CN117050165B

CN117050165B - An antibody targeting monkeypox virus, its antigen-binding fragment and its use

Info

Publication number: CN117050165B
Application number: CN202311022296.2A
Authority: CN
Inventors: 黄亮; 郎国竣; 张星辰; 闫鑫甜; 王文蓉; 胡宇豪; 尚瑞沙; 曹俊强
Original assignee: Sanyou Biopharmaceuticals Co Ltd
Current assignee: Sanyou Biopharmaceuticals Co Ltd
Priority date: 2023-08-14
Filing date: 2023-08-14
Publication date: 2025-03-25
Anticipated expiration: 2043-08-14
Also published as: CN117050165A

Abstract

本发明公开了一种靶向猴痘病毒的抗体、其抗原结合片段及其用途。所述抗体或其抗原结合片段包含重链可变区和轻链可变区或包含重链可变区但不包含轻链可变区。本发明的抗体或其抗原结合片段靶向人类和非人灵长类M1R，所述抗M1R抗体或其抗原结合片段具有提高或降低的亲和力，可以达到提高药效或降低毒性的目的，在组成双特异性抗体时可以根据靶点的不同进行选择性适配，为药物研发提供了灵活的适配方案。The present invention discloses an antibody targeting monkeypox virus, an antigen-binding fragment thereof and uses thereof. The antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region or comprises a heavy chain variable region but does not comprise a light chain variable region. The antibody or its antigen-binding fragment of the present invention targets human and non-human primate M1R, and the anti-M1R antibody or its antigen-binding fragment has an increased or decreased affinity, which can achieve the purpose of increasing drug efficacy or reducing toxicity. When forming a bispecific antibody, selective adaptation can be performed according to different targets, providing a flexible adaptation scheme for drug development.

Description

Antibody targeting monkey poxvirus, antigen binding fragment thereof and application thereof

Technical Field

The invention relates to the field of antibody medicaments, in particular to an antibody targeting a monkey pox virus, an antigen binding fragment thereof and application thereof, and particularly relates to an antibody specifically binding to a monkey pox virus surface antigen M1R, and a conjugate and a fusion containing the anti-M1R antibody or the antigen binding fragment thereof. Furthermore, the invention relates to nucleic acids encoding said anti-M1R antibodies and host cells comprising said nucleic acids, as well as methods for preparing said antibodies. The invention also relates to pharmaceutical compositions comprising said anti-M1R antibodies and to the medical use of said anti-M1R antibodies.

Background

Monkey pox (Monkeypox, MPXV) was first shown in 1958 in outbreaks of rhesus macaques raised by the institute of denmark, and over 99 countries and regions have been reported worldwide to the world-guard organization for over 51257 cases of monkey pox since 2017. At 7 and 23 2022, WHO announced that monkey pox constituted an internationally interesting global sudden Public Health Event (PHEIC).

Monkey pox is an acute self-limiting disease with a latency period of 5 to 21 days. The febrile stage of the disease usually lasts for 1 to 3 days, and symptoms include fever, severe headache, lymphadenectasis, back pain, muscle pain and severe weakness. The fever phase is followed by a eruption phase of the skin for 2-4 weeks. Lesions progress from spots (basal flat lesions) to papules (protruding hard painful lesions), to vesicles (full of clear fluid), to pustules (full of pus), and then crusting. In the recorded cases, the proportion of deaths was between 0 and 11% with higher infant deaths.

Monkey Poxvirus (MPXV) is a double stranded DNA virus of the genus orthopoxvirus (Orthopoxvirus) of the family poxviridae (Poxviridae). Two genetic clades of monkey poxvirus have been characterized as western africa (mortality 10%) and congo basin (mortality 3.6%). The virus produces two different forms of virus particles with infection capability in an infected host, namely extracellular enveloped virus EEV (Extracellular Enveloped Virion) with a double-layer membrane structure and intracellular mature virus particle IMV (Intracellular Enveloped Virion) with a single-layer membrane structure. Members of the family Poxviridae (the largest animal virus currently known) include Variola virus (VARV, smallpox virus), cowpox virus (CPV, vaccinia virus) and Vaccinia virus (VACV, vaccinia virus). The life cycle of monkey poxviruses involves adsorption, membrane fusion, capsid removal, DNA replication, transcription and translation, assembly, release, etc., depending on the co-action of multiple proteins. Antigens that have been reported to produce neutralizing antibodies include B6R and A35R on the EEV surface, and M1R, A L and A30L on the IMV surface. Wherein, M1R binds to an unknown receptor on the surface of a host cell, is highly conserved, participates in mediating virus infection and is necessary for inducing low-pH triggered cell-cell fusion, so that the M1R can be used as a key drug target and detection index.

Currently, two prophylactic vaccines against monkey pox virus have been approved for emergency use by the FDA, the second generation VACV live vaccine ACAM2000 and the third generation Modified Vaccinia Ankara (MVA) attenuated vaccine JYNNEOS, respectively. The former shows clinically significant side effects such as myocarditis and pericarditis as a live vaccine, while the latter is vaccinated at 28 day intervals with two doses, possibly at risk of pre-exposure infection. Therapeutic drugs against monkey poxvirus infection currently have two major small molecule inhibitors, tecovirimat and Brincidofovir. The former targets VP37 membrane proteins of MPXV, preventing the formation of virus particles that can be enveloped, thus disrupting viral transmission, and the latter inhibits orthopoxvirus DNA polymerase-mediated DNA synthesis. Thus, there is a need in the art for more specific antibody drug development targeting monkey poxviruses, and for the development of high affinity neutralizing antibodies that recognize and bind to the surface membrane proteins of monkey poxviruses to effectively diagnose, prevent and treat infections of this family of poxviridae.

Disclosure of Invention

The invention aims to solve the technical problem that antibodies with good effects on monkey pox viruses and variant strains thereof are lacking in the prior art, and provides an antibody targeting the monkey pox viruses or an antigen binding fragment thereof and application thereof. The antibody or antigen binding fragment thereof has good binding activity on all variants of the monkey pox virus, provides more choices for preventing and treating virus infection, and has important clinical value.

In a first aspect the invention provides an antibody or antigen binding fragment thereof targeting M1R, said antibody comprising a heavy chain variable region and a light chain variable region or said antibody comprising a heavy chain variable region but not a light chain variable region;

When the antibody comprises a heavy chain variable region and a light chain variable region:

The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 respectively, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as 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 shown as 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 shown as 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 shown as 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 shown as 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 shown as 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 shown as 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 with amino acid sequences shown as SEQ ID NO. 33, SEQ ID NO. 34 and SEQ ID NO. 35 respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID NO. 36, SEQ ID NO. 37 and SEQ ID NO. 38 respectively;

When the antibody comprises a heavy chain variable region but does not comprise a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO. 41, SEQ ID NO. 42 and SEQ ID NO. 43, respectively.

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

In some embodiments of the invention, the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO. 7 or 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 light chain variable region has an amino acid sequence as shown in SEQ ID NO. 8 or 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.

In other embodiments of the invention, the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO. 15 or 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 light chain variable region has an amino acid sequence as shown in SEQ ID NO. 16 or 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.

In other embodiments of the invention, the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO. 23 or 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 light chain variable region has an amino acid sequence as shown in SEQ ID NO. 24 or 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.

In other embodiments of the invention, the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO. 31 or 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 light chain variable region has an amino acid sequence as shown in SEQ ID NO. 32 or 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.

In other embodiments of the invention, the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO 39 or 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 39, and the light chain variable region has an amino acid sequence as shown in SEQ ID NO 40 or 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 40.

In other embodiments of the invention, the antibody is a VHH antibody, the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO. 44 or 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. 44.

In the present invention, the antibody may be a full length antibody, fab ', F (ab') 2, fv, VHH, or multispecific antibody.

In some embodiments of the invention, the antibody is a full length antibody, the heavy chain constant region of which is derived from a heavy chain of a human antibody or a variant thereof, and the light chain constant region of which is derived from a kappa chain or a lambda chain of a human antibody or a variant thereof. In some embodiments of the invention, when the antibody is a VHH antibody, the antibody further comprises an Fc region having an amino acid sequence as shown in SEQ ID NO. 46, SEQ ID NO. 49 or SEQ ID NO. 50

In some embodiments of the invention, the amino acid sequence of the heavy chain constant region is shown as SEQ ID NO. 45, and the amino acid sequence of the light chain constant region is shown as SEQ ID NO. 47 or SEQ ID NO. 48.

In a second aspect the invention provides an antibody combination comprising one or more antibodies or antigen binding fragments thereof as described in the first aspect.

In some embodiments of the invention, the antibody combination comprises a first antibody and a second antibody.

In some embodiments of the invention, the heavy chain variable region of the first antibody comprises the amino acid sequences of HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO. 33, SEQ ID NO. 34 and SEQ ID NO. 35, respectively, the light chain variable region comprises the amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO. 36, SEQ ID NO. 37 and SEQ ID NO. 38, respectively, and the heavy chain variable region of the second antibody comprises the amino acid sequences of HCDR1, HCDR2 and HCDR3 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 of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO. 20, SEQ ID NO. 21 and SEQ ID NO. 22, respectively.

In other embodiments of the invention, the heavy chain variable region of the first antibody comprises the amino acid sequences of HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO. 33, SEQ ID NO. 34 and SEQ ID NO. 35, respectively, the light chain variable region comprises the amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO. 36, SEQ ID NO. 37 and SEQ ID NO. 38, respectively, and the second antibody comprises the heavy chain variable region but not the light chain variable region, and the heavy chain variable region comprises the amino acid sequences of HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO. 41, SEQ ID NO. 42 and SEQ ID NO. 43, respectively.

A third aspect of the invention provides an isolated nucleic acid encoding an antibody or antigen binding fragment thereof according to the first aspect, or a combination of antibodies according to the second aspect.

In a fourth aspect the present invention provides a recombinant expression vector comprising a nucleic acid as described in the third aspect.

In some embodiments of the invention, the recombinant expression vector may be a vector conventional in the art, such as a plasmid, cosmid, phage, or viral vector.

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

In a fifth aspect the present invention provides a transformant comprising a recombinant expression vector according to the fourth aspect in a host cell.

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

In some embodiments of the invention, the host cell is selected from a yeast cell, a mammalian cell, or other cell suitable for the preparation of antibodies or antigen binding fragments thereof.

In some embodiments of the invention, the mammalian cell is a HEK293 cell.

In a sixth aspect the invention provides a method of preparing an antibody or antigen-binding fragment thereof that targets M1R, the method comprising culturing a transformant according to the fifth aspect, and obtaining the antibody or antigen-binding fragment thereof that targets M1R from the culture. A seventh aspect of the invention provides a pharmaceutical composition comprising an antibody or 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.

An eighth aspect of the invention provides a kit comprising an antibody or antigen binding fragment thereof according to the first aspect, a combination of antibodies according to the second aspect, a nucleic acid according to the third aspect, a recombinant expression vector according to the fourth aspect, a transformant according to the fifth aspect, or a pharmaceutical composition according to the seventh aspect.

In some embodiments of the invention, the kit further comprises a reagent for detecting binding of the antibody or antigen binding fragment thereof to a monkey poxvirus.

The ninth aspect of the present invention provides the use of an antibody or antigen binding fragment thereof according to the first aspect, an antibody combination according to the second aspect, a nucleic acid according to the third aspect, a recombinant expression vector according to the fourth aspect, a transformant according to the fifth aspect, a pharmaceutical composition according to the seventh aspect or a kit according to the eighth aspect for the preparation of a medicament for the diagnosis, prevention and/or treatment of a viral infection.

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

In some embodiments of the invention, the poxvirus infection is a monkey poxvirus infection.

A tenth aspect of the invention provides the use of an antibody or antigen binding fragment thereof according to the first aspect, an antibody combination according to the second aspect, a nucleic acid according to the third aspect, a recombinant expression vector according to the fourth aspect, a transformant according to the fifth aspect, a pharmaceutical composition according to the seventh aspect or a kit according to the eighth aspect for the preparation of a medicament for the diagnosis, prevention and/or treatment of cancer.

An eleventh aspect of the invention provides a method of diagnosing and/or treating a viral infection comprising administering to a subject in need thereof an effective amount of an antibody or antigen binding fragment thereof according to the first aspect, a combination of antibodies according to the second aspect, a nucleic acid according to the third aspect, a recombinant expression vector according to the fourth aspect, a transformant according to the fifth aspect, a pharmaceutical composition according to the seventh aspect or a kit according to the eighth aspect.

In some embodiments of the invention, the method is in vivo or in vitro.

In some embodiments of the invention, the viral infection is as described in the ninth aspect.

Definition of the definition

The term "complementarity determining region" or "CDR" as used herein is a region of an antibody variable domain that is hypervariable in sequence and forms structurally defined loops ("hypervariable loops") and/or contains antigen-contacting residues ("antigen-contacting points"). The CDRs are mainly responsible for binding to the epitope, and include CDR1, CDR2 and CDR3 sequentially numbered from the N-terminus. In a given heavy chain variable region amino acid sequence, the exact amino acid sequence boundaries of each CDR can be determined using any one of a number of well-known antibody CDR assignment systems, or a combination thereof. It is well known to those skilled in the art that CDRs of an antibody can be defined in a variety of ways, such as Chothia (Chothia et al (1989) Nature 342:877-883, al-Lazikani et al, journal of Molecular Biology,273,927-948 (1997)), kabat (Kabat et al ,U.S.Department of Health and Human Services,National Institutes of Health(1987))、AbM(University of Bath),Contact(University College London)、 International ImMunoGeneTics database (IMGT) (world Wide Web IMGT. Cis. Fr /), and North CDR definitions based on neighbor-propagating clusters (affinity propagation clustering) using a large number of crystal structures, based on the topology of the CDR loops of an antibody.

Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. However, although CDRs vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. Using at least two of the Kabat, chothia, IMGT, abM and Contact methods, the minimum overlap region can be determined, providing a "minimum binding unit" for antigen binding. The minimum binding unit may be a sub-portion of the CDR. As will be apparent to those skilled in the art, the residues in the remainder of the CDR sequences can be determined by the structure of the antibody and the protein folding. Thus, the present invention also contemplates variants of any of the CDRs presented herein. For example, in a variant of one CDR, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia or AbM may be replaced by conserved amino acid residues.

As used herein, "percent (%) sequence identity" of amino acid sequences, sequence identity "has art-recognized definitions that refer to the percentage of identity between two polypeptide sequences as determined by sequence alignment (e.g., by manual inspection or by a known algorithm). The determination may be made using methods known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, clustal Omega and FASTA software.

In the present invention, unless the context clearly indicates otherwise, when referring to the term "antibody" it includes not only whole antibodies but also antigen-binding fragments of antibodies.

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 epitopes. The multispecific antibody may be, for example, a bispecific, trispecific or tetraspecific antibody, which is capable of specifically binding 2, 3 or 4 epitopes, respectively. As used herein, the term "epitope" or "antigenic determinant" refers to a region of an antigen that specifically binds to an antigen binding site of an antibody.

The term "isolated" as used herein refers to being obtained from a natural state by artificial means. If a "isolated" substance or component occurs in nature, it may be that the natural environment in which it is located is altered, or that the substance is isolated from the natural environment, or both. For example, a polynucleotide or polypeptide that has not been isolated naturally occurs in a living animal, and the same polynucleotide or polypeptide is said to be "isolated" in a high purity from its natural state. The term "isolated" does not exclude the incorporation of artificial or synthetic substances, nor the presence of other impure substances that do not affect the activity of the substance.

As used herein, "vector" refers to a construct 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, cosmid 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 producer cells.

The term "host cell" as used herein refers to a cell that can be used to introduce a vector, and includes, but is not limited to, a prokaryotic cell such as E.coli, a fungal cell such as a yeast cell, an insect cell such as S2 Drosophila cell or Sf9, or an animal cell such as a fibroblast, CHO cell, COS cell, NSO cell, heLa cell, BHK cell, HEK293 cell or human cell.

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.

The invention has the positive progress effects that:

The antibody or the antigen binding fragment thereof targets human and non-human primate M1R, and the anti-M1R antibody or the antigen binding fragment thereof has improved or reduced affinity, can achieve the aim of improving the drug effect or reducing the toxicity, can be selectively adapted according to different targets when forming a bispecific antibody, and provides a flexible adaptation scheme for drug research and development.

Drawings

FIG. 1 shows ELISA detection results of antigen M1R, negative control is isotype antibody of irrelevant target, blank control is no antibody control.

FIG. 2 shows ELISA detection results of binding of the whole human library antibody to the antigen M1R, wherein negative control is an isotype antibody of an irrelevant target, and blank control is an antibody-free control.

FIG. 3 shows ELISA detection results of the binding of mouse immune library antibodies to the antigen M1R, wherein negative control is an isotype antibody of an irrelevant target, and blank control is an antibody-free control.

FIG. 4 shows ELISA detection results of the binding of the nanobody library antibody and the antigen M1R, wherein the negative control is an homotype antibody of an irrelevant target, and the blank control is an antibody-free control.

FIG. 5 shows the results of antibody pairing assays, negative controls being isotype antibodies to unrelated targets, blank controls being no antibody controls.

Fig. 6 shows the colloidal gold detection results.

Detailed Description

The following describes exemplary embodiments of the invention, and it will be understood by those skilled in the art that the disclosure is illustrative only and that various other substitutions, adaptations, and modifications may be made within the scope of the invention. Therefore, the present invention is not limited to the specific embodiments set forth herein.

The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

Example 1 preparation and identification of antigenic proteins

Antigenic protein preparation by genetic manipulation at the genetic level, the sequence C-terminal of the extracellular domain of the human M1R protein (M1R-ECD, see: uniprot ID Q8V502 AA: 1-181) is tagged with mFc (SEQ ID NO: 50) or His tag. The obtained nucleic acid sequence was constructed into GSV0 vector, then transformed into E.coli DH 5. Alpha. And cultured overnight at 37℃before plasmid extraction using endotoxin-free plasmid extraction kit (OMEGA, D6950-01). The resulting plasmid was transiently transferred to HEK293 cells using ExpiFectamine ^TM transfection kit (Gibco ^TM, A14524)CRL-1573 ^TM), after 5 days of expression, cell culture supernatants were harvested and affinity purified on protein containing Fc tags by protein a/G affinity chromatography columns. After purification, the target protein was eluted with 100mM glycinate (ph=3.0), concentrated and buffer was replaced. His-tagged proteins were affinity purified using Ni Smart Beads 6FF (Sedum Palustre and Biotech Co., ltd., SA 036050) and then the target proteins were eluted with an imidazole gradient. The eluted proteins were each changed to PBS buffer by ultrafiltration concentration tube (Millipore, UFC 901096). Finally, M1R antigen proteins (M1R-ECD-mFc and M1R-ECD-His) with mFc tag and His tag respectively are obtained.

Antigen identification the activity of the prepared antigen protein was detected with the purchased Anti-Monkeypox virus/MPXV M1R Antibody (SAA 0283) protein. The specific method is that ELISA plates are coated with 2 mug/mL M1R-ECD-mFc and M1R-ECD-His, the ELISA plates are subjected to 4 ℃ overnight, after 3 times of washing, 5% skimmed milk prepared by PBS is used for sealing for 1 hour at room temperature, after 3 times of washing, antibodies Anti-Monkeypox virus/MPXV M1R Anti-body (SAA 0283) diluted by 1% PBSM gradient are added for incubation for 1 hour at room temperature, after washing, a second Antibody Goat-Anti-mouse-IgG-Fc-HRP (abcam; ab 97265) diluted by 1% PBSM (1:8000) is added, anti-mouse-Fab-HRP (sigma; M4155-1 ML) is added for incubation for 1 hour at room temperature, after 6 times of washing, TMB is added for development for 5min-20min, the color development reaction is stopped, OD ₄₅₀ data is read by an ELISA reader, and data processing and mapping is performed by using GRAPHPAD PRISM. As shown in FIG. 1, the antibodies Anti-Monkeypox virus/MPXV M1R Antibody (SAA 0283) bound well to the antigens M1R-ECD-mFc and M1R-ECD-His expressed in the construction of example 1.1, and had an antigen binding activity of EC ₅₀ of 0.007442. Mu.g/mL.

Example 2 construction and characterization of M1R-FL-EGFP-HEK293 overexpressing cell lines

The coding nucleotide sequence of M1R-FL-EGFP (SEQ ID NO: 51) was constructed onto pLVX-puro plasmid (Clontech, cat# 632164). The resulting plasmid was then electrotransformed into HEK293 cells by electrotransformation apparatus (Invitrogen, neonTM Transfection System, MP 922947)CRL-1573 ^TM). After electrotransformation, the resulting cells were transferred to DMEM medium (Gibco, 11995065) containing 10% FBS (Gibco, 15140-141) by volume and no antibiotics, respectively. Cells were then transferred to a 10X 10cm cell culture dish for 48 hours, then seeded into 96 well cell culture plates at an average cell/well density of 0.51E+4 cells/well, puromycin at a final concentration of 2. Mu.g/mL was added as a selection pressure, and cell lines forming clones were picked up for about 2 weeks for identification.

Flow cytometry identification of M1R-FL-EGFP-HEK293 overexpressing cells of the above cell lines in log phase were digested and plated into 96-well plates, washed with FACS buffer (1 XPBS buffer containing 2% FBS by volume), incubated for 30min with commercial primary Antibody (Anti-Monkeypox virus/MPXV M1R Antibody (SAA 0283)) diluted gradient in PBS at 4 ℃, washed, and then with formulated fluorescent secondary Antibody Anti human IgG Fc (abcam, 98596), incubated for 30min at 4℃and finally detected by flow cytometry (Beckman, cytoFLEXAOO-1-1102). The detection result shows that the M1R-FL-EGFP-HEK293 cell strain with high expression of human M1R on the cell surface is obtained.

EXAMPLE 3 construction and screening of a recombinant human phage display antibody library

In this example, a phage display antibody gene library was constructed and screened using M1R-ECD-His and M1R-ECD-mFc as screening antigens to obtain a plurality of antibody molecules having specific binding to the M1R-ECD-His protein. In this example, the construction and screening method of a library of humanized phage display recombinant antibodies is described in example 2 of patent CN 112250763B.

After a total of three rounds of screening, a second and third rounds of positive clone ELISA screening were selected. Finally, 207 positive clones capable of binding to the M1R-ECD-His protein were screened out in 444 clones, and after sequencing analysis, the sequences of 48 clones were finally selected to construct the full length for the next experiment.

The specific implementation method is as follows:

After completion of the primary screening work, positive clones were numbered, 2. Mu.L of the bacterial liquid was aspirated into 2mL of 2YT medium, cultured overnight at 37℃at 220rpm, and plasmids were extracted for second generation sequencing. Sequencing results the original AB1 file was integrated, aligned, and the non-antibody gene sequences were removed by SeqMan to generate the fasta file of the antibody gene integrated version. The DNA sequence is then translated into an amino acid sequence by MEGA6, and the fasta file containing the terminator, an unconventional sequence, etc., is found from the amino acid sequence, and the amino acid sequence is derived.

Cloning NL-M1R-A139, NL-M1R-A185, NL-M1R-A198, NL-M1R-A98 are preferred molecules, the CDR amino acid sequences of the resulting antibody Fab are shown in the sequence list and sequence table, and the CDR sequences are determined by defining the CDRs by AbM.

EXAMPLE 4 mouse immunization and immune repertoire construction screening

4.1 Immunization protocol

2 Balb/C and 2C 57 mice (Mouse #1 and Mouse # 2) were immunized by subcutaneous and intraperitoneal injection with M1R-ECD-His antigen, 2 Balb/C and 2C 57 mice (Mouse #3 and Mouse # 4) were cross-immunized by subcutaneous and intraperitoneal injection with M1R-ECD-mFc and M1R-ECD-His antigen, and a total of 4 immunizations were performed every two weeks (Zhejiang Vetolihua laboratory animal technologies Co.). One week after the 4 th immunization, the mouse blood was taken for the immunization titer detection, and finally, the immunization was boosted once again with M1R-ECD-mFc.

4.2 Detection of serum antibody titers of mice after immunization

ELISA plates were coated with 2. Mu.g/mL M1R-ECD-His overnight (30. Mu.L/well) at 4℃and blocked with PBS-formulated 5% skim milk (5% PBSM) for 1 hour at room temperature after 3 plate washes, mouse serum diluted with 5% PBSM gradient was added after 3 plate washes while antibody 7D11-chimera (see: PMID: 17688903) was added as positive control and incubated for 1 hour at room temperature, secondary antibody Goat-anti-mouse-lgG (1+2a+2b+3) -HRP (Jackson, 115-035-164) or Goat-anti-human-kappa+lambda-HRP (lipore, AP502P+AP 506P) was added after 6 plate washes and incubated for 1 hour at room temperature, and the data was read with a microplate reader OD450 after termination of the chromogenic reaction. The results are shown in tables 1-2, and the serum titers of 8 mice reach the standard.

TABLE 1 final serum titer assay (ELISA)

TABLE 2 final serum titer assay (ELISA)

4.3 Construction of phage display antibody Gene library

After immunization, the spleen of the mice was taken, collected after grinding and filtration, the spleen cells were lysed by adding 1mL of TRIzol ^TM Reagent (Thermo Fisher, 15596026), total RNA was extracted by phenol chloroform method, and the extracted RNA was reverse transcribed into cDNA by reverse transcription kit (TaKaRa, 6210A). And then, respectively amplifying the light chain variable region genes and the heavy chain variable region genes of the antibody by using cDNA as a PCR template and adopting specific primers of a murine antibody sequence. The PCR product was digested with NcoI and NotI to give antibody gene fragments, which were inserted into phage display vectors, ligated by T4 ligase, the ligation products were recovered by DNA recovery kit (Omega, D6492-02), and finally transformed into competent E.coli SS320 (Lucigen, MC 1061F) by electrotransfer (Bio-Rad, micropulser), the electrotransferred bacteria were plated on 2-YT (C ⁺/K⁺ 2-YT) solid plates containing ampicillin and tetracycline, SS320 bacteria of correctly transformed antibody plasmids were amplified, and packaged with VSCM helper phage (purchased from Stratagene), obtaining phage display libraries containing Fab sequences.

4.4 Screening phage display antibody Gene libraries by cell methods

M1R-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 (Source culture, B310 KJ), then 2mL of 4% paraformaldehyde (Bio-technology, E672002-0500) was added for fixation for 0.5 hours, and finally washed twice with PBS as a screening raw material. In screening, phage display library and immobilized M1R-FL-EGFP-HEK293 cells were incubated for 1 hour at room temperature, washed three times with 1 XPBS, 2mL glycine-HCl (pH=2.0) was added and gently mixed for 10 minutes to elute phage specifically binding to human ROR1, then the eluted supernatant was infected with SS320 cells (Lucigen, 60512-1) in logarithmic phase, allowed to stand for 30 minutes, then cultured for 1 hour at 37℃at 220rpm, then VSCM helper phage was added, allowed to stand for 30 minutes, continued to be cultured for 1 hour at 37℃at 220rpm, centrifuged and replaced into C ⁺/K⁺ 2-YT medium, and the phage finally obtained was continued for the second round of screening. The screening was repeated multiple times, 10 clones were randomly selected for sequence analysis per round to evaluate the pool, and after 3 rounds of screening, the sequence enrichment in the pool was evident.

4.5 Screening phage display antibody Gene library by Immunotube method

The immune tube method and the magnetic bead method are adopted to enrich specific antibodies aiming at antigens, and the two methods are mutually supplemented and verified.

The immune tube method screening is to coat antigen protein M1R-ECD-His or M1R-ECD-mFc on the surface of an immune tube with high adsorption force, and the phage display antibody library is added into the immune tube and undergoes the panning process of incubation, washing and elution with the antigen protein adsorbed on the surface of the immune tube, and the panned antigen protein is subjected to 2-4 rounds of panning, so that the specific monoclonal antibody Fab aiming at the antigen is finally enriched. In this example, monoclonal antibody Fab against M1R-ECD-His was enriched after 3 rounds of panning, for specific procedures as described in example 2.4.2 of patent CN 112250763B.

ELISA detection is carried out on the phage pools eluted from each round to evaluate the enrichment effect, and the result shows that the sequence enrichment is obvious after the second round of screening, so that the second round and the third round of screening are selected for positive clone screening of ELISA.

4.6 Selection of monoclonal

After a total of three rounds of screening, a second and third rounds of positive clone ELISA screening were selected. Finally, 200 positive clones capable of binding to the M1R-ECD-His protein were screened out in total from 354 clones, and after sequencing analysis, the sequences of 6 clones were finally selected to construct the full length for the next experiment.

The specific implementation method is as follows:

Clone m1r_m_pr_a139 is a preferred molecule, the CDR amino acid sequences of the resulting antibody Fab are listed in the sequence list and sequence listing, and the CDR sequences are determined by defining CDRs by AbM.

EXAMPLE 5 construction and screening of humanized backbone nanobody phage display libraries

In this example, antibody screening was performed using a phage display library constructed and prefabricated in this department, using a library dAb-15 as a humanized backbone nanobody library with a library capacity of 2.52X10 ¹¹. The screening route used included screening the library with M1R-ECD-His and M1R-ECD-mFc. An antibody molecule that specifically binds to M1R-ECD-His was obtained.

5.1 Phage display library construction

DAb-15 is a humanized skeleton nano antibody library, based on the fully humanized nano antibody skeleton, random primers with different lengths are designed, gene fragments of different CDR regions are respectively amplified and recombined, and a fully synthetic phage display antibody library is obtained by recombining 3 CDRs in the humanized skeleton nano antibody library (library construction method refers to example 8.1 in patent CN 112625136A). The library capacity was measured by gradient dilution plating as an antibody gene library of 2.52X10 ¹¹, i.e. 2.52X10 ¹¹ antibody genes (for the method of calculation of the library capacity, see example 2.2 in patent CN 112250763B). Packaging with VSCM helper phage (available from Stratagene) resulted in an antibody gene phage display library (see example 2.3 in patent CN112250763B for preparation of antibody gene phage display library).

5.2 Screening of phage display libraries

The library is screened based on an immune tube method, and M1R-ECD-His and M1R-ECD-mFc are adopted for screening, so that antibodies with relatively conserved binding sequences and relatively good affinity activity are obtained.

5.2.1 Screening of antibody Gene phage display library by Immunotube method

The principle of the immune tube screening is that M1R-ECD-His or M1R-ECD-mFc is coated on the surface of an immune tube with high adsorption capacity, and a phage display antibody library is added into the immune tube and undergoes the panning process of incubation, washing and elution with antigen proteins adsorbed on the surface of the immune tube, and then the monoclonal antibody is subjected to 3 rounds of panning, and finally, the monoclonal antibody specific to the antigen is enriched.

The specific implementation method is as follows:

In the first round of screening, 1mL of 100. Mu.g/mL M1R-ECD-His or M1R-ECD-mFc was added to the immune tube, the coating was left overnight at 4 ℃, the coating was discarded the next day, PBS containing 5% milk was added to block for 2 hours, PBS was added to wash twice, the constructed phage library of nanobody genes of 200 alpaca was added, incubated for 2 hours, washed 8 times with PBST and then 2 times with PBS to remove non-specifically bound phage, then 0.8mL of 0.05% EDTA pancreatin digest was added to the immune tube for eluting phage specifically bound to the antigen of interest, then it was infected with logarithmic phase SS320 phage (Lucigen, 60512-1), allowed to stand for 30min at 37℃and then cultured for 1 hour at 220rpm, then VSCM helper phage was added, allowed to stand for 30min, and cultured for 1 hour at 220rpm, centrifuged and replaced to C ⁺/K⁺ -YT medium, and cultured at 30℃and 220rpm overnight. The following day phages were precipitated for the following 2-4 rounds of screening. The second and third rounds of phage screening coating antigen is M1R-ECD-His or M1R-ECD-mFc, the antigen coating concentration is decreased in sequence to 30 mug/mL and 10 mug/mL respectively, in addition, the PBST rinsing intensity is increased gradually, and the PBST eluting times are 10 times and 14 times in sequence.

ELISA detection is carried out on phage pools eluted from each round by using the ELISA adsorption test and the coated M1R-ECD-His to evaluate the enrichment effect, and 10 clones are randomly selected from phage pools screened from each round for sequence analysis. The results show that the enrichment of the antibody sequences is obvious after the third round of screening, and each round of screening has better enrichment. Thus, the clones obtained from the second and third rounds were selected for positive clone screening by ELISA.

5.3 Selection of monoclonal

After the total rounds of screening, the clones obtained from the second and third rounds were selected for ELISA screening of positive clones for ELISA. Finally, 296 positive clones capable of binding to the M1R-ECD-His protein were screened out of 446 clones, and after sequencing analysis and ELISA binding detection, the sequences of 44 clones were selected to construct full length antibodies (VHH-Fc) for further experiments.

Clone M1R_N_PR_A013 is a preferred molecule, the CDR amino acid sequence of the obtained VHH is shown in a sequence list and a sequence table, and the CDR sequence is determined by adopting an AbM mode of defining the CDR.

EXAMPLE 6 candidate antibody construction, expression and purification

6.1 Construction of plasmid

The VH coding sequence in the Fab sequence of the monoclonal antibody obtained by screening is connected with the coding sequence of the heavy chain constant region (SEQ ID NO: 45) of human IgG1 to obtain the heavy chain coding sequence of the antibody, the VL coding sequence in the Fab sequence is connected with the Kappa type (SEQ ID NO: 47) or Lambda type (SEQ ID NO: 48) coding sequence of the human light chain constant region (CL) to obtain the light chain coding sequence of the antibody, and the coding sequence of the monoclonal VHH obtained by screening is connected with the coding sequence of the Fc region (IgG 1-CS mutant) (SEQ ID NO: 46) of human IgG1 to obtain the coding sequence of VHH-Fc. The constructed coding sequences are respectively inserted into eukaryotic expression vector plasmid GSV0 and transformed into escherichia coli DH5 alpha, and are cultured at 37 ℃ overnight. Plasmid extraction was performed using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free antibody plasmids for eukaryotic expression.

6.2 Expression and purification of candidate antibodies

Candidate antibodies were expressed by ExpiCHO transient transfection expression system (Thermo Fisher, A29133) by confirming that the cell density was around 7X 10 ⁶ to 1X 10 ⁷ viable cells/mL, cell viability >98% on the day of transfection, at which time the cells were adjusted to a final concentration of 6X 10 ⁶ cells/mL with fresh ExpiCHO expression medium pre-warmed at 37 ℃. The plasmid constructed in example 3.1 was diluted with OptiPROTM SFM pre-chilled at 4 ℃ (1 μg plasmid was added to 1mL of the medium), while ExpiFectamineTMCHO was diluted with OptiPROTMSFM, and then the two were mixed in equal volumes and gently swirled to prepare ExpiFectamineTMCHO/plasmid DNA mixture, incubated at room temperature for 1-5min, slowly added to the prepared cell suspension while gently shaking, and finally placed in a cell culture shaker and incubated at 37 ℃ under 8% CO ₂.

At 18-22h post-transfection, expiCHOTMEnhancer and ExpiCHOTMFeed were added to the broth and the flask was placed on a 32℃shaker and 5% CO ₂ for further culture. On day 5 post-transfection, the same volume ExpiCHOTMFeed was added, slowly while gently mixing the cell suspension. After 7 days of transfection, the cell culture supernatant expressing the protein of interest was centrifuged at 15000g for 10min at high speed, and the resulting supernatant was affinity purified with MabSelect SuRe LX (GE, 17547403), then eluted with 100mM sodium acetate (pH 3.0), then neutralized with 1M Tris-HCl, and finally the resulting protein was exchanged into PBS buffer by ultrafiltration concentration tube (Millipore, UFC 901096).

Example 7 identification of physicochemical Properties of candidate antibodies

Preparation of non-reducing solution candidate antibody of example 6 and quality control IPI (i.e., ipilimumab, ipilimumab) 1. Mu.g were added to 5 XSDS loading buffer and 40mM iodoacetamide, heated in a 75℃dry bath for 10min, cooled to room temperature, and centrifuged at 12000rpm for 5min to obtain the supernatant.

Preparation of reduction solution candidate antibody of example 6 and quality control IPI 2. Mu.g were added to 5 XSDS loading buffer and 5mM DTT, and the mixture was heated in a 100℃dry bath for 10 minutes, cooled to room temperature, and centrifuged at 12000rpm for 5 minutes to obtain a supernatant.

The supernatant was added to Bis-tris 4-15% gradient gel (gold Style biosciences Co.) for gel electrophoresis and protein bands were visualized by coomassie brilliant blue staining. Protein gels with chromogenic protein bands were scanned using an EPSON V550 color scanner (decolorized to gel background transparent) and reduced and non-reduced band purities were calculated by ImageJ according to peak area normalization.

The results are shown in Table 3, where the antibody purities were greater than 95%.

TABLE 3 physicochemical detection results of candidate antibodies

Example 8 ELISA affinity detection of horizontal candidate antibodies

In this example, the binding of the expressed candidate antibody to the M1R-ECD-His antigen protein was detected based on ELISA.

8.1 ELISA-based detection of the binding Capacity of human recombinant library candidate antibodies to M1R-ECD-His

96-Well ELISA plates (30. Mu.L/well) were coated with 2. Mu.g/mL M1R-ECD-His at 4℃overnight. The next day, the well plates were washed 3 times with PBST and blocked with 5% skimmed milk for 2h. After washing the plates 3 times with PBST, each antibody was added in a gradient dilution (3.0, 0.33, 0.11, 0.037, 0.012, 0.004, 0.0014, 0.0002. Mu.g/mL) and incubated for 1h with positive control antibody 7D11-chimera (see sequence: PMID: 17688903). The plates were then washed 3 times with PBST and secondary antibodies Goat-Anti-human Fc-HRP (abcam, ab 97225) were added and incubated for 1h. After incubation, the plates were washed 6 times with PBST and developed with TMB (SurModics, TMBS-1000-01). Based on the color development results, the reaction was stopped by adding 2M stop solution, and the absorbance was read at OD450 by a microplate reader (Molecular Devices, specterMax, 190).

As shown in FIG. 2 and Table 4, the binding activities of the antibody molecules NL-M1R-A139, NL-M1R-A185, NL-M1R-A198 and NL-M1R-A98 to the antigen protein M1R-ECD-His were strong.

TABLE 4 binding data of fully human library candidate antibodies to M1R-ECD-His

Protein name	Library sources	EC₅₀(μg/mL)
			NL-M1R-A139	Human recombinant library	0.03727
NL-M1R-A185	Human recombinant library	0.01086
			NL-M1R-A198	Human recombinant library	0.01153
NL-M1R-A98	Human recombinant library	0.02333

8.2 ELISA-based detection of the binding Capacity of mouse immune library candidate antibodies to M1R-ECD-His

96-Well ELISA plates (30. Mu.L/well) were coated with 2. Mu.g/mL M1R-ECD-His at 4℃overnight. The next day, the well plates were washed 3 times with PBST and blocked with 5% skimmed milk for 2h. After washing the plates 3 times with PBST, each antibody was added in a gradient dilution (3.0, 0.33, 0.11, 0.037, 0.012, 0.004, 0.0014, 0.0002. Mu.g/mL) and the positive control antibody 7D11-chimera and incubated for 1h. The plates were then washed 3 times with PBST and secondary antibodies Goat-Anti-human Fc-HRP (abcam, ab 97225) were added and incubated for 1h. After incubation, the plates were washed 6 times with PBST and developed with TMB (SurModics, TMBS-1000-01). Based on the color development results, the reaction was stopped by adding 2M stop solution, and the absorbance was read at OD450 by a microplate reader (Molecular Devices, specterMax, 190).

The results are shown in FIG. 3 and Table 5, in which the binding activity of the antibody molecule M1R_M_PR_A139 to the antigen protein M1R-ECD-His was strong.

TABLE 5 mouse immune candidate antibodies and M1R-ECD-His binding data

Protein name	Library sources	EC₅₀(μg/mL)
			M1R_M_PR_A139	Mouse immune repertoire	0.002317

8.3 Detection of binding ability of nanobody library candidate antibody to M1R-ECD-His based on ELISA

96-Well ELISA plates (30. Mu.L/well) were coated with 2. Mu.g/mL M1R-ECD-His at 4℃overnight. The next day, the well plates were washed 3 times with PBST and blocked with 5% skimmed milk for 2h. After washing the plates 3 times with PBST, each antibody was added in a gradient dilution (3.0, 0.33, 0.11, 0.037, 0.012, 0.004, 0.0014, 0.0002. Mu.g/mL) and the positive control antibody 7D11-chimera and incubated for 1h. The plates were then washed 3 times with PBST and secondary antibodies Goat-Anti-human Fc-HRP (abcam, ab 97225) were added and incubated for 1h. After incubation, the plates were washed 6 times with PBST and developed with TMB (SurModics, TMBS-1000-01). Based on the color development results, the reaction was stopped by adding 2M stop solution, and the absorbance was read at OD 450 by a microplate reader (Molecular Devices, specterMax, 190).

The results are shown in FIG. 4 and Table 6, in which the binding activity of the antibody molecule M1R_N_PR_A013 to the antigen protein M1R-ECD-His was strong and EC ₅₀ was 0.008817. Mu.g/mL.

TABLE 6 nanometer antibody library candidate antibodies and M1R-ECD-His binding data

Protein name	Library sources	EC₅₀(μg/mL)
			M1R_N_PR_A013	Nano antibody library	0.008817

Example 9 ELISA double anti-sandwich antibody pairing

In this example, the pairing of antibodies to the M1R-ECD-His antigen protein was detected based on ELISA double-antibody sandwich.

96-Well ELISA plates (30. Mu.L/well) were coated with 2. Mu.g/mL of antibody at 4℃overnight. The next day, the well plates were washed 3 times with PBST and blocked with 5% skimmed milk for 2h. After washing the plates 3 times with PBST, 2. Mu.g/mL of M1R-ECD-His was added and incubated for 1h. Plates were washed 3 times with PBST and gradient diluted (10, 3.33333, 1.11111, 0.37037, 0.12346, 0.04115, 0.01372, 0.00457. Mu.g/mL) of biotinylated detection antibody was added and incubated for 1h. Plates were then washed 3 times with PBST and secondary Neutravidin-HRP (Thermo, 31001) was added and incubated for 1h. After incubation, the plates were washed 6 times with PBST and developed with TMB (SurModics, TMBS-1000-01). Based on the color development results, the reaction was stopped by adding 2M stop solution, and the absorbance was read at OD450 by a microplate reader (Molecular Devices, specterMax, 190).

As a result, as shown in FIG. 5, the effect of M1R_M_PR_A139 and M1R_N_PR_A013, M1R_M_PR_A139 and NL-M1R-A198 on detection of the M1R-ECD-His antigen by the paired antibodies was more excellent. In FIG. 5, the antibodies M1R_M_PR_A139 were coated on ELISA plates, and the detection effect was good when the liquid phase was labeled with different candidate antibodies, and when M1R_M_PR_A139 was coated on ELISA plates, the liquid phase was labeled with M1R_N_PR_A013 or NL-M1R-A198, and EC ₅₀ was 0.05359. Mu.g/mL and 0.02592. Mu.g/mL, respectively.

Example 10 colloidal gold assay card for detection of M1R monkey poxvirus proteins

In this example, the antibodies selected in example 9 and having a good effect of detecting the M1R-ECD-His antigen protein were used to prepare colloidal gold detection cards for detecting the M1R monkey poxvirus proteins, such as M1R_M_PR_A139 and M1R_N_PR_A013, M1R_M_PR_A139 and NL-M1R-A198.

10.1 Preparation of colloidal gold detection card

In this example, a colloidal gold test card was prepared using the above-described paired antibodies according to the method described in the examples in chinese patent application CN102747040a, and passed each quality test. In this detection card, the antibody M1R_M_PR_A139 (1.71 mg/ml) was used as capture antibody, and the antibody M1R_N_PR_A013 (2.76 mg/ml) or NL-M1R-A198 (0.74 mg/ml) was used as detection antibody.

10.2 Sensitivity determination

In this example, the monkey poxvirus M1R protein was diluted 2-fold from 1. Mu.g/mL to 650pg/mL. The M1R proteins subjected to gradient dilution are respectively added into the sample adding holes (left lanes of the card) of the colloidal gold detection card at the volume of 120 mu L per hole, and color development is carried out for 20min, wherein a C line is a quality control line, a T line is a detection line, 3 parallel groups are arranged, and 9 blank controls (only diluent is added) are arranged. As shown in FIG. 6, the detection sensitivity of the detection card for the M1R protein of the monkey pox virus can reach 10ng/mL.

The left side of FIG. 6 is the M1R_M_PR_A139 and M1R_N_PR_A013 groups and the right side is the M1R_M_PR_A139 and NL-M1R-A198 groups.

Exemplary sequence

Claims

1. An antibody or antigen-binding fragment thereof that targets a monkey poxvirus, wherein the antibody comprises a heavy chain variable region and a light chain variable region;

the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown as SEQ ID NO. 33, SEQ ID NO. 34 and SEQ ID NO. 35 respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID NO. 36, SEQ ID NO. 37 and SEQ ID NO. 38 respectively.

2. The antibody or antigen-binding fragment thereof of claim 1,

The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 39 or has at least 80% sequence identity with SEQ ID NO. 39, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 40 or has at least 80% sequence identity with SEQ ID NO. 40.

3. The antibody or antigen-binding fragment thereof of claim 2, wherein the antibody is a full-length antibody, fab ', F (ab') 2, or Fv.

4. The antibody or antigen-binding fragment thereof of claim 3, wherein when the antibody is a full length antibody, the heavy chain constant region of the full length antibody is derived from a heavy chain of a human antibody or a variant thereof, and the light chain constant region of the full length antibody is derived from a kappa chain or a lambda chain of a human antibody or a variant thereof.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein when the antibody is a full length antibody, the amino acid sequence of the heavy chain constant region is shown as SEQ ID NO. 45 and the amino acid sequence of the light chain constant region is shown as SEQ ID NO. 47 or SEQ ID NO. 48.

6. An antibody combination comprising one or more antibodies or antigen-binding fragments thereof according to any one of claims 1-5.

7. The combination of antibodies of claim 6, wherein the combination of antibodies comprises a first antibody and a second antibody.

8. The combination of claim 7, wherein,

The heavy chain variable region of the first antibody comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown as SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35, respectively, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38, respectively, the heavy chain variable region of the second antibody comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown as 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 shown as SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, respectively, or

The heavy chain variable region of the first antibody comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences shown as SEQ ID NO. 33, SEQ ID NO. 34 and SEQ ID NO. 35, respectively, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having amino acid sequences shown as SEQ ID NO. 36, SEQ ID NO. 37 and SEQ ID NO. 38, respectively, and the second antibody comprises a heavy chain variable region but not a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences shown as SEQ ID NO. 41, SEQ ID NO. 42 and SEQ ID NO. 43, respectively.

9. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1-5, or the antibody combination of any one of claims 6-8.

10. A recombinant expression vector, which is characterized in that, the recombinant expression vector comprising the nucleic acid of claim 9.

11. The recombinant expression vector of claim 10, wherein the recombinant expression vector is a plasmid or a viral vector.

12. The recombinant expression vector of claim 11,

The plasmid is a cosmid;

the viral vector is a phage, retroviral vector, adenoviral vector or adeno-associated viral vector.

13. The recombinant expression vector of claim 12, wherein the retroviral vector is a lentiviral vector.

14. A transformant, characterized in that it is a host cell comprising the recombinant expression vector according to any one of claims 10-13.

15. The transformant of claim 14, wherein the host cell is a prokaryotic cell or a eukaryotic cell.

16. The transformant of claim 15, wherein the host cell is selected from the group consisting of a yeast cell, a mammalian cell, and other cells suitable for use in the preparation of antibodies or antigen-binding fragments thereof.

17. The transformant of claim 16, wherein the mammalian cell is a HEK293 cell.

18. A method of making an antibody or antigen-binding fragment thereof that targets M1R, comprising culturing the transformant of any one of claims 14-17, and obtaining the antibody or antigen-binding fragment thereof that targets M1R from the culture.

19. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-5, or the antibody combination of any one of claims 6-8, the nucleic acid of claim 9, the recombinant expression vector of any one of claims 10-13, or the transformant of any one of claims 14-17.

20. The kit of claim 19, further comprising a reagent for detecting binding of the antibody or antigen-binding fragment thereof to a monkey poxvirus.

21. Use of an antibody or antigen binding fragment thereof according to any one of claims 1-5, an antibody combination according to any one of claims 6-8, a nucleic acid according to claim 9, a recombinant expression vector according to any one of claims 10-13, a transformant according to any one of claims 14-17, or a kit according to any one of claims 19-20 for the preparation of a reagent for diagnosing a monkey poxvirus infection.