Antibody targeting monkey pox virus, and antigen binding fragment and application thereof

Abstract

The invention discloses a monkey pox virus targeting antibody, an antigen binding fragment thereof and application thereof. The antibody or the antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region. The antibody or the antigen binding fragment thereof targets human and non-human primate A35R, has improved or reduced affinity, can achieve the purpose of improving the drug effect or reducing the toxicity, can perform selective adaptation according to different target spots, and provides a flexible adaptation scheme for drug research and development.

Description

Technical Field

[0001] The present invention relates to the field of antibody drugs, and specifically to an antibody or an antigen-binding fragment thereof targeting monkeypox virus and its use, in particular an antibody that specifically binds to the monkeypox virus surface antigen A35R and a conjugate or fusion comprising the anti-A35R antibody or its antigen-binding fragment. In addition, the present invention also relates to a nucleic acid encoding the anti-A35R antibody and a host cell comprising the nucleic acid, as well as a method for preparing the antibody. The present invention also relates to a pharmaceutical composition comprising the anti-A35R antibody and the medical use of the anti-A35R antibody. Background Art

[0002] Monkeypox (MPXV) first broke out in macaques raised by a Danish research institute in 1958. Since 2017, more than 99 countries and regions around the world have reported more than 51,257 cases of monkeypox to the World Health Organization. On July 23, 2022, WHO announced that monkeypox constitutes a global public health emergency of international concern (PHEIC).

[0003] Monkeypox is an acute, self-limited illness with an incubation period of 5 to 21 days. The febrile phase of the illness usually lasts 1 to 3 days and symptoms include fever, severe headache, swollen lymph nodes, back pain, muscle pain, and severe weakness. The febrile phase is followed by a skin eruption phase that lasts 2-4 weeks. Lesions progress from macules (lesions with a flat base) to papules (raised, hard, painful lesions) to vesicles (filled with clear fluid) to pustules (filled with pus) and then crusts. Death has occurred in between 0 and 11% of recorded cases, with higher rates among young children.

[0004] Monkeypox virus is an enveloped double-stranded DNA virus. There are two forms of viral particles: extracellular enveloped virus (EV) and intracellular mature virion (MV). The MV form is the most abundant and the most stable in the environment. It is the main form of virus transmission between hosts. More than 20 structural proteins have been identified, among which A29L is the membrane protein of monkeypox virus MV, which binds to cell surface glycosaminoglycans and mediates the fusion of virus and cells; M1R is also a membrane protein of MV, which is highly conserved among known poxviruses; the envelope glycoprotein A35R on the surface of EV can induce the production of neutralizing antibodies and block the spread of the virus between cells. These proteins are considered to be important targets for monkeypox virus research. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the lack of antibodies with good effects on monkeypox virus and its variants in the prior art, and provide an antibody or antigen-binding fragment thereof targeting monkeypox virus and its use. The antibody or antigen-binding fragment thereof of the present invention has good binding activity to various variants of monkeypox virus, provides more options for preventing and treating viral infections, and has important clinical value.

[0006] The present invention solves the above technical problems through the following technical solutions.

[0007] The first aspect of the present invention provides an antibody or an antigen-binding fragment thereof targeting monkeypox virus, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; or,

[0008] The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively; or,

[0009] The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively; or,

[0010] The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively; or,

[0011] The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38, respectively.

[0012] In the present invention, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by AbM.

[0013] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 7, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 8, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 8.

[0014] In other embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 15 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 16; or or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16.

[0015] In other embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 23 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 23, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 24 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 24.

[0016] In other embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 31 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 31, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 32 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 32.

[0017] In other embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 39 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 39, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 40 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 40.

[0018] In the present invention, the sequence identity may be limited to the framework regions of the heavy chain variable region and the light chain variable region. The antibody or antigen-binding fragment thereof of the present invention targets an antigen amino acid sequence, and the heavy chain variable region and the light chain variable region that have at least 80% sequence identity with the framework regions of the reference sequences of the heavy chain variable region and the light chain variable region corresponding to each CDR retain the binding activity to the antigen.

[0019] In the present invention, the antibody may be a full-length antibody, Fab, Fab', F(ab')2, Fv or a multispecific antibody.

[0020] In some embodiments of the present invention, when the antibody is a full-length antibody, the heavy chain constant region of the full-length antibody is derived from the heavy chain of a human antibody or a variant thereof, and the light chain constant region of the full-length antibody is derived from the κ chain or λ chain of a human antibody or a variant thereof; when the antibody is Fv, the Fv is scFv.

[0021] In some specific embodiments of the present invention, the antibody is a full-length antibody, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 41, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 43 or SEQ ID NO: 44.

[0022] In other specific embodiments of the present invention, when the antibody is Fv, the antibody further comprises an Fc region, and the amino acid sequence of the Fc region is preferably as shown in SEQ ID NO: 42, SEQ ID NO: 45 or SEQ ID NO: 46.

[0023] The second aspect of the present invention provides an antibody combination, which comprises one or more antibodies or antigen-binding fragments thereof as described in the first aspect.

[0024] In some embodiments of the present invention, the antibody combination comprises a first antibody and a second antibody.

[0025] In some specific embodiments of the present invention, the heavy chain variable region of the first antibody comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively; the heavy chain variable region of the second antibody comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0026] In other specific embodiments of the present invention, the heavy chain variable region of the first antibody comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively; the heavy chain variable region of the second antibody comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0027] In other specific embodiments of the present invention, the heavy chain variable region of the first antibody comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively; the heavy chain variable region of the second antibody comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively.

[0028] In other specific embodiments of the present invention, the heavy chain variable region of the first antibody comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; the heavy chain variable region of the second antibody comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively.

[0029] The third aspect of the present invention provides an isolated nucleic acid encoding the antibody or antigen-binding fragment thereof as described in the first aspect, or the antibody combination as described in the second aspect.

[0030] The fourth aspect of the present invention provides a recombinant expression vector, which comprises the nucleic acid as described in the third aspect.

[0031] In some embodiments of the present invention, the recombinant expression vector may be conventional in the art, such as a plasmid, cosmid, phage or virus vector.

[0032] In some specific embodiments of the present invention, the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector or an adeno-associated viral vector.

[0033] The fifth aspect of the present invention provides a transformant, which comprises the recombinant expression vector as described in the fourth aspect in a host cell.

[0034] In the present invention, the host cell is a prokaryotic cell or a eukaryotic cell.

[0035] In some embodiments of the present invention, the host cell is selected from yeast cells, mammalian cells or other cells suitable for producing antibodies or antigen-binding fragments thereof.

[0036] In some specific embodiments of the present invention, the mammalian cell is a HEK293 cell.

[0037] The sixth aspect of the present invention provides a method for preparing an antibody or an antigen-binding fragment thereof targeting monkeypox virus, the method comprising culturing the transformant as described in the fifth aspect, and obtaining the antibody or an antigen-binding fragment thereof targeting monkeypox virus from the culture.

[0038] The seventh aspect of the present invention provides a pharmaceutical composition, which comprises the antibody or antigen-binding fragment thereof as described in the first aspect, or the antibody combination as described in the second aspect, and a pharmaceutically acceptable carrier.

[0039] The eighth aspect of the present invention provides a kit, which comprises the antibody or antigen-binding fragment thereof as described in the first aspect, the antibody combination as described in the second aspect, the nucleic acid as described in the third aspect, the recombinant expression vector as described in the fourth aspect, the transformant as described in the fifth aspect, or the pharmaceutical composition as described in the seventh aspect.

[0040] In some embodiments of the present invention, the kit further comprises a reagent for detecting the binding of the antibody or antigen-binding fragment thereof to monkeypox virus.

[0041] The ninth aspect of the present invention provides an antibody or antigen-binding fragment thereof as described in the first aspect, an antibody combination as described in the second aspect, a nucleic acid as described in the third aspect, a recombinant expression vector as described in the fourth aspect, a transformant as described in the fifth aspect, a pharmaceutical composition as described in the seventh aspect, or a kit as described in the eighth aspect for use in the preparation of a drug for diagnosing, preventing and / or treating viral infection.

[0042] In some embodiments of the invention, the viral infection is a poxvirus infection.

[0043] In some specific embodiments of the present invention, the poxvirus infection is monkeypox virus infection.

[0044] The tenth aspect of the present invention provides a method for diagnosing and / or treating viral infection, comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof as described in the first aspect, the antibody combination as described in the second aspect, the nucleic acid as described in the third aspect, the recombinant expression vector as described in the fourth aspect, the transformant as described in the fifth aspect, the pharmaceutical composition as described in the seventh aspect, or the kit as described in the eighth aspect.

[0045] In some embodiments of the invention, the diagnosis and / or treatment is in vivo or in vitro.

[0046] In some embodiments of the present invention, the viral infection is as described in the eighth aspect.

[0047] definition

[0048] The term "complementarity determining region" or "CDR region" or "CDR" as used herein is a region of an antibody variable domain that is highly variable in sequence and forms a structurally defined loop ("hypervariable loop") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigens and include CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. In a given heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of many well-known antibody CDR assignment systems. It is well known to those skilled in the art that the CDR of an antibody can be defined by a variety of methods in the art, such as Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (World Wide Web imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. It will be appreciated by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g. variable region) should be understood to encompass the complementarity determining regions as defined by any of the above-mentioned known schemes described in the present invention.

[0049] Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. However, although CDRs are different between antibodies, only a limited number of amino acid positions in CDRs are directly involved in antigen binding. Using at least two of the Kabat, Chothia, IMGT, AbM and Contact methods, the minimum overlapping region can be determined, thereby providing a "minimum binding unit" for antigen binding. The minimum binding unit can be a sub-portion of a CDR. As those skilled in the art will appreciate, by the structure of the antibody and protein folding, the residues of the rest of the CDR sequence can be determined. Therefore, the present invention also contemplates variants of any CDR provided herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit can remain unchanged, and the remaining CDR residues defined according to Kabat or Chothia or AbM can be replaced by amino acid residues.

[0050] As used herein, "percent (%) sequence identity" and "sequence identity" of an amino acid sequence have a definition recognized in the art, which refers to the percentage of identity between two polypeptide sequences determined by sequence alignment (e.g., by manual inspection or a publicly known algorithm). It can be determined using methods known to those skilled in the art, such as using publicly available computer software such as BLAST, BLAST-2, Clustal Omega and FASTA software.

[0051] In the present invention, unless the context clearly indicates otherwise, when the term "antibody" is mentioned, it includes not only intact antibodies but also antigen-binding fragments of antibodies.

[0052] As used herein, the term "multispecific antibody" refers to an antibody that is capable of specifically binding to two or more (e.g., 2, 3, 4, 5, or 6) different antigenic epitopes. A multispecific antibody may, for example, be a bispecific, trispecific, or tetraspecific antibody, which is capable of specifically binding to 2, 3, or 4 antigenic epitopes, respectively. As used herein, the term "epitope" or "antigenic determinant" refers to a region of an antigen that specifically binds to the antigen binding site of an antibody.

[0053] The term "isolated" as used herein refers to something obtained artificially from a natural state. If a certain "isolated" substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been separated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity separated from this natural state is called "isolated". The term "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0054] As used herein, "vector" refers to a construct that is capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the genes or sequences in the host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0055] The term "host cell" as used herein refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli, fungal cells such as yeast cells, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.

[0056] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0057] The positive and progressive effects of the present invention are:

[0058] The antibody or antigen-binding fragment thereof of the present invention targets human and non-human primate A35R. The anti-A35R antibody or antigen-binding fragment thereof has increased or decreased affinity, which can achieve the purpose of improving drug efficacy or reducing toxicity. When constituting a bispecific antibody, selective adaptation can be performed according to different targets, providing a flexible adaptation scheme for drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 The results of ELISA test for antigen A35R are shown.

[0060] Figure 2 The ELISA test results of the binding of the full human library antibodies to the antigen A35R are shown.

[0061] Figure 3 The ELISA test results of the mouse immune library antibodies binding to the antigen A35R are shown.

[0062] Figure 4 , Figure 5 and Figure 6 Antibody pairing assay results are shown. DETAILED DESCRIPTION

[0063] The invention generally described herein will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the scope of the invention. These examples are not intended to represent that the experiments below are all or the only experiments performed.

[0064] The experimental methods in the following examples without specifying specific conditions were carried out according to conventional methods and conditions, or selected according to the product instructions.

[0065] Example 1: Preparation and identification of raw materials

[0066] 1.1 Antigen protein preparation and identification

[0067] Preparation of antigen protein: mFc (SEQ ID NO: 46) or His tag was added to the C-terminus of the extracellular domain of human A35R protein (A35R-ECD, see: Uniprot ID Q8V4U4 AA: 58-181) through genetic manipulation at the gene level. The obtained nucleic acid sequence was constructed into the GSV0 vector, then transformed into Escherichia coli DH5α, cultured overnight at 37°C, and then the plasmid was extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01). The resulting plasmid was transiently transformed into HEK293 cells (ATCC® CRL-1573™) using the ExpiFectamine™ 293 transfection kit (Gibco™, A14524). After 5 days of expression, the cell culture supernatant was collected and the protein containing the Fc tag was affinity purified using a ProteinA / G affinity chromatography column. After purification, the target protein was eluted with 100 mM glycine (pH=3.0), concentrated, and replaced with buffer. The protein containing the His tag was affinity purified with Ni Smart Beads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd., SA036050), and then the target protein was eluted with an imidazole gradient. The eluted proteins were exchanged into PBS buffer through ultrafiltration concentration tubes (Millipore, UFC901096). Finally, A35R antigen proteins with mFc tags and His tags (A35R-ECD-mFc, A35R-ECD-His) were obtained.

[0068] Antigen identification: The activity of the prepared antigen protein was detected using the purchased Anti-Monkeypox virus / MPXV A35R Antibody (SAA0287) antibody protein. The specific method is as follows: ELISA plates were coated with 2 μg / mL A35R-ECD-mFc and A35R-ECD-His at 4°C overnight; after washing the plates three times, they were blocked with 5% skim milk prepared in PBS at room temperature for 1 hour; after washing the plates three times, the antibodies Anti-Monkeypox virus / MPXV A35R Antibody (SAA0287) diluted in 1% PBSM were added and incubated at room temperature for 1 hour; after washing the plates, the secondary antibodies Goat-Anti-mouse-IgG-Fc-HRP (abcam; ab97265) and Anti-mouse-Fab-HRP (sigma; M4155-1ML) diluted in 1% PBSM (1:8000) were added and incubated at room temperature for 1 hour, the plates were washed six times, and then TMB was added for color development for 5 min-20 min, the color development reaction was terminated, and the OD was read using an enzyme reader. 450 The data were processed and plotted using Graphpadprism. Figure 1 As shown, the antibody Anti-Monkeypox virus / MPXV A35RAntibody (SAA0287) binds well to the antigens A35R-ECD-mFc and A35R-ECD-His constructed and expressed in Example 1.1, and has EC 50 The antigen binding activity was 0.007442 μg / mL.

[0069] Example 2: Construction and identification of overexpression cell lines

[0070] 2.1. Construction and identification of A35R-FL-EGFP-HEK293 overexpression cell line

[0071] The coding nucleotide sequence of A35R-FL-EGFP (A35R-FL, see: Uniprot ID Q8V4U4, EGFP see SEQ ID NO: 47) was constructed into the pLVX-puro plasmid (Clontech, Cat#632164). Then, the resulting plasmid was electroporated into HEK293 cells (ATCC® CRL-1573™) by an electroporator (Invitrogen, NeonTM Transfection System, MP922947). After electroporation, the resulting cells were transferred to DMEM medium (Gibco, 11995065) containing 10% FBS (Gibco, 15140-141) by volume and without antibiotics. The cells were then transferred to a 10×10 cm cell culture dish and cultured for 48 hours. The cells were then seeded into a 96-well cell culture plate at an average density of 0.51E+4 cells / well. Puromycin with a final concentration of 2 µg / mL was added as a screening pressure. After about 2 weeks, the cell lines that formed clones were picked for identification.

[0072] Flow cytometric identification of A35R-FL-EGFP-HEK293 overexpressing cells: The cells of the above cell lines in the logarithmic growth phase were digested and plated into 96-well plates, washed with FACS buffer (1×PBS buffer containing 2% FBS by volume), and then added with commercially available first antibody (Anti-Monkeypox virus / MPXV A35RAntibody (SAA0287)) diluted in gradients with PBS and incubated at 4°C for 30 min; after washing, the prepared fluorescent second antibody Anti humanIgG Fc (abcam, 98596) was added and incubated at 4°C for 30 min; finally, the cells were detected by flow cytometry (Beckman, CytoFLEXAOO-1-1102). The detection results showed that the A35R-FL-EGFP-HEK293 cell line with high expression of human A35R on the cell surface was obtained.

[0073] Example 3: Construction and screening of a fully human natural antibody phage display library

[0074] In this example, a phage display antibody gene library was constructed, and the library was screened using A35R-ECD-His as a screening antigen to obtain a plurality of antibody molecules that specifically bind to the A35R-ECD-His protein.

[0075] 3.1 Construction of human antibody gene library

[0076] Take 15 mL of Ficoll-Paque density gradient separation solution (purchased from GE, catalog number: 17144003S) and slowly add it to a 50 mL centrifuge tube. Tilt the centrifuge tube and slowly add 15 mL of collected normal human blood along the tube wall in batches, so that the Ficoll-Paque density gradient separation solution and normal human blood maintain a clear separation interface. Centrifuge the 50 mL centrifuge tube containing the blood and separation solution at about 15°C for 20 min, wherein the centrifuge is set to 400 g, the acceleration is 3, and the deceleration is 0. After centrifugation, the entire liquid surface is divided into four layers, the upper layer is a plasma mixture, the lower layer is red blood cells and granulocytes, and the middle layer is Ficoll-Paque liquid. At the junction of the upper and middle layers, there is a narrow band of white cloud layer dominated by PBMC, that is, the PBMC cell layer. Carefully aspirate the upper plasma mixture with a sterile Pasteur pipette, and then aspirate PBMC with a new sterile Pasteur pipette to obtain separated PBMC. The separated PBMCs were first rinsed twice with PBS, then centrifuged at 1500 rpm for 10 min at 4°C, and finally resuspended with 1.5 mL of PBS and counted using a cell counter (CountStar, CountStar Altair).

[0077] Total RNA was extracted from isolated PBMC cells by conventional methods. The extracted total RNA was reverse transcribed into cDNA using a reverse transcription kit (purchased from TaKaRa, catalog number: 6210A). Based on the sequence similarity of the heavy chain and light chain germline genes, degenerate primers were designed at the front end of the V region of the heavy chain and light chain and the back end of the first constant region, respectively (Li Xiaolin, Construction and preliminary screening of a large-capacity non-immune human Fab phage antibody library, Master's thesis of Peking Union Medical College), and the heavy chain variable region gene fragment and light chain variable region gene fragment of the antibody were obtained after PCR. After recovering the heavy chain variable region gene fragment and light chain variable region gene fragment of the antibody, amplification was performed by fusion PCR to obtain a fragment containing the variable regions of the heavy and light chains of the antibody. Then, the PCR product and the phage display vector were digested, recovered and connected, and the connection product was recovered by a recovery kit (Omega, catalog number: D6492-02). For specific materials and methods, please refer to Li Xiaolin's paper above. Finally, the cells were transformed into competent Escherichia coli SS320 (Lucigen, MC1061 F) using an electroporator (Bio-Rad, MicroPulser), and the transformed Escherichia coli SS320 culture medium was spread on ampicillin-resistant 2-YT solid plates (solid plates were prepared with 1.5% tryptone, 1% yeast extract, 0.5% NaCl, and 1.5% agar at a mass volume of g / mL).

[0078] 3.2 Calculation of antibody gene library capacity

[0079] Take the transformed E. coli SS320 bacterial solution and inoculate it with antibiotic-free 2YT culture medium at a volume of 1:50. Cultivate at 37°C, 220 rpm for 1.5-2 h until OD600 reaches 0.5-0.6, then take it out to room temperature. Add the bacterial solution to a 96-well round-bottom dilution plate at 90 μL / well. Each bacterial solution sample is diluted 10 times, for a total of 12 dilution gradients. Use an 8-channel 10 μL range pipette to draw 2 μL of the diluted sample and add it to C in the order of the dilution gradient from low to high. + / T + Place the cells on a plate (with carbenicillin and tetracycline antibiotic concentrations of 50 μg / mL and 50 μg / mL, respectively), place upright for 5 min, then invert and incubate at 37°C overnight. Observe the growth of clones on the second day and calculate the reservoir capacity. The reservoir capacity is calculated as follows: starting from row A, mark them as 1, 2, 3, 4, 5, 6, 7, 8 to row X. First, select the counting wells, select the counting wells with 3-20 clones, obtain the row number X, and count the number of clones n in the corresponding wells. The calculation formula is 5×100×10 X ×n, through calculation, the total storage capacity is 2.61×10 11 cfu, i.e. 2.61×10 11 Antibody genes.

[0080] 3.3 Preparation of antibody gene phage display library

[0081] Based on the antibody gene library capacity, 50 OD (1 OD is 5×10 8 cfu) of the whole human phage library was added to fresh 2-YT liquid culture medium, so that the initial OD value was 0.1. The culture medium was placed in a shaker at 37°C and 220 rpm to the logarithmic growth phase (OD600 = about 0.6), and then VSCM13 helper phage (purchased from Stratagene) was added at a quantity 50 times the number of bacteria (i.e., the multiplicity of infection (MOI) was about 50), mixed thoroughly, and continued to be cultured in a shaker at 220 rpm for 1 hour after standing for 30 minutes. Subsequently, the culture was centrifuged at 10,000 rpm for 5 minutes, the supernatant was discarded, and the culture medium was replaced with 2-YT medium with dual resistance of carbenicillin 50 μg / mL / kanamycin 40 μg / mL (hereinafter referred to as C + / K +2-YT medium) and continued to culture overnight at 30°C and 220 rpm. The next day, the bacterial solution was centrifuged at 13000g for 10 min, and the supernatant was collected and added with 20% PEG / NaCl (prepared with PEG6000 with a volume concentration of 20% and 2.5 M NaCl) to make the final concentration of PEG / NaCl 4%. The mixture was mixed and placed on ice for 1 hour, and then centrifuged at 13000g for 10 min. The precipitated phages were rinsed with PBS and stored for subsequent phage screening.

[0082] 3.4 Screening of antibody gene phage display library

[0083] 3.4.1 Immunotube method for screening phage display antibody gene library

[0084] The purpose of both the immunotube method and the magnetic bead method is to enrich specific antibodies against antigens, and they are two complementary experimental methods.

[0085] The principle of immunotube screening is to coat A35R-ECD-His on the surface of an immunotube with high adsorption capacity, add the phage display antibody library to the immunotube and perform a panning process of incubation, washing and elution with the antigen protein adsorbed on the surface of the immunotube. After three rounds of panning, specific monoclonal antibodies against the antigen are finally enriched.

[0086] The specific implementation methods are as follows:

[0087] In the first round of screening, 1 mL of 100 μg / mL A35R-ECD-His was added to the immunotube and coated overnight at 4°C. The next day, the coating solution was discarded and 5% milk in PBS was added for blocking for 2 h. After rinsing twice with PBS, a total of 1.20×10 13 The phages of the human antibody library were incubated for 2 h, rinsed with PBS and PBST for 2 and 6 times respectively to remove non-specifically bound phages, and then 0.8 mL of 0.05% EDTA trypsin digestion solution was added to the immune tube to elute the phages that specifically bound to the target antigen, and then infected with SS320 bacteria (Lucigen, 60512-1) in the logarithmic phase, and allowed to stand at 37°C for 30 min, and then cultured at 220 rpm for 1 h, and then VSCM13 helper phages were added, allowed to stand for 30 min, and continued to be cultured at 220 rpm for 1 h, centrifuged and replaced to C + / K +2-YT medium, and continue to culture overnight at 30°C, 220 rpm. The phages were precipitated the next day and used for the next three rounds of screening. The antigen coating concentrations used in the second and third rounds were usually reduced to 30 μg / mL and 10 μg / mL respectively. In addition, the PBST washing intensity was gradually increased, and the number of PBST elutions was 10 and 14 times respectively.

[0088] The phage pool eluted in each round was tested by ELISA to evaluate the enrichment effect. The results showed that the sequence was significantly enriched after the second round of screening. Therefore, the second and third rounds of ELISA positive clone screening were selected.

[0089] 3.5 Selection of single clones

[0090] After three rounds of screening, the second and third rounds of positive clones were selected for ELISA screening. Finally, 337 positive clones that could bind to A35R-ECD-His protein were screened out of 360 clones. After sequencing analysis, 43 clones were selected to construct the full length sequence for the next experiment.

[0091] The specific implementation methods are as follows:

[0092] Sequencing and analysis of positive clones

[0093] After the initial screening, the positive clones were numbered, 2 μL of bacterial solution was pipetted into 2 mL of 2 YT medium, cultured overnight at 37°C and 220 rpm, and the plasmid was extracted for next-generation sequencing. The sequencing results were integrated, aligned, and non-antibody gene sequences were removed from the original AB1 file using SeqMan to generate a fasta file of the antibody gene integration version. The DNA sequence was then translated into an amino acid sequence using MEGA6, and the amino acid sequence was used to find out the terminator, unconventional sequences, etc., and the fasta file of the amino acid sequence was exported.

[0094] Clone NL-A35R-A253 is the preferred molecule. The CDR amino acid sequence of the obtained antibody Fab is shown in Table 6 and the sequence table. The CDR sequence was determined using the AbM definition method.

[0095] Example 4: Mouse immunization and immune library construction and screening

[0096] 4.1 Immunization regimen

[0097] A35R-ECD-His antigen was used to cross-immunize 2 Balb / C and 2 C57 mice by subcutaneous injection and intraperitoneal injection. A35R-ECD-mFc and A35R-ECD-His antigen were cross-immunized by subcutaneous injection and intraperitoneal injection. 2 Balb / C and 2 C57 mice (Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.) were immunized once every two weeks, for a total of 4 times. One week after the 4th immunization, the mouse blood was taken for immune titer detection, and finally, A35R-ECD-mFc was used for booster immunization once.

[0098] 4.2 Detection of serum antibody titers in mice after immunization

[0099] ELISA plates were coated with 2 μg / mL A35R-ECD-His at 4°C overnight (30 μL / well), washed 3 times, and blocked with 5% skim milk (5% PBSM) at room temperature for 1 hour; washed 3 times, mouse serum diluted with 5% PBSM was added, and the antibody Anti-Monkeypox virus / MPXV A35R Antibody (SAA0287) was added as a positive control, and incubated at room temperature for 1 hour; washed 6 times, the secondary antibody Goat-anti-mouse-lgG (1+2a+2b+3)-HRP (Jackson, 115-035-164) diluted with 5% PBSM was added, and incubated at room temperature for 1 hour, washed 6 times, TMB was added for color development for 5-20 minutes, and the color development reaction was terminated and the data was read by OD450 using an enzyme reader. The results are shown in Tables 1 and 2, and the titers of the sera of 8 mice were all up to standard.

[0100] Table 1: Final immunization serum titer test (ELISA)

[0101]

[0102] Table 2: Final immunization serum titer test (ELISA)

[0103]

[0104] 4.3 Construction of phage display antibody gene library

[0105] After immunization, the spleen of the mouse was taken, and the spleen cells were collected after grinding and filtering. 1 mL of TRIzol™ Reagent (Thermo Fisher, 15596026) was added to lyse the spleen cells, and the total RNA was extracted by the phenol chloroform method. The extracted RNA was reverse transcribed into cDNA using a reverse transcription kit (TaKaRa, 6210A). Then, cDNA was used as a PCR template, and specific primers of the mouse antibody sequence were used to amplify the light chain and heavy chain variable region genes of the antibody. The PCR product was double-digested with NcoI and NotI to obtain the antibody gene fragment, which was inserted into the phage display vector and ligated with T4 ligase. The ligation product was recovered using a DNA recovery kit (Omega, D6492-02) and finally transformed into competent Escherichia coli SS320 (Lucigen, MC1061F) using an electroporator (Bio-Rad, MicroPulser). The electroporated bacteria were spread on a 2-YT (C+ / K+ 2-YT) solid plate containing ampicillin and tetracycline, and the SS320 bacteria that were correctly transformed with the antibody plasmid were amplified and packaged using VSCM13 helper phage (purchased from Stratagene) to obtain a phage display library containing Fab sequences.

[0106] 4.4 Cell-based screening of phage-displayed antibody gene libraries

[0107] A35R-FL-EGFP-HEK293 cells were cultured in T25 culture flasks. When the cell growth density was close to 90%, the culture supernatant was removed and washed once with PBS (Yuanpei, B310KJ), then 2 mL of 4% paraformaldehyde (Shenggong, E672002-0500) was added for fixation for 0.5 hours, and finally washed twice with PBS and used as the screening raw material. During screening, the phage display library was incubated with fixed A35R-FL-EGFP-HEK293 cells at room temperature for 1 hour, washed three times with 1×PBS, and then 2 mL of glycine-HCl (pH=2.0) was added and gently mixed for 10 minutes to elute the phage that specifically binds to human ROR1. The eluted supernatant was then infected with SS320 bacteria (Lucigen, 60512-1) in the logarithmic phase, and then cultured at 37°C and 220 rpm for 1 hour. VSCM13 helper phage was added, and the cells were allowed to stand for 30 minutes. The cells were further cultured at 37°C and 220 rpm for 1 hour, centrifuged and replaced in C+ / K+ 2-YT medium, and the phages obtained were used for the second round of screening. The screening was repeated many times, and 10 clones were randomly selected for sequence analysis in each round to evaluate the library. After 3 rounds of screening, the sequence enrichment in the library was obvious.

[0108] 4.5 Immunotube screening of phage display antibody gene libraries

[0109] The immunotube method and magnetic bead method were used to enrich specific antibodies against antigens, and the two methods complemented and verified each other.

[0110] The immunotube screening is to coat the antigen protein A35R-ECD-His or A35R-ECD-mFc on the surface of an immunotube with high adsorption capacity, add the phage display antibody library to the immunotube and incubate, wash and elute with the antigen protein adsorbed on the surface of the immunotube, undergo 2-4 rounds of panning, and finally enrich the specific monoclonal antibody Fab for the antigen. In this embodiment, the monoclonal antibody Fab for A35R-ECD-His was enriched after 3 rounds of panning. The specific method refers to Example 2.4.2 in patent CN112250763B.

[0111] The phage pool eluted in each round was tested by ELISA to evaluate the enrichment effect. The results showed that the sequence was significantly enriched after the second round of screening. Therefore, the second and third rounds of ELISA positive clone screening were selected.

[0112] 4.6 Selection of single clones

[0113] After three rounds of screening, the second and third rounds of positive clones were selected for ELISA screening. Finally, 167 positive clones that could bind to the A35R-ECD-His protein were screened out of 296 clones. After sequencing analysis, 28 clones were selected to construct the full length sequence for the next experiment.

[0114] The specific implementation methods are as follows:

[0115] Sequencing and analysis of positive clones

[0116] After completing the initial screening, the positive clones were numbered, 2 μL of bacterial solution was pipetted into 2 mL of 2 YT medium, cultured overnight at 37°C and 220 rpm, and the plasmid was extracted for next-generation sequencing. The sequencing results were integrated, aligned, and non-antibody gene sequences were removed from the original AB1 file using SeqMan to generate a fasta file of the antibody gene integration version. The DNA sequence was then translated into an amino acid sequence using MEGA6, and the amino acid sequence was used to find out the terminator, unconventional sequences, etc., and the fasta file of the amino acid sequence was exported.

[0117] The clones A35R_M_PR_A142, A35R_M_PR_A064, A35R_M_PR_A094 and A35R_M_PR_A145 are preferred molecules. The CDR amino acid sequences of the obtained antibody Fab are shown in the sequence listing and sequence table. The CDR sequences were determined by using the AbM definition method.

[0118] Example 5: Construction, expression and purification of candidate antibodies

[0119] 5.1 Plasmid construction

[0120] The VH coding sequence in the Fab sequence of the screened monoclonal antibody was connected to the coding sequence of the heavy chain constant region (SEQ ID NO: 41) of human IgG1 to obtain the heavy chain coding sequence of the fully human antibody, and the VL coding sequence in the Fab sequence was connected to the coding sequence of the Kappa type (SEQ ID NO: 43) or Lambda type (SEQ ID NO: 44) of the human light chain constant region (CL) to obtain the light chain coding sequence of the fully human antibody. The coding sequences of the antibody heavy chain and light chain were respectively inserted into the eukaryotic expression vector plasmid GSV0, transformed into Escherichia coli DH5α, and cultured overnight at 37°C. The plasmid was extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain an endotoxin-free antibody plasmid for eukaryotic expression.

[0121] 5.2 Expression and purification of candidate antibodies

[0122] The candidate antibodies were expressed using the ExpiCHO transient transfection expression system (Thermo Fisher, A29133) as follows: On the day of transfection, the cell density was confirmed to be 7 × 10 6 Up to 1×10 7 The cell viability was >98%. At this time, the cells were adjusted to a final concentration of 6×10 cells / mL using fresh ExpiCHO expression medium pre-warmed at 37°C. 6 Cells / mL. Dilute the plasmid constructed in Example 3.1 with OptiPROTM SFM precooled at 4°C (add 1 μg of plasmid to 1 mL of the culture medium), and dilute ExpiFectamineTM CHO with OptiPROTM SFM, then mix the two in equal volumes and gently pipette to mix to prepare ExpiFectamineTM CHO / plasmid DNA mixture, incubate at room temperature for 1-5 min, slowly add to the prepared cell suspension and gently shake at the same time, and finally place in a cell culture shaker and culture at 37°C, 8% CO2.

[0123] 18-22 h after transfection, add ExpiCHOTMEnhancer and ExpiCHOTMFeed to the culture medium, and continue to culture in a 32°C shaker with 5% CO2. On the 5th day after transfection, add the same volume of ExpiCHOTMFeed, and gently mix the cell suspension while slowly adding it. 7 days after transfection, the cell culture supernatant expressing the target protein was centrifuged at 15000 g for 10 min, and the resulting supernatant was affinity purified using MabSelect SuRe LX (GE, 17547403), and then the target protein was eluted with 100 mM sodium acetate (pH3.0), followed by neutralization with 1 M Tris-HCl, and finally the resulting protein was exchanged into PBS buffer through an ultrafiltration concentration tube (Millipore, UFC901096).

[0124] Example 6: Identification of candidate antibody physicochemical properties

[0125] 6.1 SDS-PAGE identification of candidate antibodies

[0126] Preparation of non-reducing solution: 1 µg of the candidate antibody in Example 3 and the quality control product IPI (i.e., Ipilimumab) were added to 5× SDS loading buffer and 40 mM iodoacetamide, heated in a 75°C dry bath for 10 min, cooled to room temperature, and centrifuged at 12,000 rpm for 5 min to obtain the supernatant.

[0127] Preparation of reducing solution: 2 µg of the candidate antibody in Example 3 and the quality control product IPI were added with 5×SDS loading buffer and 5 mM DTT, heated in a 100°C dry bath for 10 min, cooled to room temperature, and centrifuged at 12,000 rpm for 5 min to obtain the supernatant.

[0128] The supernatant was added to Bis-tris 4-15% gradient gel (GenScript Biotech Co., Ltd.) for gel electrophoresis and the protein bands were visualized by Coomassie Brilliant Blue staining. The protein gel with the visualized protein bands was scanned using an EPSON V550 color scanner (decolorization with destaining solution until the gel background was transparent), and the purity of the reduced and non-reduced bands was calculated using ImageJ according to the peak area normalization method.

[0129] The results are shown in Table 3. The band of the candidate antibody in non-reducing gel is around 104 kD, and the band of reducing gel is around 60 kD, which is consistent with the expected size. The purity of the antibody molecule can be detected by reducing gel, and the purity of the antibody is greater than 93%.

[0130] Table 3. Physical and chemical test results of candidate antibodies

[0131]

[0132] Example 7: Affinity detection of candidate antibodies at the ELISA level

[0133] In this example, the binding of expressed candidate antibodies (whole human library, mouse immune library) to A35R-ECD-His antigen protein was detected based on the ELISA method.

[0134] 7.1 ELISA-based detection of the binding ability of candidate antibodies in the human library to A35R-ECD-His

[0135] 96-well ELISA plates (30 μL / well) were coated with 2 μg / mL A35R-ECD-His at 4°C overnight. The next day, the plates were washed 3 times with PBST and blocked with 5% skim milk for 2 h. After washing the plates 3 times with PBST, the antibodies with gradient dilutions (3.0, 0.33, 0.11, 0.037, 0.012, 0.004, 0.0014, 0.0002 μg / mL) and the positive control antibody Anti-Monkeypox virus / MPXV A35R Antibody (SAA0287) were added and incubated for 1 h. After that, the plates were washed 3 times with PBST and the secondary antibody Goat-Anti-human Fc-HRP (abcam, ab97225) was added and incubated for 1 h. After incubation, the plate was washed 6 times with PBST and TMB (SurModics, TMBS-1000-01) was added for color development. According to the color development results, 2 M stop solution was added to terminate the reaction, and the absorbance was read at OD450 using a microplate reader (Molecular Devices, SpecterMax 190).

[0136] The results are as follows Figure 2 As shown in Table 4, the antibody molecule NL-A35R-A253 has strong binding activity with the antigen protein A35R-ECD-His. 50 Range: 0.00490 μg / mL.

[0137] Table 4. Binding data of candidate antibodies in the full human library to A35R-ECD-His

[0138]

[0139] 7.2 ELISA-based detection of the binding ability of candidate antibodies from the mouse immune library to A35R-ECD-His

[0140] 96-well ELISA plates (30 μL / well) were coated with 2 μg / mL A35R-ECD-His at 4°C overnight. The next day, the plates were washed 3 times with PBST and blocked with 5% skim milk for 2 h. After washing the plates 3 times with PBST, the antibodies with gradient dilutions (3.0, 0.33, 0.11, 0.037, 0.012, 0.004, 0.0014, 0.0002 μg / mL) and the positive control antibody A35R_N_PR_A145 (self-produced) were added and incubated for 1 h. After that, the plates were washed 3 times with PBST and the secondary antibody Goat-Anti-human Fc-HRP (abcam, ab97225) was added and incubated for 1 h. After the incubation, the plates were washed 6 times with PBST and TMB (SurModics, TMBS-1000-01) was added for color development. According to the color development results, 2 M stop solution was added to terminate the reaction, and the OD 450 Read the absorbance at

[0141] The results are as follows Figure 3 As shown in Table 5, all four antibody molecules have strong binding activity with the antigen protein A35R-ECD-His.

[0142] Table 5. Binding data of mouse immunized candidate antibodies to A35R-ECD-His

[0143]

[0144] Example 8: ELISA double antibody sandwich method antibody pairing

[0145] In this example, the antibodies against the A35R-ECD-His antigen protein were paired based on the ELISA double antibody sandwich method.

[0146] 96-well ELISA plates (30 μL / well) were coated with 2 μg / mL antibody at 4°C overnight. The next day, the plates were washed 3 times with PBST and blocked with 5% skim milk for 2 h. After washing the plates 3 times with PBST, 2 μg / mL A35R-ECD-His was added and incubated for 1 h. After washing the plates 3 times with PBST, a gradient dilution (10, 3.33333, 1.11111, 0.37037, 0.12346, 0.04115, 0.01372, 0.00457 μg / mL) of biotinylated detection antibody was added and incubated for 1 h. After that, the plates were washed 3 times with PBST and the secondary antibody NeutrAvidin-HRP (Thermo, 31001) was added and incubated for 1 h. After incubation, the plate was washed 6 times with PBST and TMB (SurModics, TMBS-1000-01) was added for color development. According to the color development results, 2 M stop solution was added to terminate the reaction, and the absorbance was read at OD450 using a microplate reader (Molecular Devices, SpecterMax 190).

[0147] The results are as follows Figure 4~Figure 6 As shown: the four pairs of paired antibodies A35R_M_PR_A064 and A35R_M_PR_A142, A35R_M_PR_A094 and A35R_M_PR_A142, A35R_M_PR_A094 and A35R_M_PR_A064, A35R_M_PR_A142 and A35R_M_PR_A064 are more effective in detecting A35R-ECD-His antigen. Figure 4 The antibody A35R_M_PR_A064 was coated on the ELISA plate, and the liquid phase was biotin-labeled with different candidate antibodies. It can be seen from the figure that when A35R_M_PR_A064 was coated on the ELISA plate and the liquid phase was biotin-labeled with A35R_M_PR_A142, the detection effect was better, and its EC50 was 0.1320 μg / mL. Figure 5 The antibody A35R_M_PR_A094 was coated on the ELISA plate, and the liquid phase was biotin-labeled with different candidate antibodies. It can be seen from the figure that when A35R_M_PR_A094 was coated on the ELISA plate and the liquid phase was biotin-labeled with A35R_M_PR_A142 and A35R_M_PR_A064, the detection effect was better, and their EC50 were 0.1407 μg / mL and 0.2991 μg / mL, respectively. Figure 6The antibody A35R_M_PR_A142 was coated on the ELISA plate, and the liquid phase was biotin-labeled with different candidate antibodies. It can be seen from the figure that when A35R_M_PR_A142 was coated on the ELISA plate and the liquid phase was biotin-labeled with A35R_M_PR_A064, the detection effect was better, and its EC50 was 0.2622 μg / mL.

[0148] The exemplary embodiments of the present invention are described above, and it should be understood by those skilled in the art that these disclosures are only exemplary, and various other substitutions, adaptations and modifications can be made within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments listed herein.

[0149] Table 6 Sequence Listing

[0150]

[0151]

[0152] Table 7

[0153]

Claims

1. An antibody or an antigen-binding fragment thereof targeting monkeypox virus, the antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; the light chain variable region comprising LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; or, The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively; or, The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively; or, The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively; or, The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 7, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 8, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO: 8; or, The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO: 15, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 16; or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO: 16; or, The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 23, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO: 23, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 24, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO: 24; or The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 31, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO: 31, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 32, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO: 32; or, The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 39, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 39; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 40, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:

40.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein: The antibody is a full-length antibody, Fab, Fab', F(ab')2, Fv or a multispecific antibody; Preferably, when the antibody is a full-length antibody, the heavy chain constant region of the full-length antibody is derived from the heavy chain of a human antibody or a variant thereof, and the light chain constant region of the full-length antibody is derived from the κ chain or λ chain of a human antibody or a variant thereof; When the antibody is an Fv, the Fv is a scFv; More preferably, when the antibody is a full-length antibody, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO: 41, and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO: 43 or SEQ ID NO: 44; when the antibody is Fv, the antibody further comprises an Fc region, and the amino acid sequence of the Fc region is preferably as shown in SEQ ID NO: 42, SEQ ID NO: 45 or SEQ ID NO:

46.

4. An antibody combination, characterized in that The antibody combination comprises one or more antibodies or antigen-binding fragments thereof according to any one of claims 1 to 3; preferably, the antibody combination comprises a first antibody and a second antibody; more preferably: The heavy chain variable region of the first antibody comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively; the heavy chain variable region of the second antibody comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; or, The heavy chain variable region of the first antibody comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively; the heavy chain variable region of the second antibody comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; or, The heavy chain variable region of the first antibody comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively; the heavy chain variable region of the second antibody comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively; or, The heavy chain variable region of the first antibody comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; the heavy chain variable region of the second antibody comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively.

5. An isolated nucleic acid, characterized in that The nucleic acid encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the antibody combination according to claim 4.

6. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid according to claim 5; Preferably, the recombinant expression vector is a plasmid, cosmid, phage or viral vector; More preferably, the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector or an adeno-associated viral vector.

7. A transformant, characterized in that: The transformant comprises the recombinant expression vector according to claim 6 in a host cell; Preferably, the host cell is a prokaryotic cell or a eukaryotic cell; More preferably, the host cell is a cell suitable for preparing an antibody or an antigen-binding fragment thereof, for example, selected from yeast cells and mammalian cells; the mammalian cell is, for example, a HEK293 cell.

8. A method for preparing an antibody or an antigen-binding fragment thereof targeting monkeypox virus, characterized in that: The method comprises culturing the transformant according to claim 7, and obtaining an antibody or an antigen-binding fragment thereof targeting monkeypox virus from the culture.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the antibody combination according to claim 4, and a pharmaceutically acceptable carrier.

10. A kit, characterized in that: The kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the antibody combination according to claim 4, the nucleic acid according to claim 5, the recombinant expression vector according to claim 6, the transformant according to claim 7, or the pharmaceutical composition according to claim 9; Preferably, the kit further comprises a reagent for detecting the binding of the antibody or antigen-binding fragment thereof to monkeypox virus.

11. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the antibody combination according to claim 4, the nucleic acid according to claim 5, the recombinant expression vector according to claim 6, the transformant according to claim 7, the pharmaceutical composition according to claim 9 or the kit according to claim 10 in the preparation of a drug for diagnosing, preventing and / or treating viral infection; Preferably, the viral infection is a poxvirus infection; More preferably, the poxvirus infection is monkeypox virus infection.

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