以使用氨基酸置换得到具有进一步提高的亲和力的抗体。可选地，可以使用核苷酸序列的密码子优化来提高在用于生产抗体的表达系统中的翻译效率。这样的变体抗体序列与在本发明中列举的序列具有80％或更高的(即，85％、90％、95％、96％、97％、98％、99％或更大)序列同一性。相对于在本发明中列举的序列，计算所述序列同一性。或进行最佳比对时，如通过程序GAP或使用默认值间隙权重的BESTFIT。
在本文中，两氨基酸序列之间的百分比同源性等于两序列之间的百分比同一性。两序列间的百分比同一性为序列共有的相同位置数的函数(即％同源性＝相同位置数/位置总数×100)，其中需考虑产生两序列的最优比对需要引入的缺口数和每个缺口的长度。可以使用数学算法完成序列的比较和两序列间百分比同一性的测定。可以使用E.Meyers和W.Miller的算法(Comput.Appl.Biosci.,4:11-17(1988))测定两氨基酸序列间的百分比同一性。此外，可以使用Needleman和Wunsch的算法(J.Mol.Biol.48:444-453(1970))测定两氨基酸序列间的百分比同一性。
本发明的抗体还包括氨基酸序列修饰的抗体，优选地，本发明的抗体包含含有CDR1、CDR2和CDR3序列的重链可变结构域和含有CDR1、CDR2和CDR3序列的轻链可变结构域，其中这些CDR序列中的一个或多个包含基于本文所述抗体的特定氨基酸序列或其修饰，且经过修饰的抗体保留本发明抗新型冠状病毒 抗体的期望的功能特性。
本文所用术语“修饰”意指氨基酸修饰不会显著影响或改变含有该氨基酸序列的抗体的结合特征。此类修饰包括氨基酸的取代、添加和缺失。优选地，不相同的残基位置因保守氨基酸取代而不同。本发明的抗体可包括糖基化、乙酰化、磷酸化、酰胺化、通过已知的保护/封闭基团衍生化、蛋白水解切割或非天然发生的氨基酸修饰等。
保守氨基酸取代指的是具有类似侧链的残基的可互换性。例如，具有脂肪族侧链的氨基酸组为甘氨酸、丙氨酸、缬氨酸、亮氨酸和异亮氨酸；具有脂肪族-羟基侧链的氨基酸组为丝氨酸及苏氨酸；具有含酰胺侧链的氨基酸组为天冬酰胺和谷氨酰胺；具有芳香族侧链的氨基酸组为苯丙氨酸、酪氨酸和色氨酸；具有碱性侧链的氨基酸组为赖氨酸、精氨酸和组氨酸；以及具有含硫侧链的氨基酸组为半胱氨酸及甲硫氨酸。优选的保守氨基酸取代组为：缬氨酸-亮氨酸-异亮氨酸、苯丙氨酸-酪氨酸、赖氨酸-精氨酸、丙氨酸缬氨酸、谷氨酸-天门冬酸和天冬酰胺-谷氨酰胺。因此，可以用来自同一侧链家族的其它氨基酸残基替换本发明抗体CDR区中的一个或多个氨基酸残基。
如本文所述，本发明涵盖抗体或免疫球蛋白分子的氨基酸序列的微小变异，条件是氨基酸序列维持至少75％、更优选地至少80％、90％、95％，且最优选地99％的变异。特别地，预期保守氨基酸取代。保守取代是氨基酸家族中发生的那些，其与他们的侧链相关。遗传编码的氨基酸通常被分为以下家族：(1)酸性氨基酸为天冬氨酸、谷氨酸；(2)碱性氨基酸为赖氨酸、精氨酸、组氨酸；(3)非极性氨基酸为丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸和(4)不带电极性氨基酸为甘氨酸、天冬酰胺、谷氨酰胺、半胱氨酸、丝氨酸、苏氨酸、酪氨酸。亲水性氨基酸包括精氨酸、天冬酰胺、天冬氨酸、谷氨酰胺、谷氨酸、组氨酸、赖氨酸、丝氨酸和苏氨酸。疏水性氨基酸包括丙氨酸、半胱氨酸、异亮氨酸、亮氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、色氨酸、酪氨酸和缬氨酸。氨基酸的其他族包括(i)丝氨酸及苏氨酸，其为脂肪族-羟基家族； (ii)天冬酰胺及谷氨酰胺，其为含酰胺家族；(iii)丙氨酸、缬氨酸、亮氨酸及异亮氨酸，其为脂肪族家族；和(iv)苯丙氨酸、色氨酸和酪氨酸，其为芳香族家族。例如，合理预期以异亮氨酸或缬氨酸对亮氨酸分离取代、以谷氨酸对天冬氨酸分离取代、以丝氨酸对苏氨酸分离取代或以结构相关的氨基酸类似取代氨基酸将不会对所得分子的结合或特性具有重大影响，特别是如果取代不涉及框架位置中的氨基酸。氨基酸改变是否导致功能肽可以容易地通过测定多肽衍生物的特异性活性进行确定。测定方法不特别限定，可通过本领域已知方法进行测定。抗体或免疫球蛋白分子的片段或类似物可以通过本领域技术人员容易地制备。优选片段或类似物的氨基-和羧基-端发生在功能域边界附近。结构域和功能域可以通过将核苷酸和/或氨基酸序列数据与公开或私人序列数据库比较来鉴定。优选地，使用计算机化比较方法来鉴定序列基序或预测的蛋白质构形域(其发生在已知结构和/或功能的其他蛋白质中)。鉴定折叠成已知三维结构的蛋白质序列的方法是已知的。
优选的氨基酸取代为以下这些：(1)降低对蛋白酶解的敏感性；(2)降低对氧化的敏感性；(3)改变对形成蛋白质复合物的结合亲和力；(4)改变结合亲和力和(4)给予或修饰此类似物的物化或功能特性。类似物可以包括除了天然发生的肽序列以外的序列的各种突变蛋白。例如，单一或多氨基酸取代(优选地保守氨基酸取代)可在天然发生的序列中进行(优选地在形成分子间接触的结构域以外的多肽部分中)。保守氨基酸取代不应实质上改变亲代序列的结构特征(例如：取代氨基酸不应当趋于破坏亲代序列中发生的螺旋，或中断表征亲代序列的其他类型的二级结构)。
另一类可能存在的可变区修饰是突变VH和/或VK CDR1、CDR2和/或CDR3区中的氨基酸残基以改进目的抗体的一种或多种结合特性(例如亲和力)。可以通过定点诱变或PCR介导的诱变来导入突变。优选导入(如上所述的)保守修饰。突变可以是氨基酸的取代、添加或缺失，但是优选为取代。此外，CDR区中残基变化通常不超过一个、两个、三个、四个或五个。
本发明中，单克隆抗体32C7或其抗原结合片段的重链可变区的氨基酸序列包含SEQ ID NO：2、SEQ ID NO：4、SEQ ID NO：6所示的抗原互补决定区且与SEQ ID NO：14所示序列具有至少80％的同一性；和其轻链可变区的氨基酸序列包含SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12所示的抗原互补决定区且与SEQ ID NO：17所示序列具有至少80％的同一性。这里的同一性变体可以是突变发生在可变区CDR区，也可以是发生在框架区FR区，需要说明的是，无论发生在CDR区，还是框架区FR区的突变，该突变至少保留突变前序列的功能，即发生在上述区域的突变并不会影响该抗体的结合特性、结合表位以及表位的空间构象，进而并不会影响本发明抗体的中和效果。
核酸分子
本发明提供涉及编码本发明的抗体的核酸分子。核酸可以存在于完整的细胞中、存在于细胞溶胞物中、或者以部分纯化或基本纯的形式存在。当通过标准技术，包括碱/SDS处理、CsCl分带(banding)、柱层析、琼脂糖凝胶电泳和本领域众所周知的其它技术纯化除去其它细胞组分或其它污染物，例如其它细胞核酸或蛋白质时，核酸是“分离的”。本发明的核酸可以是例如DNA或RNA，而且可以含有或不含内含子序列。在优选的实施方案中，核酸是cDNA分子。
本发明的核酸包含编码选自SEQ ID NO.:2、4、6、8、10、12中任一项所示氨基酸序列的核酸，或编码与选自SEQ ID NO.:2、4、6、8、10、12中任一项所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的氨基酸序列的核酸。优选地，所述编码序列由SEQ ID NO.:1、3、5、7、9、11所示的序列组成。
可以使用标准分子生物学技术来获得本发明的核酸。一旦获得编码VH和VL区段的DNA片段，进一步通过标准重组DNA技术操作这些DNA片段，以例如将可变区基因转变为全长抗体链基因、Fab片段基因或scFv基因。在这些操作中，将编码VL或VH的DNA片段可操作地连接至编码另一蛋白质，诸如抗体恒定区或柔性接头的另一DNA片段。术语“可操作的连接”在用于本文时旨 在表示连接两DNA片段从而使得这两个DNA片段所编码的氨基酸序列保持在同一读码框中(in-frame)。
通过将编码VH的DNA可操作地连接至编码重链恒定区(CH1、CH2和CH3)的另一DNA分子可以将编码VH区的分离的DNA转变为全长重链基因。人重链恒定区基因的序列是本领域已知的。
通过将编码VL的DNA可操作的连接至编码轻链恒定区CL的另一DNA分子可以将编码VL区的分离的DNA转变为全长轻链基因(以及Fab轻链基因)。人轻链恒定区基因的序列是本领域已知的。
本发明还提供了编码本发明抗体或其抗原结合片段的重链和轻链的肽序列的多核苷酸变体。这些多核苷酸变体与本发明的多核苷酸序列相比可以具有至少70％、或至少75％、或至少80％、或至少85％、或至少90％、或至少95％、或至少96％、或至少97％、或至少98％、或至少99％或更大的序列同一性。这样的连续序列可以编码CDR序列，或可以编码完整可变区。如本领域已知的，可变区序列可以与任何适当的恒定区序列融合。如本领域技术人员所理解，通过考虑密码子简并性、氨基酸相似性、读码框定位等，可以适当地调节这些值以确定由两个核苷酸序列编码的蛋白的对应同一性。
本领域可以理解的是，在本发明所公开的抗体编码序列的基础上经宿主密码子偏好性改造的序列可适用于本发明。为了适应于不同宿主的需要，可根据简并密码子来对本发明的碱基序列进行偏好性改造。密码子偏好性改造一般不改变产物蛋白或多肽的序列。
术语“核酸”和“多核苷酸”在本文中互换地用于表示单链或双链RNA、DNA或混合的聚合物。
对于抗体的重组生产，将编码它的核酸插入载体中用于进一步克隆(DNA的扩增)或用于表达。根据在实施例中阐述的方法，分离编码本发明的抗体的DNA。如本领域技术人员所理解，许多载体可供用在抗体的重组生产中。载体组分通常包括但不限于以下的一种或多种：信号序列、复制起点、一个或多个标记基因、 增强子元件、启动子和转录终止序列。
在本发明中，载体是指表达载体，包括但不限于质粒、逆转录病毒、YAC、EBV衍生的游离基因等。合适的载体是编码功能完全的人CH或CL免疫球蛋白序列的载体，其具有经工程改造的合适的限制位点使得任何VH或VL序列可容易地插入并表达。所得的嵌合抗体可结合到任何强启动子，包括逆转录病毒LTR，例如，SV-40早期启动子、劳斯氏肉瘤病毒LTR和莫洛尼氏鼠白血病病毒LTR。同样地，可使用天然Ig启动子等。
优选的载体包括病毒载体、融合蛋白质和化学缀合物。逆转录病毒载体包括莫洛尼氏鼠白血病病毒。DNA病毒载体是优选的。这些载体包括痘载体诸如：正天花或禽痘载体、疱疹病毒载体诸如：单纯疱疹I病毒(HSV)载体。具体载体的选择将取决于靶细胞和所处理的条件。导入可通过标准技术例如，感染、转染、转导或转化。基因转移模式的实例包括例如，裸DNA、CaPO4沉淀、DEAE聚葡糖、电穿孔、原生质体融合、脂质体转染、细胞显微注射和病毒载体。
用于克隆或表达DNA的合适宿主细胞是原核细胞、酵母细胞或高等真核细胞。常用的原核宿主细胞的例子包括大肠杆菌、枯草杆菌等。常用的真核宿主细胞包括酵母细胞、昆虫细胞、哺乳动物细胞等。将用于本发明抗体生产的、用上述表达或克隆载体转化的宿主细胞在适当修饰的常规营养物培养基中培养，用于诱导启动子、选择转化体或扩增编码期望序列的基因。使用本领域普通技术人员已知的纯化技术，可以纯化从所述细胞制备的抗体。
本文所用术语“受试者”和“患者”在本文中互换地用于指可能需要本文描述的抗体相关制剂或药物、治疗和疫苗的任何动物。受试者和患者因此包括但不限于，灵长类动物(包括人类)、犬科动物、猫科动物、鼠和其它哺乳动物受试者。优选地，所述受试者是人类。从在其中使用所述术语的上下文显而易见，受试者和患者表示易于被冠状病毒感染的受试者或患者和/或被冠状病毒感染的受试者或患者。
在本发明中，术语“治疗”是指治疗性治疗和预防性或防治性措施，其目的是 预防或减缓(减少)不期望发生的生理改变或紊乱，例如自身免疫性疾病的进程。有益的或期望的临床结果包括但不限于以下无论是可检测还是不可检测的结果，包括症状的缓解、疾病程度的减小、疾病状态的稳定(即不恶化)、疾病进展的延迟或减缓、疾病状态的改善或缓和以及减轻(无论是部分还是全部)。“治疗”还意指与不接受治疗时预期的生存期限相比所延长的生存期限。需要治疗的包括那些已经患有病症或紊乱的人，以及那些容易患有病症或紊乱的人，或者那些需要预防该病症或紊乱的人。
组合物
本发明提供一种组合物，其含有本发明的抗体32C7或其抗原结合片段。作为组合物的实例，所述组合物可以是偶联物，所述偶联物是将前面所述的单克隆抗体或其抗原结合部分与其他物质偶联而成，所述其他物质包括细胞毒素、药物、放射性毒素。
细胞毒素或细胞毒剂包括对细胞有害(例如杀伤细胞)的任何试剂。实例包括紫杉醇、松胞菌素B、短杆菌肽D、溴化乙啶、依米丁、丝裂霉素、依托泊苷、替尼泊苷、长春新碱、长春碱、秋水仙素、多柔比星、柔红霉素、二羟基炭疽菌素二酮、米托蒽醌、光神霉素、放线菌素D、1-去氢睾酮、糖皮质激素类、普鲁卡因、丁卡因、利多卡因、普萘洛尔和嘌呤霉素，及其类似物或同系物。
所述组合物还可以包括例如抗代谢物类(例如甲氨蝶呤、6-巯基嘌呤、6-硫鸟嘌呤、阿糖胞苷、5-氟尿嘧啶、达卡巴嗪)、烷化剂类(例如双氯乙基甲胺、塞替哌、苯丁酸氮芥、美法仑、卡莫司汀和洛莫司汀、环磷酰胺、白消安、二溴甘露醇、链唑霉素、丝裂霉素C、和顺式二氯二胺铂(II)(DDP)顺铂)、蒽环类抗生素(例如柔红霉素(以前称为道诺霉素)和多柔比星)、抗生素类(例如放线菌素D)、博来霉素、光神霉素、和氨茴霉素(AMC))、和抗有丝分裂剂(例如长春新碱和长春碱)、duocarmycin、加利车霉素、美登素和auristatin，及其衍生物。
药物
在一个实施方案中，本发明的抗体、其片段可以用做治疗性药物。此类药物 通常将用于诊断、预测、监控、治疗、缓解、预防和/或延迟与刺突蛋白相关的疾病或病理学的进展。在某些实施方案在，其能够治疗或者预防新冠病毒感染引起的呼吸系统损伤。将抗体制剂，优选地为具有对其标靶抗原具有高特异性和高亲和力的抗体，施用至受试者并且通常因其与靶标结合而将具有效果。抗体的施用可以降低、拮抗、中和、消除或抑制或干扰靶标与其天然结合的内源性配体的结合。
本发明的药物包含本发明的抗体或其抗原结合部分，以及药学上可接受的载体。“药学上可接受的载体”包括任何和所有溶剂、分散介质、包衣、抗细菌和抗真菌剂、等渗和吸收延迟剂、等等生理学相容的载体。本发明的药物还可以以联合疗法施用，即联合其它治疗剂及其药剂。此类治疗剂包括任何抗感染活性剂、小分子药物，包括但不限于瑞德西韦、利巴韦林、奥司他韦、扎那米韦、羟氯喹、干扰素、镇痛药、阿奇霉素和皮质类固醇。
本文所用术语“有效量”表示引发例如研究者或临床医师所追求的组织、系统、动物或人的生物学或药学响应的药物或药剂的量。此外，术语“治疗有效量”表示，与没有接受该量的相应受试者相比，引起疾病、病症或副作用的改进治疗、治愈、预防或减轻的量，或者使疾病或病况的进展速率降低的量。该术语在其范围内还包括有效增强正常生理功能的量。通常，本文中的有效量根据各种因素而变化，所述因素例如给定的药物或化合物、药学制剂、给药途径、疾病或病症的类型、被治疗的受试者等等，但仍然可以由本领域技术人员常规地确定。本发明的化合物的有效量可以由本领域技术人员通过本领域已知的常规方法容易地确定。
用途
本发明提供单克隆抗体32C7或其抗原结合片段在制备用于治疗或者预防人类新型冠状病毒感染的药物或试剂中的用途。其中，新型冠状病毒包括但不限于原始株Original病毒株、B.1.351、B.1.1.7、P1、B.1.617.2等突变病毒株。更具体地，所述突变病毒株具有选自下述的突变：B.1.1.7(N501Y)；B.1.351(K417N，E484K，N501Y)；P1(K417T，E484K，N501Y)；B.1.617.2(L452R，T478K)。
本文所用术语“原始株”是指2020年1月份序列公开，与GISAID编号为EPI_ISL_403934的毒株具有相同序列的SARS-CoV-2(新冠)病毒毒株。
本文所用术语“突变株”是指与EPI_ISL_403934毒株相比较、在序列上发生突变的新冠病毒毒株。
实施例1
本实施例为单克隆抗体32C7的制备，具体如下。
一、实验材料
1、实验所需细胞，病毒和转基因小鼠、Vero E6细胞、SARS-CoV-2、人源化ACE2转基因小鼠(hACE2-mice)。
2、实验所需试剂和材料
S5芯片购于GE公司，滤器购于PALL公司，DMEM培养基，胎牛血清(FBS)均购自于GIBCO公司。
3、实验所需仪器
SPR表面等离子共振小分子互作仪、多功能酶标仪、q-RT PCR仪、CO 2细胞培养箱购自Thermo公司。
二、实验方法
1)首先，收集26名新型冠状病毒肺炎康复患者的外周血，并分离血清与淋巴细胞进行保存，其中新型冠状病毒肺炎康复患者的病原为新冠原始病毒毒株；
2)接着，通过体外ELISA试验筛选了26名康复患者血清中的新型冠状病毒RBD蛋白特异性中和抗体活性，发现其中3名康复患者存在较高的中和抗体滴度；
3)通过细胞相关标记及流式细胞分选技术，制备该3名康复患者的新型冠状病毒RBD蛋白特异性B细胞的单细胞转录本，并通过PCR技术扩增得到对应的抗体重链基因和抗体轻链基因；
4)将测序验证的抗体重链基因和抗体轻链基因在真核细胞内表达，生产并纯化新型冠状病毒特异性抗体,最终得到候选抗体200个；
5)最后，通过体外ELISA实验、假病毒中和实验及动物实验，筛选及组合具有高亲和力的新型冠状病毒中和性抗体。
三、实验结果
本发明得到针对于新冠病毒的抗体32C7。该抗体对应的重链基因序列及轻链基因序列是由RBD蛋白特异性B细胞的单细胞转录本经过抗体基因特异性PCR扩增及PCR产物测序而获得，并通过IMGT网站(http://www.imgt.org)分析得出重链基因及轻链基因对应的可变区序列。抗体32C7具有SEQ ID NO：13所示的重链可变区核酸序列，SEQ ID NO：14所示的重链可变区氨基酸序列以及SEQ ID NO：15所示的重链恒定区氨基酸序列；SEQ ID NO：16所示的轻链可变区核酸序列，SEQ ID NO：17所示的轻链可变区氨基酸序列以及SEQ ID NO：18所示的轻链恒定区氨基酸序列。
抗体32C7具有如SEQ ID NO：1所示的重链CDR1区域核苷酸序列，SE Q ID NO：2所示的氨基酸序列；SEQ ID NO：3所示的CDR2区域核苷酸序列，SEQ ID NO：4所示的氨基酸序列；SEQ ID NO：5所示的CDR3区域核苷酸序列，SEQ ID NO：6所示的氨基酸序列。抗体32C7具有如SEQ ID NO：7所示的轻链CDR1区域核苷酸序列，SEQ ID NO：8所示的氨基酸序列；SEQ ID NO：9所示的CDR2区域核苷酸序列，SEQ ID NO：10所示的氨基酸序列；SEQ ID NO：11所示的CDR3区域核苷酸序列，SEQ ID NO：12所示的氨基酸序列。
实施例2
本实施例为单克隆抗体32C7体外的抗新冠病毒效果，具体如下。
1.实验方法与结果
1.1单克隆抗体32C7与新冠病毒RBD结构域等离子共振互作检测
1)将表达纯化后的RBD蛋白与芯片偶联，然后用缓冲液稳定过夜。
2)用缓冲液将单克隆抗体32C7从120nM开始两倍梯度稀释，使用0.2滤 器过滤之后离心除去气泡。
3)上样检测单克隆抗体32C7和新冠病毒RBD的亲和常数和解离常数。
结果如图1中A所示：单克隆抗体32C7可以高效迅速的和新冠病毒的RBD结构域结合，亲和常数达到1.00×10 5。其解离常数为1.09×10 -8，通过分析和计算二者之间的平衡解离常数为1.09×10 -8。说明单克隆抗体32C7和新冠病毒的RBD结构域之间具有很高的亲和力，并且解离的十分缓慢。
1.2单克隆抗体32C7抗体中和实验
1)将Vero E6细胞铺于48孔板中，37℃孵育24小时。
2)以MOI＝0.005感染(1)中的细胞，将单克隆抗体32C7用含有2％胎牛血清的培养基从10μg/mL开始5倍梯度稀释，加入到(1)中的细胞。
3)感染48小时后将上清取出，用空斑试验检测病毒滴度。
4)将Vero E6细胞铺于96孔板中于37℃孵育24小时，然后将上述病毒上清梯度稀释加到96孔板中，在37℃孵育1小时，然后弃掉上清再加入含有1.6％的羟甲基纤维素的培养基200μL。
5)培养24小时后用4％的多聚甲醛固定细胞并加入0.5％的吐温X-100通透细胞。随后加入抗SARS-CoV-2核衣壳的抗体孵育，再加入HRP标记的二抗。
结果如图1中B所示。单克隆中和抗体32C7可以有效地抑制SARS-CoV-2活毒的感染，对于原始株以及SARS-CoV-2D614G突变株(即B.1.1.7毒株)都具有很好地中和抑制效果，其IC 50分别为4.588ng/mL，和127.6ng/mL。对于新型冠状病毒南非突变株501Y.V2，中和抗体32C7仍然具有一定的中和效果，其IC 50为1.42μg/mL。然而目前已经公布的代表性的抗体CC12.1和REGN10987针对于D614G突变株的IC 50分别是22ng/mL和19.4ng/mL，他们对于南非突变株中和效果很微弱。CV07-270的IC 50是82.3ng/mL。
实施例3
本实施例为抗体32C7在人源化小鼠模上的抗病毒效果，具体如下。
1.实验流程：
1)将8周龄人源ACE2转基因(hACE)小鼠随机分为药物评价组，4只小鼠。阴性对照组3只小鼠。正式实验前，小鼠适应环境2～3d。
2)攻毒当天，小鼠经1％戊巴比妥钠轻度麻醉(每克体重约0.1ml麻醉药)，然后用移液枪采用滴鼻方式感染4×10 4PFU的SARS-CoV-2病毒液。
3)感染病毒后第6小时腹腔内注射20mg/kg的抗体。每天定时称量记录每组动物体重变化，观察小鼠生存状况。
4)感染后第五天处死小鼠，取出肺脏并研磨。用qRT-PCR的方法分别检测肺研磨液的病毒滴度和相关炎症因子的表达情况。同时取出小鼠肺脏进行组织病理学检测。
5)根据统计结果，绘制出体重变化曲线和存活率曲线。
2、实验结果
结果如图2中A所示，抗体32C7在转基因小鼠模型上可以有效的降低SARS-CoV-2的病毒滴度，病毒感染五天后抗体治疗组相对于病毒对照组小鼠肺脏中的病毒滴度降低了90％。同时，如图2中B所示，抗体32C7处理后肺部相关炎症因子的表达也明显减少。如图2中C所示，肺部的验证浸润情况也得到改善。综上所述，抗体32C7在SARS-CoV-2转基因小鼠模型上仍然具有优秀的保护效果，明显地抗病毒能力。
实施例4
本实施例为抗体32C7和S蛋白结合的结构解析，具体如下。
1、实验流程
1)蛋白的表达和纯化：表达SARS-CoV-2的S-6P突变体(其中，该突变体是指spike蛋白的6个丝氨酸突变成6个脯氨酸的突变体)。HEK293F细胞在含8％CO 2的SMM 293T-I培养基中培养，温度为37℃。当细胞密度达到2×10 6个/mL时，在PEI:DNA质量比为3:1，每升培养1mg DNA的情况下，用25-kDa 线性聚乙烯亚胺将S-2P和S-6P质粒瞬时转染HEK293F细胞。收集细胞培养上清，10000×g离心30分钟。使用HisPurTM钴树脂和StrepTactin树脂纯化分泌的S-2P和S-6P蛋白。采用Superose 6 10/300色谱柱，在含有20mM HEPES pH 7.2、150mM NaCl和10％海藻糖的缓冲液中进行纯化。木瓜蛋白酶在含20mM HEPES pH 7.2、150mM NaCl、5mM EDTA和5mM l-半胱氨酸的缓冲液中，37℃消化40min后获得抗体32C7的Fab区。所得Fab用脱盐柱纯化l-半胱氨酸，然后用HiTrap Q柱进一步纯化。收集纯化的Fabs，浓缩至0.6mg/mL。
2)2μL的S-6P(1.2mg/mL)和2μL的32C7 Fab(0.6mg/mL)在室温下孵育3min，然后装载到发光放电的金栅格上。用含有20mM HEPES、pH 7.2和150mM NaCl的缓冲液清洗栅格。使用Mark IV Vitrobot在100％湿度和16℃下印迹网格3s，然后通过浸入式冷冻将栅格浸入液体乙烷中。S-6P-32C7 Fab复合物的显微照片记录在300kV的FEI Titan Krios电子显微镜上。在K3 Summit直接电子探测器上，以标称放大81,000倍的超分辨率模式，离焦范围为1.2至1.3μm，共记录了3740个动态过程。探测器采用狭缝宽度为20ev的GIF量子能量滤波器。显微照片的剂量分成32帧，总电子曝光约50个电子每
Figure PCTCN2021125690-appb-000001
2、实验结果
结果如图3中A所示，通过结构解析发现单克隆抗体32C7可以与新型冠状病毒“down”状态下的RBD结合，导致新冠病毒失去感染能力。同时发现，32C7与目前报道的三类的抗原表位类似。其抗原表位包含S蛋白的345，346，347，348，349，351，352，354，440，441，442，443，444，445，448，450，451，452，466，499，509位的氨基酸残基(结果如图3中B所示)。32C7与新冠病毒RBD结构域有21个相互作用的氨基酸残基，并且都避开了目前新冠病毒突变株的突变位点，使得其对于多种突变株都有很好的抑制效果。
目前已知的突变毒株在RBD上的突变情况概括在此：B.1.1.7(N501Y)；B.1.351(K417N,E484K,N501Y)；P1(K417T,E484K,N501Y)；B.1.617.2(L452R,T478K)，32C7能对上述新冠病毒突变株都具有很好的中和效果。
尽管已经参考示例性实施方案对本发明进行了描述，但应理解本发明不限于公开的示例性实施方案。在不背离本发明的范围或精神的情况下，可对示例性实施方案做多种调整或变化。本发明的范围应基于最宽的解释以涵盖所有修改和等同结构与功能。

Claims (19)

  1. 一种抗体32C7或其抗原结合片段，其特征在于，其能够特异性结合刺突蛋白，并且其能够结合选自刺突蛋白的第345、346、347、348、349、351、352、354、440、441、442、443、444、445、448、450、451、452、466、499、509位的氨基酸残基的表位，或者所述表位选自刺突蛋白的第T345、R346、F347、S349、N440、L441、K444、N450、Y351和Y451，以抑制或阻断所述刺突蛋白与受体的结合。
  2. 一种抗体32C7或其抗原结合片段，其特征在于，其包含重链和轻链，并且表现出对刺突蛋白的受体结构域小于120nM的Kd。
  3. 一种抗体32C7或其抗原结合片段，其特征在于，其含有重链可变区的抗原互补决定区CDR1、CDR2和CDR3，其氨基酸序列分别为SEQ ID NO：2、SEQ ID NO：4、SEQ ID NO：6；和
    轻链可变区的抗原互补决定区CDR1、CDR2和CDR3，其氨基酸序列分别为SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12，所述抗体32C7或其抗原结合片段能够特异性结合刺突蛋白的表位。
  4. 根据权利要求3所述的抗体32C7或其抗原结合片段，其特征在于，所述抗体具有SEQ ID NO：14所示的重链可变区氨基酸序列和SEQ ID NO：17所示的轻链可变区氨基酸序列。
  5. 根据权利要求3或4任一项所述的抗体32C7或其抗原结合片段，其特征在于，所述抗体或其片段还包含重链恒定区、轻链恒定区、Fc区或其组合。
  6. 根据前述权利要求任一项所述的抗体32C7或其抗原结合片段，其特征在于，其重链可变区的氨基酸序列包含SEQ ID NO：2、SEQ ID NO：4、SEQ ID NO：6所示的抗原互补决定区且与SEQ ID NO：14所示序列具有至少80％，优选至少90％，还优选至少95％，进一步优选至少99％的同一性；和其轻链可变区的氨基酸序列包含SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12所示的抗原 互补决定区且与SEQ ID NO：17所示序列具有至少80％，优选至少90％，还优选至少95％，进一步优选至少99％的同一性，并且同时至少保留突变前序列的功能。
  7. 根据权利要求6所述的抗体32C7或其抗原结合片段，其特征在于，其中所述抗体32C7或其抗原结合片段是单克隆抗体、嵌合抗体、纳米抗体、全人源化抗体，优选为单克隆抗体。
  8. 根据权利要求7所述的抗体32C7或其抗原结合片段，其特征在于，其中所述抗体32C7或其抗原结合片段是Fab片段、Fab’、F(ab’) 2片段、单链可变片段scFv、scFv-Fc片段或单链抗体ScAb。
  9. 一种用于制备治疗或者预防ACE2相关疾病的组合物，其包含前述权利要求中任一项所述的抗体32C7或其抗原结合片段，优选地，所述疾病选自冠状病毒感染。
  10. 一种分离的核酸分子，其特征在于，其编码根据前述权利要求中任一项所述的抗体32C7或其抗原结合片段。
  11. 一种载体，其特征在于，其包含权利要求10所述的核酸分子。
  12. 一种细胞系，其特征在于，其能够产生根据前述权利要求中任一项所述的抗体32C7或其抗原结合片段。
  13. 一种药物组合物，其含有根据权利要求1-8任一项所述的抗体32C7或其抗原结合片段，权利要求10所述的核酸分子，或权利要求11所述的载体，和药学可接受载体和/或赋形剂。
  14. 根据权利要求13所述的药物组合物，其特征在于，所述药物组合物为疫苗，优选为亚单位疫苗，所述赋形剂为佐剂。
  15. 根据权利要求14所述的药物组合物，其特征在于，所述佐剂包括铝佐剂、弗氏佐剂、磷酸铝、磷酸钙、石蜡油、羊毛脂、表面活性剂、藻酸钙、多聚核苷酸、胞壁肽、皂苷、RIBI佐剂系统、霍乱毒素、丙烯酸或甲基丙烯酸的聚合物、油包水乳剂、水包油乳剂中的一种或几种，优选为铝佐剂。
  16. 一种抗体32C7或其抗原结合片段的制备方法，其特征在于，其通过培养细胞从而产生抗体的步骤，其中所述细胞包含根据权利要求11所述的载体，或者所述细胞为权利要求12所述的细胞系。
  17. 根据前述权利要求任一项所述的抗体32C7或其抗原结合片段在制备用于治疗或者预防ACE2相关疾病的药物或试剂中的用途，优选地，所述疾病选自冠状病毒感染。
  18. 根据权利要求17所述的用途，其特征在于，所述药物或试剂进一步与小分子抗病毒组合物或抗病毒剂联合使用。
  19. 一种预防或治疗受试者中冠状病毒感染的方法，其特征在于，经口服、肌肉注射或鼻腔滴注给予所述受试者有效量的根据权利要求13-15任一项所述的药物组合物。
PCT/CN2021/125690 2021-08-17 2021-10-22 单克隆抗体32c7及其制备方法和用途 WO2023019723A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110940792.0 2021-08-17
CN202110940792.0A CN113388030B (zh) 2021-08-17 2021-08-17 单克隆抗体32c7及其制备方法和用途

Publications (1)

Publication Number Publication Date
WO2023019723A1 true WO2023019723A1 (zh) 2023-02-23

Family

ID=77622625

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/125690 WO2023019723A1 (zh) 2021-08-17 2021-10-22 单克隆抗体32c7及其制备方法和用途

Country Status (2)

Country Link
CN (1) CN113388030B (zh)
WO (1) WO2023019723A1 (zh)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113388030B (zh) * 2021-08-17 2021-11-23 上海浙江大学高等研究院 单克隆抗体32c7及其制备方法和用途
WO2023046057A1 (zh) * 2021-09-24 2023-03-30 南京金斯瑞生物科技有限公司 抗SARS-CoV-2 L452R刺突蛋白的单克隆抗体及其应用
CN116406375A (zh) * 2021-11-05 2023-07-07 上海科技大学 抗SARS-CoV-2刺突蛋白的三聚体抗体
CN114057870B (zh) * 2021-11-23 2023-04-28 武汉奥科博泰生物科技有限公司 用于新型冠状病毒Delta突变株检测的标记抗体及其应用
CN115028715B (zh) * 2022-06-23 2023-08-18 生工生物工程（上海）股份有限公司 一种抗新型冠状病毒的抗体或其抗原结合片段、试剂盒及应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111423508A (zh) * 2020-03-31 2020-07-17 江苏省疾病预防控制中心(江苏省公共卫生研究院) 一种分离的抗病毒感染的SARS-CoV-2蛋白结合分子
CN111909263A (zh) * 2020-08-19 2020-11-10 重庆医科大学 新冠病毒rbd特异性单克隆抗体和应用
CN112175073A (zh) * 2020-09-30 2021-01-05 上海市公共卫生临床中心 冠状病毒的中和抗体或其抗原结合片段
CN113388030A (zh) * 2021-08-17 2021-09-14 上海浙江大学高等研究院 单克隆抗体32c7及其制备方法和用途

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2605928C2 (ru) * 2010-03-02 2016-12-27 Эббви Инк. Терапевтические dll4-связывающие белки
CN106350486A (zh) * 2016-09-13 2017-01-25 青海七彩花生物科技有限公司 一种提高dc‑cik细胞杀伤力的细胞培养方法
CN111592594B (zh) * 2020-03-13 2022-05-10 北京大学 一种抗新型冠状病毒的单克隆抗体及其应用
CN113388031B (zh) * 2021-08-17 2021-11-09 上海浙江大学高等研究院 单克隆抗体35b5及其制备方法和用途

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111423508A (zh) * 2020-03-31 2020-07-17 江苏省疾病预防控制中心(江苏省公共卫生研究院) 一种分离的抗病毒感染的SARS-CoV-2蛋白结合分子
CN111909263A (zh) * 2020-08-19 2020-11-10 重庆医科大学 新冠病毒rbd特异性单克隆抗体和应用
CN112175073A (zh) * 2020-09-30 2021-01-05 上海市公共卫生临床中心 冠状病毒的中和抗体或其抗原结合片段
CN113388030A (zh) * 2021-08-17 2021-09-14 上海浙江大学高等研究院 单克隆抗体32c7及其制备方法和用途

Also Published As

Publication number Publication date
CN113388030A (zh) 2021-09-14
CN113388030B (zh) 2021-11-23

Similar Documents

Publication Publication Date Title
WO2023019724A1 (zh) 单克隆抗体35b5及其制备方法和用途
WO2023019723A1 (zh) 单克隆抗体32c7及其制备方法和用途
CA2908878C (en) Human antibodies binding to rsv g protein
CA2908921C (en) Human antibodies binding to rsv g protein
JP6483337B2 (ja) デングウイルス血清型１のｅタンパク質に対して特異性を有するヒトモノクローナル抗体およびその使用
KR20100091195A (ko) 항ｒｓｖ ｇ 단백질 항체
CN111417651B (zh) 多瘤病毒中和抗体
TWI785583B (zh) 一種抗新型冠狀病毒的單克隆抗體及其應用
MX2010009885A (es) Composiciones y métodos para la terapia y diagnóstico de citomegalovirus.
JP2023534923A (ja) ＳＡＲＳ－ＣｏＶ－２を標的にする抗原結合分子
JP2022541652A (ja) エンテロウイルスｄ６８に対するヒトモノクローナル抗体
WO2022044573A1 (ja) コロナウイルススパイク蛋白に対するヒト抗体またはその抗原結合断片
WO2014115893A1 (ja) ヒト・メタニューモウイルスに特異的なヒト抗体もしくはその抗原結合性断片
CN115087667B (zh) 特异性结合SARS-CoV-2的抗原结合蛋白
JP2024518335A (ja) ＳＡＲＳ－ＣｏＶ－２に対するヒト中和モノクローナル抗体、及びそれらの使用
WO2022148374A1 (zh) 抗冠状病毒的全人广谱中和抗体76e1及其应用
WO2024053719A1 (ja) コロナウイルス変異株に対するヒト抗体またはその抗原結合断片
WO2022095045A1 (zh) SARS-CoV-2抗体及其应用
WO2023145859A1 (ja) 変異株交差性を有する抗ＳＡＲＳ－ＣｏＶ－２ヒト中和抗体およびその抗原結合性断片
WO2022020327A1 (en) Broadly neutralizing binding molecules against marburgviruses
CN117683123A (zh) 抗狂犬病毒人源化抗体和抗体组合及其用途
CN116964103A (zh) SARS-CoV-2病毒的广谱抗体及其应用
AU2020273365A1 (en) Human antibodies to Ross River virus and methods of use therefor
CN115304672A (zh) 冠状病毒抗体及其应用
CN114174331A (zh) 结合人类偏肺病毒融合蛋白的抗体及其用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21953959

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE