Antibody targeting monkeypox virus, antigen-binding fragment thereof, and use thereof

Abstract

Disclosed are an antibody targeting the monkeypox virus, an antigen-binding fragment thereof, and a use 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 of the present invention targets A35R of humans and non-human primates.

Description

An antibody targeting monkeypox virus, its antigen-binding fragment and its uses

[0001] This application claims priority to Chinese patent application 2024119060309, filed on 2024 / 12 / 23. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of antibody drugs, specifically to an antibody targeting monkeypox virus or its antigen-binding fragment and its use, particularly an antibody specifically binding to the monkeypox virus surface antigen A35R, and conjugates and fusions comprising the anti-A35R antibody or its antigen-binding fragment. Furthermore, this invention 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. This invention also relates to pharmaceutical compositions comprising the anti-A35R antibody and the medical use of the anti-A35R antibody. Background Technology

[0003] Monkeypox (MPXV) first broke out in macaques at a Danish research institution in 1958. Since 2017, more than 51,257 cases of monkeypox have been reported to the World Health Organization (WHO) by more than 99 countries and regions worldwide. On July 23, 2022, the WHO declared monkeypox a Public Health Emergency of International Concern (PHEIC).

[0004] Monkeypox is an acute, self-limiting disease with an incubation period of 5 to 21 days. The febrile phase typically lasts 1 to 3 days, with symptoms including fever, severe headache, swollen lymph nodes, back pain, muscle aches, and severe weakness. Following the febrile phase is the rash phase, which lasts 2–4 weeks. The lesions progress from spots (flat-based lesions) to papules (raised, hard, painful lesions), to vesicles (filled with clear fluid), to pustules (filled with pus), and finally to crusts. In recorded cases, the mortality rate ranges from 0% to 11%, with a higher mortality rate in young children.

[0005] Monkeypox virus (MPV) is an enveloped double-stranded DNA virus with two infectious forms: extracellular enveloped virus (EV) and mature intracellular virus (MV). MV is the most abundant and stable form in the environment, and is the main form of viral transmission between hosts. More than 20 structural proteins have been identified, including A29L, a membrane protein of MPV MV, which binds to glycosaminoglycans on the cell surface and mediates virus-cell fusion; M1R, another membrane protein of MV, is highly conserved among known poxviruses; and A35R, an envelope glycoprotein on the surface of EV, can induce the production of neutralizing antibodies, blocking viral transmission between cells. These proteins are considered important targets for MPV research. Summary of the Invention

[0006] The technical problem this invention aims to solve is to overcome the lack of antibodies with good efficacy against monkeypox virus and its variants in the prior art, and to provide an antibody or its antigen-binding fragment targeting monkeypox virus and its uses. The antibody or its antigen-binding fragment of this invention exhibits good binding activity against various variants of monkeypox virus, providing more options for the prevention and treatment of viral infections and possessing significant clinical value.

[0007] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0008] A first aspect of the present invention provides an antibody or 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 amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; the light chain variable region comprising amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; or,

[0009] The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively; the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, respectively; or,

[0010] The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, respectively; the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22, respectively; or...

[0011] The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively; the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; or,

[0012] The heavy chain variable region comprises amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35, respectively; the light chain variable region comprises amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38, respectively.

[0013] In this invention, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined using AbM.

[0014] 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 with 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 with SEQ ID NO:8.

[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: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 with 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%, or at least 99% sequence identity with SEQ ID NO:16.

[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: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 with 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 with SEQ ID NO:24.

[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: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 with 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 with SEQ ID NO:32.

[0018] 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 with 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 with SEQ ID NO:40.

[0019] In this invention, the sequence identity may be limited to the frame regions of the heavy chain variable region and the light chain variable region. The antibody or its antigen-binding fragment of this invention targets the antigen amino acid sequence, and the heavy chain variable regions and light chain variable regions that have at least 80% sequence identity with the frame regions of the reference sequences of the heavy chain variable regions and light chain variable regions corresponding to each CDR retain their binding activity with the antigen.

[0020] In this invention, the antibody may be a full-length antibody, Fab, Fab', F(ab')2, Fv, or a multispecific antibody.

[0021] 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 an Fv, the Fv is scFv.

[0022] 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.

[0023] In some 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.

[0024] A second aspect of the present invention provides an antibody combination comprising one or more antibodies or antigen-binding fragments thereof as described in the first aspect.

[0025] In some embodiments of the present invention, the antibody combination includes a first antibody and a second antibody.

[0026] In some specific embodiments of the present invention, the heavy chain variable region of the first antibody contains amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively, and the light chain variable region contains amino acid sequences LCDR1, LCDR2, and LCDR3 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 contains amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the light chain variable region contains amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0027] In some other specific embodiments of the present invention, the heavy chain variable region of the first antibody comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 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 the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0028] In some other specific embodiments of the present invention, the heavy chain variable region of the first antibody comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 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 the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, respectively.

[0029] In some other specific embodiments of the present invention, the heavy chain variable region of the first antibody comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as 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 the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, respectively.

[0030] A third aspect of the present invention provides an isolated nucleic acid that encodes an antibody or an antigen-binding fragment thereof as described in the first aspect, or a combination of antibodies as described in the second aspect.

[0031] A fourth aspect of the present invention provides a recombinant expression vector comprising the nucleic acid as described in the third aspect.

[0032] In some embodiments of the present invention, the recombinant expression vector may be conventional in the art, such as plasmids, granules, bacteriophages or viral vectors.

[0033] In some specific embodiments of the present invention, the viral vector is a retroviral vector, a lentiviral vector, an adenovirus vector, or an adeno-associated virus vector.

[0034] A fifth aspect of the present invention provides a transformant comprising, in a host cell, a recombinant expression vector as described in the fourth aspect.

[0035] In this invention, the host cell is a prokaryotic cell or a eukaryotic cell.

[0036] In some embodiments of the present invention, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparing antibodies or their antigen-binding fragments.

[0037] In some specific embodiments of the present invention, the mammalian cell is a HEK293 cell.

[0038] A sixth aspect of the present invention provides a method for preparing an antibody or antigen-binding fragment thereof targeting monkeypox virus, the method comprising culturing a transformant as described in the fifth aspect and obtaining an antibody or antigen-binding fragment thereof targeting monkeypox virus from the culture.

[0039] A seventh aspect of the present invention provides a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in the first aspect, or a combination of antibodies as described in the second aspect, and a pharmaceutically acceptable carrier.

[0040] An eighth aspect of the present invention provides a kit comprising 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, or a pharmaceutical composition as described in the seventh aspect.

[0041] In some embodiments of the present invention, the kit further includes a reagent for detecting the binding of the antibody or its antigen-binding fragment to monkeypox virus.

[0042] The ninth aspect of the present invention provides the use of 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 in the preparation of medicaments for diagnosing, preventing, and / or treating viral infections.

[0043] In some embodiments of the present invention, the viral infection is a poxvirus infection.

[0044] In some specific embodiments of the present invention, the poxvirus infection is monkeypoxvirus infection.

[0045] The tenth aspect of the present invention provides a method for diagnosing and / or treating a viral infection, comprising administering to a subject in need an effective amount of 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.

[0046] In some embodiments of the present invention, the diagnosis and / or treatment are in vivo or in vitro.

[0047] In some embodiments of the present invention, the viral infection is as described in aspect eight.

[0048] definition

[0049] As used herein, the term "complementarity-determining region" or "CDR region" or "CDR" refers to a region within the antibody variable domain that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). CDRs are primarily responsible for antigen binding and are sequentially numbered from the N-terminus as CDR1, CDR2, and CDR3. Within a given heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any of many known antibody CDR assignment systems or combinations thereof. It is well known to those skilled in the art that antibody CDRs can be defined in various ways, such as Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., Journal of Molecular Biology, 273, 927-948 (1997) based on the three-dimensional structure of the antibody and the topology of the CDR rings; Kabat et al. (1987) based on antibody sequence variability; AbM (University of Bath), Contact (University College London); the international ImMunoGeneTics database (IMGT) (imgt.cines.fr / ); and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. Those skilled in the art will understand that, unless otherwise specified, the terms “CDR” and “complementary determination region” for a given antibody or its region (e.g., variable region) should be understood to encompass the complementary determination region defined by any of the above-described known schemes as described in this invention.

[0050] Antibodies with different specificities (i.e., different binding sites against different antigens) have different core binding receptors (CDRs). However, although CDRs differ between antibodies, only a limited number of amino acid sites within a CDR are directly involved in antigen binding. Minimal overlapping regions can be determined using at least two of the Kabat, Chothia, IMGT, AbM, and Contact methods, thus providing a “minimum binding unit” for antigen binding. The minimum binding unit can be a sub-part of a CDR. As will be apparent to those skilled in the art, the residues of the remaining CDR sequence can be determined by the antibody’s structure and protein folding. Therefore, the present invention also contemplates any variants of the CDRs given herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat, Chothia, or AbM may be substituted with amino acid residues.

[0051] As used herein, “percentage (%) sequence identity” or “sequence identity” has a generally accepted definition in the art, referring to the percentage of identical amino acid sequences between two polypeptide sequences as determined by sequence alignment (e.g., by manual inspection or a known algorithm). This can be determined using methods known to those skilled in the art, such as publicly available computer software like BLAST, BLAST-2, Clustal Omega, and FASTA software.

[0052] In this invention, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0053] As used herein, the term "multispecific antibody" refers to an antibody that can specifically bind to two or more (e.g., 2, 3, 4, 5, or 6) different antigenic epitopes. Multispecific antibodies can be, for example, bispecific, trispecific, or tetraspecific antibodies, which can specifically bind to 2, 3, or 4 antigenic epitopes, respectively. As used herein, the term "antigenic epitope" or "antigenic determinant" refers to a region of an antigen that specifically binds to an antibody's antigen-binding site.

[0054] As used in this article, the term "isolated" refers to substances obtained artificially from their natural state. If an "isolated" substance or component appears in nature, it may be due to an alteration of its natural environment, the isolation of the substance from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide isolated 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 impurities that do not affect the substance's activity.

[0055] As used in this invention, "vector" refers to a construct capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing said 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, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0056] As used in this article, the term "host cell" refers to cells that can be used to introduce the 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 cells, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

[0057] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0058] The positive and progressive effects of this invention are as follows:

[0059] The antibody or its antigen-binding fragment of the present invention targets human and non-human primate A35R. The anti-A35R antibody or its antigen-binding fragment has increased or decreased affinity, which can improve efficacy or reduce toxicity. When forming bispecific antibodies, selective adaptation can be performed according to different targets, providing a flexible adaptation scheme for drug development. Attached Figure Description

[0060] Figure 1 shows the ELISA results of antigen A35R.

[0061] Figure 2 shows the ELISA results of binding of the fully human library antibody to antigen A35R.

[0062] Figure 3 shows the ELISA results of the binding of mouse immune repositories antibodies to antigen A35R.

[0063] Figures 4, 5, and 6 show the antibody pairing detection results. Detailed Implementation

[0064] The invention generally described herein will be more readily understood by referring to the following embodiments, which are provided by way of example and are not intended to limit the scope of the invention. These embodiments are not intended to represent all or only the experiments conducted below.

[0065] For experimental methods in the following examples where specific conditions are not specified, follow conventional methods and conditions, or select according to the product instructions.

[0066] Example 1: Raw material preparation and identification

[0067] 1.1 Preparation and Identification of Antigen Proteins

[0068] Antigen protein preparation: An mFc (SEQ ID NO:46) or His tag was added to the C-terminus of the extracellular domain (A35R-ECD, see: Uniprot ID Q8V4U4 AA:58-181) of the human A35R protein via genetic manipulation at the gene level. The obtained nucleic acid sequence was constructed into the GSV0 vector, then transformed into *E. coli* DH5α, and cultured overnight at 37°C. Plasmids were then extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01). The obtained plasmids were analyzed using ExpiFectamine. TM 293 Transfection Kit (Gibco) TM A14524) was transiently transfected into HEK293 cells. CRL-1573 TM Five days after expression, the cell culture supernatant was collected. Proteins containing the Fc tag were purified by affinity chromatography using a Protein A / G column. After purification, the target protein was eluted with 100 mM glycine (pH 3.0), concentrated, and the buffer was replaced. Proteins containing the His tag were purified by affinity chromatography using a Ni Smart Beads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd., SA036050), followed by elution with an imidazole gradient. The eluted proteins were then transferred to PBS buffer via ultrafiltration concentrators (Millipore, UFC901096). Finally, A35R antigen proteins (A35R-ECD-mFc and A35R-ECD-His) with mFc and His tags, respectively, were obtained.

[0069] 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: Coat the ELISA plate with 2 μg / mL A35R-ECD-mFc and A35R-ECD-His and incubate overnight at 4°C; after washing the plate 3 times, block it with 5% skim milk prepared with PBS at room temperature for 1 hour; after washing the plate 3 times, add the antibody Anti-Monkeypox virus / MPXV A35R Antibody (SAA0287) serially diluted with 1% PBSM and incubate at room temperature for 1 hour; after washing the plate, add the secondary antibody Goat-Anti-mouse-IgG-Fc-HRP (abcam; ab97265) and Anti-mouse-Fab-HRP (sigma; M4155-1mL) diluted with 1% PBSM (1:8000) and incubate at room temperature for 1 hour, wash the plate 6 times, then add TMB for color development for 5-20 minutes, terminate the color development reaction, and read the OD using an ELISA reader. 450 Data were processed and plotted using GraphPad Prism. The results are shown in Figure 1. The Anti-Monkeypox virus / MPXV A35R Antibody (SAA0287) binds well to the antigens A35R-ECD-mFc and A35R-ECD-His expressed in Example 1.1, exhibiting EC50-like activity. 50 The antigen-binding activity was 0.007442 μg / mL.

[0070] Example 2: Construction and identification of overexpression cell lines

[0071] 2.1. Construction and identification of A35R-FL-EGFP-HEK293 overexpression cell lines

[0072] The encoding 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). The resulting plasmid was then electroporated into HEK293 cells using an Invitrogen, Neon™ Transfection System, MP922947. CRL-1573 TMAfter electroporation, the resulting cells were transferred to DMEM medium (Gibco, 11995065) containing 10% FBS (Gibco, 15140-141) and free of antibiotics. The cells were then cultured in 10×10 cm cell culture dishes for 48 hours. Following this, cells were seeded into 96-well cell culture plates at an average density of 0.51E+4 cells / well, and puromycin was added to a final concentration of 2 μg / mL as a selection pressure. Cell lines that formed clones were picked for identification after approximately two weeks.

[0073] Flow cytometry identification of A35R-FL-EGFP-HEK293 overexpressing cells: Cells of the above-mentioned cell line in logarithmic growth phase were digested and plated into 96-well plates. After washing with FACS buffer (1×PBS buffer containing 2% FBS), the cells were incubated at 4°C for 30 min with serially diluted commercial primary antibody (Anti-Monkeypox virus / MPXV A35R Antibody (SAA0287)) at 4°C. After washing, the prepared fluorescent secondary antibody Anti human IgG 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 results showed that an A35R-FL-EGFP-HEK293 cell line with high expression of human A35R on the cell surface was obtained.

[0074] Example 3: Construction and screening of a fully human natural antibody phage display library

[0075] In this embodiment, a phage display antibody gene library was constructed, and the library was screened using A35R-ECD-His as the screening antigen to obtain multiple antibody molecules that specifically bind to the A35R-ECD-His protein.

[0076] 3.1 Construction of a gene library of human antibodies

[0077] 15 mL of Ficoll-Paque density gradient separation solution (purchased from GE, catalog number: 17144003S) was slowly added to a 50 mL centrifuge tube. The centrifuge tube was tilted, and 15 mL of collected normal human blood was slowly added in batches along the tube wall, ensuring a clear separation interface between the Ficoll-Paque density gradient separation solution and the normal human blood. The 50 mL centrifuge tube containing the blood and separation solution was centrifuged at approximately 15°C for 20 minutes, with the centrifuge set to 400 g, acceleration of 3, and deceleration of 0. After centrifugation, the liquid surface separated into four layers: an upper layer of plasma mixture, a lower layer of erythrocytes and granulocytes, a middle layer of Ficoll-Paque liquid, and a narrow band of white, cloudy layer dominated by PBMCs (PBMC cell layer) at the boundary between the upper and middle layers. The upper plasma mixture was carefully aspirated using a sterile Pasteur pipette, and then the PBMCs were aspirated using a new sterile Pasteur pipette to obtain the separated PBMCs. The isolated PBMCs were first rinsed twice with PBS, then centrifuged at 1500 rpm for 10 min at 4 °C, and finally resuspended in 1.5 mL of PBS and counted using a cell counter (CountStar, CountStar Altair).

[0078] Total RNA was extracted from PBMCs isolated using standard methods. The extracted total RNA was reverse transcribed into cDNA using a reverse transcription kit (TaKaRa, catalog number: 6210A). Based on the sequence similarity of the heavy and light chain germline genes, degenerate primers were designed at the front end of the V region and the back end of the first constant region of the heavy and light chains, respectively (Li Xiaolin, Construction and Preliminary Screening of a Large-Capacity Non-Immune Human Fab Phage Antibody Library, Master's Thesis, Peking Union Medical College). PCR was performed to obtain the variable region gene fragments of the antibody's heavy and light chains. After recovering the variable region gene fragments of the antibody's heavy and light chains, fusion PCR was used to amplify fragments containing the variable regions of both the heavy and light chains. Subsequently, the PCR product and the phage display vector were digested with enzymes, recovered, and ligated. The ligation product was recovered using a recovery kit (Omega, catalog number: D6492-02). Specific materials and methods are described in Li Xiaolin's thesis above. Finally, the bacteria were transformed into competent Escherichia coli SS320 (Lucigen, MC1061 F) using an electroporator (Bio-Rad, MicroPulser), and the transformed E. coli SS320 culture was spread on ampicillin-resistant 2-YT solid plates (the solid plates were prepared by mixing 1.5% tryptone, 1% yeast extract, 0.5% NaCl, and 1.5% agar, at a mass-volume ratio of g / mL).

[0079] 3.2 Calculation of antibody gene library capacity

[0080] Transformed *E. coli* SS320 bacterial suspension was inoculated with antibiotic-free 2YT medium at a volume ratio of 1:50 and incubated at 37°C and 220 rpm for 1.5-2 hours until the OD600 reached 0.5-0.6, then cooled to room temperature. The bacterial suspension was added to 90 μL / well of a 96-well round-bottom dilution plate, with each sample serially diluted 10-fold, for a total of 12 dilutions. Using an 8-channel 10 μL pipette, 2 μL of the diluted sample was added to the wells in ascending order of dilution. + / T + The culture medium was placed on plates (carbenicillin and tetracycline antibiotic concentrations of 50 μg / mL and 50 μg / mL, respectively), incubated upright for 5 min, then inverted and incubated overnight at 37°C. The next day, clonal growth was observed, and library volume was calculated. The library volume was calculated as follows: starting from row A, rows were sequentially labeled 1, 2, 3, 4, 5, 6, 7, 8 to X. First, counting wells were selected, choosing those with 3-20 clones to determine the row number X. The number of clones n in each well was then counted using the formula 5 × 100 × 10⁻⁶. X ×n, through calculation, the total storage capacity is 2.61 × 10⁻⁶. 11 CFU, i.e., 2.61 × 10 11 One antibody gene.

[0081] 3.3 Preparation of antibody gene phage display library

[0082] Based on the antibody gene library capacity, 50 ODs were extracted (1 OD is 5 × 10⁻⁶). 8 The bacterial culture of a fully human phage library (CFU) was added to fresh 2-YT liquid medium to bring the initial OD value to 0.1. The medium was incubated at 37°C and 220 rpm in a shaker until the logarithmic growth phase (OD600 = approximately 0.6). Then, VSCM13 helper phage (purchased from Stratagene) was added at 50 times the bacterial count (i.e., the multiple of infection (MOI) was approximately 50), thoroughly mixed, and allowed to stand for 30 min before being incubated for another hour at 220 rpm in a shaker. Subsequently, the culture was centrifuged at 10,000 rpm for 5 min, the supernatant was discarded, and the culture medium was replaced with 2-YT medium containing carbenicillin 50 μg / mL / kanamycin 40 μg / mL (hereinafter also referred to as CFU). + / K +The culture was prepared in 2-YT medium and incubated overnight at 30°C and 220 rpm. The next day, the bacterial culture was centrifuged at 13000g for 10 min, and the supernatant was collected. 20% PEG / NaCl (prepared from 20% PEG6000 and 2.5M NaCl) was added to make the final PEG / NaCl concentration 4%. The mixture was mixed and placed on ice for 1 hour. Then, it was centrifuged at 13000g for 10 min. The precipitated phages were rinsed with PBS and stored for subsequent phage screening.

[0083] 3.4 Screening of antibody gene phage display libraries

[0084] 3.4.1 Screening of phage display antibody gene libraries using the immunotube method

[0085] Both the immunotube method and the magnetic bead method aim to enrich specific antibodies against antigens, and are two complementary experimental methods.

[0086] The principle of immunotube screening is to coat the surface of the immunotube with A35R-ECD-His, which has a high adsorption capacity. The phage display antibody library is added to the immunotube and incubated with the antigen protein adsorbed on the surface of the immunotube. The screening process involves incubation, washing and elution. After three rounds of screening, the specific monoclonal antibodies against the antigen are finally enriched.

[0087] The specific implementation method is as follows:

[0088] In the first round of screening, 1 mL of 100 μg / mL A35R-ECD-His was added to the immunoassay tube, and the tube was coated overnight at 4°C. The coating solution was discarded the next day, and the tube was blocked with 5% milk PBS for 2 hours. After washing twice with PBS, a total of 1.20 × 10⁻⁶ ppm was added. 13 Phages from a fully human antibody library were incubated for 2 hours. The cells were then washed twice with PBS and six times with PBST to remove non-specifically bound phages. 0.8 mL of 0.05% EDTA trypsin digest was added to the immunotherapy tube to elute phages specifically bound to the target antigen. The tubes were then used to infect logarithmic-phase SS320 cells (Lucigen, 60512-1), incubated at 37°C for 30 minutes, and then cultured at 220 rpm for 1 hour. VSCM13 helper phages were added, incubated for 30 minutes, and then cultured at 220 rpm for another 1 hour. The tubes were then centrifuged and the culture was transferred to a C14 container. + / K + In 2-YT medium, the phages were cultured overnight at 30°C and 220 rpm. The next day, the phages were precipitated and used for the next three rounds of screening. The antigen coating concentrations used in the second and third rounds were typically decreased sequentially to 30 μg / mL and 10 μg / mL, respectively. In addition, the PBST rinsing intensity was gradually increased, with 10 and 14 PBST elutions, respectively.

[0089] The enrichment effect was evaluated by ELISA detection of the phage pools eluted in each round. The results showed that the sequence enrichment was obvious after the second round of screening. Therefore, the second and third rounds were selected for ELISA screening of positive clones.

[0090] 3.5 Selection of Monoclonal Cells

[0091] After three rounds of screening, the second and third rounds were used for ELISA screening of positive clones. Ultimately, 337 positive clones capable of binding to the A35R-ECD-His protein were selected from 360 clones. After sequencing analysis, the full-length sequences of 43 clones were finally constructed for further experiments.

[0092] The specific implementation method is as follows:

[0093] Positive clone sequencing and analysis

[0094] After initial screening, positive clones were numbered, and 2 μL of bacterial culture was added to 2 mL of 2YT medium and incubated overnight at 37°C and 220 rpm. Plasmids were then extracted for next-generation sequencing. The sequencing results were used to integrate, align, and remove non-antibody gene sequences from the original AB1 file using SeqMan, generating an antibody gene-integrated FASTA file. Subsequently, the DNA sequence was translated into amino acid sequences using MEGA6, and the presence of terminators and non-standard sequences was identified through the amino acid sequences, leading to the export of the amino acid sequence FASTA file.

[0095] Cloning NL-A35R-A253 is the preferred molecule. The CDR amino acid sequence of the obtained antibody Fab is shown in Tables 6 and 7. The CDR sequence was determined using the AbM definition method.

[0096] Example 4: Mouse immunization and immune library construction and screening

[0097] 4.1 Immunization regimen

[0098] Two Balb / C and two C57 mice were cross-immunized with A35R-ECD-His antigen via subcutaneous and intraperitoneal injection. The mice (Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) were immunized every two weeks. A total of four immunizations were administered. One week after the fourth immunization, blood was collected from the mice for immunotiter testing. Finally, a booster immunization with A35R-ECD-mFc was given.

[0099] 4.2 Detection of serum antibody titers in mice after immunization

[0100] ELISA plates were coated with 2 μg / mL A35R-ECD-His solution and incubated overnight at 4°C (30 μL / well). After washing three times, the plates were blocked with 5% skim milk (5% PBSM) prepared in PBS at room temperature for 1 hour. After washing three times, mouse serum serially diluted with 5% PBSM was added, along with the antibody Anti-Monkeypox virus / MPXV A35R Antibody (SAA0287) as a positive control, and incubated at room temperature for 1 hour. After washing six times, the secondary antibody Goat-anti-mouse-IgG (1+2a+2b+3)-HRP (Jackson, 115-035-164) diluted in 5% PBSM was added and incubated at room temperature for 1 hour. After washing six times, TMB was added for color development for 5-20 min. After stopping the color development reaction, the data were read using an OD450 microplate reader. The results are shown in Tables 1 and 2. The serum titers of all eight mice reached the target.

[0101] Table 1: Final Immunization Serum Titer Detection (ELISA)

[0102] Table 2: Final Immunization Serum Titer Detection (ELISA)

[0103] 4.3 Construction of Phage Display Antibody Gene Library

[0104] After immunization, the spleen of the mouse was harvested, and the spleen cells were collected after grinding and filtration. 1 mL of TRIzol was added. TM Reagent (Thermo Fisher, 15596026) lysed spleen cells, and total RNA was extracted using the phenol-chloroform method. The extracted RNA was then reverse transcribed into cDNA using a reverse transcription kit (TaKaRa, 6210A). Subsequently, using the cDNA as a PCR template, specific primers for the mouse antibody sequence were used to amplify the variable regions of the antibody's light and heavy chains. The PCR product was digested with NcoI and NotI to obtain the antibody gene fragment, which was then inserted into a phage display vector and ligated using T4 ligase. The ligation product was recovered using a DNA recovery kit (Omega, D6492-02) and finally transformed into competent Escherichia coli SS320 cells (Lucigen, MC1061F) using an electroporator (Bio-Rad, MicroPulser). The electroporated bacteria were plated on 2-YT (C+ / K+2-YT) solid plates containing ampicillin and tetracycline to amplify the correctly transformed SS320 cells with the antibody plasmid. The cells were then packaged using VSCM13 helper phage (purchased from Stratagene) to obtain a phage display library containing the Fab sequence.

[0105] 4.4 Cellular screening of phage display antibody gene libraries

[0106] A35R-FL-EGFP-HEK293 cells were cultured in T25 culture flasks. When the cell density approached 90%, the culture supernatant was removed, and the cells were washed once with PBS (source culture, B310KJ). Then, 2 mL of 4% paraformaldehyde (Sangon Biotech, E672002-0500) was added for fixation for 0.5 hours. Finally, the cells were washed twice with PBS and used as screening raw materials. During screening, the phage display library was incubated with immobilized A35R-FL-EGFP-HEK293 cells at room temperature for 1 hour. After washing three times with 1×PBS, 2 mL of glycine-HCl (pH=2.0) was added and gently mixed for 10 minutes to elute phages that specifically bind to human ROR1. The eluted supernatant was then used to infect logarithmic-phase SS320 cells (Lucigen, 60512-1), incubated for 30 minutes, and then cultured at 37°C and 220 rpm for 1 hour. VSCM13 helper phage was then added, incubated for 30 minutes, and cultured again at 37°C and 220 rpm for 1 hour. The cells were then centrifuged and transferred to C+ / K+2-YT medium. The resulting phages were used for a second round of screening. Screening was repeated multiple times, with 10 clones randomly selected in each round for sequence analysis to evaluate the library. After three rounds of screening, significant sequence enrichment was observed in the library.

[0107] 4.5 Screening of phage display antibody gene libraries using the immunotube method

[0108] Specific antibodies against antigens were enriched using both the immunotube method and the magnetic bead method, with the two methods complementing and validating each other.

[0109] Immunotube screening involves coating the antigen protein A35R-ECD-His or A35R-ECD-mFc onto the surface of an immunotube with high adsorption capacity. A phage display antibody library is added to the immunotube, and the protein adsorbed on the immunotube surface is incubated, washed, and eluted. This process involves 2-4 rounds of panning to ultimately enrich Fab monoclonal antibodies specific to the antigen. In this example, after 3 rounds of panning, Fab monoclonal antibodies against A35R-ECD-His were enriched. The specific method is described in Example 2.4.2 of patent CN112250763B.

[0110] The enrichment effect was evaluated by ELISA detection of the phage pools eluted in each round. The results showed that the sequence enrichment was obvious after the second round of screening. Therefore, the second and third rounds were selected for ELISA screening of positive clones.

[0111] 4.6 Selection of Monoclonal Cells

[0112] After three rounds of screening, the second and third rounds were used for ELISA screening of positive clones. Ultimately, 167 positive clones capable of binding to the A35R-ECD-His protein were selected from 296 clones. After sequencing analysis, the full-length sequences of 28 clones were finally constructed for further experiments.

[0113] The specific implementation method is as follows:

[0114] Positive clone sequencing and analysis

[0115] After initial screening, positive clones were numbered, and 2 μL of bacterial culture was added to 2 mL of 2YT medium and incubated overnight at 37°C and 220 rpm. Plasmids were then extracted for next-generation sequencing. The sequencing results were used to integrate, align, and remove non-antibody gene sequences from the original AB1 file using SeqMan, generating an antibody gene-integrated FASTA file. Subsequently, the DNA sequence was translated into amino acid sequences using MEGA6, and the presence of terminators and non-standard sequences was identified through the amino acid sequences, leading to the export of the amino acid sequence FASTA file.

[0116] 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 list and sequence listing. The CDR sequence was determined using the AbM definition method.

[0117] Example 5: Construction, expression, and purification of candidate antibodies

[0118] 5.1 Plasmid Construction

[0119] The VH coding sequence in the Fab sequence of the screened monoclonal antibody was ligated 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. The VL coding sequence in the Fab sequence was ligated 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 heavy and light chain coding sequences of the antibody were inserted into the eukaryotic expression vector plasmid GSV0, respectively, and transformed into *E. coli* DH5α, cultured overnight at 37°C. Endotoxin-free plasmid extraction was performed using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free antibody plasmids for eukaryotic expression.

[0120] 5.2 Expression and purification of candidate antibodies

[0121] The candidate antibody was expressed using the ExpiCHO transient transfection system (Thermo Fisher, A29133), as follows: On the day of transfection, the cell density was confirmed to be 7 × 10⁶ cells / year. 6 Up to 1×10 7 With approximately 100 viable cells / mL and a cell viability >98%, the cells were adjusted to a final concentration of 6 × 10⁶ cells / mL using fresh ExpiCHO expression medium pre-warmed to 37°C. 6 Cells / mL. Dilute the plasmid constructed in Example 3.1 with OptiPRO™ SFM pre-cooled to 4°C (add 1 μg of plasmid to 1 mL of the culture medium described above), and simultaneously dilute ExpiFectamine™ CHO with OptiPRO™ SFM. Mix the two in equal volumes and gently pipette to prepare the ExpiFectamine™ CHO / plasmid DNA mixture. Incubate at room temperature for 1-5 min, slowly add the mixture to the prepared cell suspension while gently shaking, and finally place it in a cell culture shaker and culture at 37°C and 8% CO2.

[0122] 18-22 h post-transfection, ExpiCHOTMEnhancer and ExpiCHOTMFeed were added to the culture medium, and the shake flasks were incubated at 32°C on a shaker with 5% CO2. On day 5 post-transfection, the same volume of ExpiCHOTMFeed was added slowly while gently mixing the cell suspension. Seven days post-transfection, the cell culture supernatant expressing the target protein was centrifuged at 15000g for 10 min. The supernatant was then purified by affinity using MabSelect SuRe LX (GE, 17547403), followed by elution with 100 mM sodium acetate (pH 3.0), neutralization with 1 M Tris-HCl, and finally, the protein was transferred to PBS buffer via ultrafiltration concentrator (Millipore, UFC901096).

[0123] Example 6: Identification of Physicochemical Properties of Candidate Antibodies

[0124] 6.1 SDS-PAGE identification of candidate antibodies

[0125] Preparation of non-reducing solution: 1 μg of the candidate antibody and quality control IPI (i.e., ipilimumab) from Example 3 were added to 5×SDS loading buffer and 40 mM iodoacetamide. The mixture was heated in a dry bath at 75°C for 10 min, cooled to room temperature, and centrifuged at 12,000 rpm for 5 min to collect the supernatant.

[0126] Preparation of reduction solution: 2 μg of the candidate antibody and quality control IPI from Example 3 were added to 5×SDS loading buffer and 5 mM DTT, heated in a dry bath at 100°C for 10 min, cooled to room temperature, and centrifuged at 12000 rpm for 5 min to collect the supernatant.

[0127] The supernatant was added to a Bis-tris 4-15% gradient gel (GenScript Biotechnology Co., Ltd.) for gel electrophoresis and the protein bands were stained with Coomassie Brilliant Blue. The protein gels with stained protein bands were scanned using an EPSON V550 color scanner (after destaining with destaining solution until the gel background was transparent). The purity of reduced and non-reduced bands was calculated using ImageJ according to the peak area normalization method.

[0128] The results are shown in Table 3. The candidate antibody bands on the non-reducing gel were around 104 kDa, and the bands on the reducing gel were around 60 kDa, which is consistent with the expected size. The antibody molecules could be detected by the reducing gel, and the antibody purity was greater than 93% in all cases.

[0129] Table 3. Physicochemical Detection Results of Candidate Antibodies

[0130] Example 7: Affinity detection of candidate antibodies at the ELISA level

[0131] In this embodiment, the binding of expressed candidate antibodies (whole human library and mouse immune library) to A35R-ECD-His antigen protein was detected using the ELISA method.

[0132] 7.1 ELISA-based detection of the binding affinity of candidate antibodies from a fully human library to A35R-ECD-His

[0133] 96-well ELISA plates were coated with 2 μg / mL A35R-ECD-His (30 μL / well) and incubated overnight at 4°C. The next day, the plates were washed three times with PBST and blocked with 5% skim milk for 2 h. After washing three more times with PBST, serially diluted (3.0, 0.33, 0.11, 0.037, 0.012, 0.004, 0.0014, 0.0002 μg / mL) of each antibody and the positive control antibody Anti-Monkeypox virus / MPXV A35RAntibody (SAA0287) were added and incubated for 1 h. After washing three times with PBST, 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 then developed with TMB (SurModics, TMBS-1000-01). Based on the color development results, the reaction was terminated by adding 2M stop solution, and the absorbance was read at OD450 using a microplate reader (Molecular Devices, SpecterMax 190).

[0134] The results are shown in Figure 2 and Table 4: the antibody molecule NL-A35R-A253 exhibits strong binding activity to the antigen protein A35R-ECD-His, EC 50 Range 0.00490 μg / mL.

[0135] Table 4. Binding data of fully human library candidate antibodies with A35R-ECD-His

[0136] 7.2 ELISA-based detection of the binding affinity of candidate antibodies from a mouse immune library to A35R-ECD-His

[0137] 96-well ELISA plates were coated with 2 μg / mL A35R-ECD-His (30 μL / well) and incubated overnight at 4°C. The next day, the plates were washed three times with PBST and blocked with 5% skim milk for 2 h. After washing three more times with PBST, serially diluted (3.0, 0.33, 0.11, 0.037, 0.012, 0.004, 0.0014, 0.0002 μg / mL) of each antibody and the positive control antibody A35R_N_PR_A145 (self-produced) were added and incubated for 1 h. After washing three times with PBST, the secondary antibody Goat-Anti-human Fc-HRP (abcam, ab97225) was added and incubated for 1 h. After incubation, the plates were washed six times with PBST and then developed with TMB (SurModics, TMBS-1000-01). Based on the colorimetric results, the reaction was terminated by adding 2M stop solution. The result was then measured using a microplate reader (Molecular Devices, SpecterMax 190) at OD500.450 Read the absorbance at the location.

[0138] The results are shown in Figure 3 and Table 5: all four antibody molecules showed strong binding activity to the antigen protein A35R-ECD-His.

[0139] Table 5. Binding data of mouse immune candidate antibodies with A35R-ECD-His

[0140] Example 8: Antibody pairing in ELISA double-antibody sandwich method

[0141] In this embodiment, the pairing of antibodies against the A35R-ECD-His antigen protein was detected using the ELISA double-antibody sandwich method.

[0142] 96-well ELISA plates were coated with 2 μg / mL antibody (30 μL / well) and incubated overnight at 4°C. The next day, the plates were washed three times with PBST and blocked with 5% skim milk for 2 h. After washing three more times with PBST, 2 μg / mL of A35R-ECD-His was added and incubated for 1 h. After washing three times with PBST, serially diluted (10, 3.33333, 1.11111, 0.37037, 0.12346, 0.04115, 0.01372, 0.00457 μg / mL) biotinylated detection antibody was added and incubated for 1 h. After washing three times with PBST, 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 then developed with TMB (SurModics, TMBS-1000-01). Based on the color development results, the reaction was terminated by adding 2M stop solution, and the absorbance was read at OD450 using a microplate reader (Molecular Devices, SpecterMax 190).

[0143] The results are shown in Figures 4-6: 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, and A35R_M_PR_A142 and A35R_M_PR_A064, showed superior detection of the A35R-ECD-His antigen. Figure 4 shows the antibody A35R_M_PR_A064 coated on an ELISA plate. Different candidate antibodies were used for biotin labeling in the liquid phase. As can be seen from the figure, when A35R_M_PR_A064 was coated on the ELISA plate and A35R_M_PR_A142 was used for biotin labeling in the liquid phase, the detection effect was better, with an EC50 of 0.1320 μg / mL. Figure 5 shows the application of antibody A35R_M_PR_A094 to an ELISA plate. Different candidate antibodies were used for biotin labeling in the liquid phase. The figure shows that when A35R_M_PR_A094 was coated on the ELISA plate, and the liquid phase was labeled with biotin using A35R_M_PR_A142 and A35R_M_PR_A064, the detection performance was good, with EC50 values ​​of 0.1407 μg / mL and 0.2991 μg / mL, respectively. Figure 6 shows the application of antibody A35R_M_PR_A142 to an ELISA plate. Different candidate antibodies were used for biotin labeling in the liquid phase. The figure shows that when A35R_M_PR_A142 was coated on the ELISA plate, and the liquid phase was labeled with biotin using A35R_M_PR_A064, the detection performance was good, with an EC50 of 0.2622 μg / mL.

[0144] Table 6 Sequence List

[0145] Table 7

[0146] The foregoing describes exemplary embodiments of the present invention. Those skilled in the art should understand that these disclosures are merely 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.

[0147] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

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

2. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, 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 with SEQ ID NO:7; 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 with 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%, or at least 99% sequence identity with SEQ ID NO:15; 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%, or at least 99% sequence identity with 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%, or at least 99% sequence identity with SEQ ID NO:23; 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 with 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%, or at least 99% sequence identity with SEQ ID NO:31; 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 with 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 with 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 with SEQ ID NO:

40.

3. The antibody or its antigen-binding fragment as described in claim 2, characterized in that, 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 Fv, the Fv is 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 as described in any one of claims 1-3; preferably, the antibody combination comprises a first antibody and a second antibody; more preferably: The heavy chain variable region of the first antibody contains amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively, and the light chain variable region contains amino acid sequences LCDR1, LCDR2, and LCDR3 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 contains amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the light chain variable region contains amino acid sequences LCDR1, LCDR2, and LCDR3 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 contains amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, respectively, and the light chain variable region contains amino acid sequences LCDR1, LCDR2, and LCDR3 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 contains amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the light chain variable region contains amino acid sequences LCDR1, LCDR2, and LCDR3 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 contains amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, respectively, and the light chain variable region contains amino acid sequences LCDR1, LCDR2, and LCDR3 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 contains amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively, and the light chain variable region contains amino acid sequences LCDR1, LCDR2, and LCDR3 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 contains amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the light chain variable region contains amino acid sequences LCDR1, LCDR2, and LCDR3 as 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 contains amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively, and the light chain variable region contains amino acid sequences LCDR1, LCDR2, and LCDR3 as 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 its antigen-binding fragment as described in any one of claims 1 to 3, or the antibody combination as described in claim 4.

6. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid as described in claim 5; Preferably, the recombinant expression vector is a plasmid, granule, bacteriophage, or viral vector; More preferably, the viral vector is a retroviral vector, a lentiviral vector, an adenovirus vector, or an adeno-associated virus vector.

7. A transformant, characterized in that, The transformant contains the recombinant expression vector as described in claim 6 in the 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 antibodies or their antigen-binding fragments, such as yeast cells and mammalian cells; the mammalian cell is, for example, HEK293 cells.

8. A method for preparing an antibody or antigen-binding fragment targeting monkeypox virus, characterized in that, The method includes culturing the transformant as described in claim 7, and obtaining an antibody or antigen-binding fragment targeting monkeypox virus from the culture.

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

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

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

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