CN117050165A - Antibody targeting monkey poxvirus, antigen binding fragment thereof and application thereof - Google Patents

Abstract

The invention discloses an antibody targeting monkey poxvirus, an antigen binding fragment thereof and application thereof. The antibody or antigen binding fragment thereof 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 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.

Description

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

Technical Field

The present invention relates to the field of antibody drugs, and specifically to an antibody targeting monkeypox virus, an antigen-binding fragment thereof and uses thereof, in particular an antibody specifically binding to the surface antigen M1R of the monkeypox virus and a conjugate or fusion comprising the anti-M1R antibody or its antigen-binding fragment. In addition, the present invention relates to a nucleic acid encoding the anti-M1R antibody and a host cell comprising the nucleic acid, as well as a method for preparing the antibody. The present invention also relates to a pharmaceutical composition comprising the anti-M1R antibody and the medical use of the anti-M1R antibody.

Background Art

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

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

Monkeypox virus (MPXV) is a double-stranded DNA virus of the genus Orthopoxvirus in the family Poxviridae. Two genetic clades of MPXV have been characterized: West African (10% case fatality rate) and Congo Basin (3.6% case fatality rate). The virus produces two different forms of infectious virus particles in the infected host, the extracellular enveloped virus EEV (Extracellular Enveloped Virion) with a double-layer membrane structure and the intracellular mature virion IMV (Intracellular Enveloped Virion) with a single-layer membrane structure. Members of its family Poxviridae (the largest animal virus known so far) include: Variola virus (VARV, smallpox virus), Cowpox virus (CPV, cowpox virus) and Vacciniavirus (VACV, vaccinia virus). The life cycle of MPXV includes processes such as adsorption, membrane fusion, uncoating, DNA replication, transcription and translation, assembly, and release, which depends on the joint action of multiple proteins. Antigens that have been reported to produce neutralizing antibodies include B6R and A35R on the surface of EEV, and M1R, A29L and A30L on the surface of IMV. Among them, M1R binds to an unknown receptor on the surface of host cells, is highly conserved, participates in mediating viral infection, and is necessary for inducing low pH-triggered cell-cell fusion, so it can be used as a key drug target and detection indicator.

At present, two preventive vaccines against monkeypox virus have been approved for emergency use by the FDA, namely the second-generation VACV live vaccine ACAM2000 and the third-generation modified vaccinia Ankara (MVA) attenuated vaccine JYNNEOS. The former, as a live vaccine, has clinically shown obvious side effects such as myocarditis and pericarditis, while the latter, which is administered in two doses at an interval of 28 days, may have a risk of pre-exposure infection. The main therapeutic drugs for monkeypox virus infection are currently Tecovirimat and Brincidofovir, two small molecule inhibitors. The former targets the VP37 membrane protein of MPXV, preventing the formation of enveloped virus particles, thereby destroying the spread of the virus; the latter inhibits DNA synthesis mediated by orthopoxvirus DNA polymerase. Therefore, there is an urgent need for the development of more antibody drugs that specifically target monkeypox virus. This field needs to develop high-affinity neutralizing antibodies that recognize and bind to the surface membrane protein of monkeypox virus to effectively diagnose, prevent and treat infections of this type of poxvirus family.

Summary of the invention

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

The first aspect of the present invention provides an antibody or an antigen-binding fragment thereof targeting M1R, wherein the antibody comprises a heavy chain variable region and a light chain variable region or the antibody comprises a heavy chain variable region but does not comprise 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 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; the light chain variable region comprises 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 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 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 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 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 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 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 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 LCDR1, LCDR2 and LCDR3 as shown in 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 whose amino acid sequences are 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 according to AbM.

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

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

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

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

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

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

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

In some embodiments of the present invention, 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. In some specific embodiments of the present invention, when the antibody is a VHH antibody, the antibody further comprises an Fc region, and the amino acid sequence of the Fc region is shown in SEQ ID NO: 46, SEQ ID NO: 49 or SEQ ID NO: 50

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

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

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

In some specific embodiments of the present invention, the heavy chain variable region of the first antibody comprises HCDR1, HCDR2 and HCDR3 as shown in 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 as shown in 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 as shown in SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, respectively.

In other specific embodiments of the present invention, the heavy chain variable region of the first antibody comprises HCDR1, HCDR2 and HCDR3 as shown in 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 as shown in SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38, respectively; the second antibody comprises a heavy chain variable region but does not comprise a light chain variable region, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43, respectively.

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

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

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

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

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

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

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

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

The sixth aspect of the present invention provides a method for preparing an antibody or an antigen-binding fragment thereof targeting M1R, the method comprising culturing the transformant as described in the fifth aspect, and obtaining the antibody or an antigen-binding fragment thereof targeting M1R from the culture. The seventh aspect of the present invention provides a pharmaceutical composition, the pharmaceutical composition comprising the antibody or an antigen-binding fragment thereof as described in the first aspect, or the antibody combination as described in the second aspect, and a pharmaceutically acceptable carrier.

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

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

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

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

In some specific embodiments of the invention, the poxvirus infection is monkeypox virus infection.

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

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

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

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

definition

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

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

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

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

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

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

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

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

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

The positive and progressive effects of the present invention are:

The antibody or antigen-binding fragment thereof of the present invention targets human and non-human primate M1R. The anti-M1R antibody or antigen-binding fragment thereof has increased or decreased affinity, which can achieve the purpose of improving drug efficacy or reducing toxicity. When constituting a bispecific antibody, selective adaptation can be performed according to different targets, providing a flexible adaptation scheme for drug development.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the ELISA test results of antigen M1R, the negative control is the same type antibody of irrelevant target, and the blank control is the no antibody control.

Figure 2 shows the ELISA test results of the binding of the full human library antibody to the antigen M1R. The negative control is an isotype antibody of an irrelevant target, and the blank control is a control without antibody.

Figure 3 shows the ELISA test results of the mouse immune library antibodies binding to the antigen M1R, the negative control is the same type antibody of an irrelevant target, and the blank control is the no antibody control.

Figure 4 shows the ELISA test results of the binding of the nanobody library antibodies to the antigen M1R, the negative control is the same type antibody of an irrelevant target, and the blank control is a no-antibody control.

Figure 5 shows the results of the antibody pairing test, where the negative control is an isotype antibody of an irrelevant target and the blank control is a control without antibody.

FIG6 shows the colloidal gold detection results.

DETAILED DESCRIPTION

The following describes exemplary embodiments of the present invention, and it should be understood by those skilled in the art that these disclosures are merely exemplary, and various other substitutions, adaptations, and modifications may be made within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments listed herein.

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

Example 1 Preparation and identification of antigen protein

Preparation of antigen protein: Through genetic manipulation at the gene level, mFc (SEQ ID NO: 50) or His tag was added to the C-terminus of the extracellular domain of human M1R protein (M1R-ECD, see: Uniprot ^ID Q8V502 AA: 1–181). The obtained nucleic acid sequence was constructed into the GSV0 vector, then transformed into Escherichia coli DH5α, cultured overnight at 37°C, and then the plasmid was extracted using an endotoxin-free plasmid extraction kit ( ^OMEGA , D6950-01). The obtained plasmid was transiently transfected into HEK293 cells ( CRL-1573 ^TM ), after 5 days of expression, the cell culture supernatant was collected, and the protein containing the Fc tag was affinity purified by ProteinA/G affinity chromatography column. After purification, the target protein was eluted with 100mM glycine salt (pH=3.0), concentrated and replaced with buffer. The protein containing the His tag was affinity purified with Ni Smart Beads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd., SA036050), and then the target protein was eluted with imidazole gradient. The eluted proteins were exchanged into PBS buffer by ultrafiltration concentration tubes (Millipore, UFC901096). Finally, M1R antigen proteins with mFc tags and His tags (M1R-ECD-mFc, M1R-ECD-His) were obtained.

Antigen identification: The activity of the prepared antigen protein was detected using the purchased Anti-Monkeypox virus/MPXV M1R Antibody (SAA0283) antibody protein. The specific method is as follows: coat the ELISA plate with 2 μg/mL M1R-ECD-mFc and M1R-ECD-His at 4°C overnight; wash the plate three times, and block it with 5% skim milk prepared in PBS at room temperature for 1 hour; wash the plate three times, add the antibody Anti-Monkeypox virus/MPXV M1R Antibody (SAA0283) diluted in a gradient with 1% PBSM and incubate it 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 it at room temperature for 1 hour, wash the plate six times, then add TMB for color development for 5min-20min, terminate the color development reaction, read the OD ₄₅₀ data with an enzyme reader, and use Graphpad prism for data processing and drawing. The results are shown in Figure 1. The antibody Anti-Monkeypox virus/MPXV M1R Antibody (SAA0283) binds well to the antigens M1R-ECD-mFc and M1R-ECD-His constructed and expressed in Example 1.1, and has an antigen binding activity with an EC ₅₀ of 0.007442 μg/mL.

Example 2 Construction and identification of M1R-FL-EGFP-HEK293 overexpression cell line

The coding nucleotide sequence of M1R-FL-EGFP (SEQ ID NO: 51) was constructed into pLVX-puro plasmid (Clontech, Cat#632164). Then, the obtained plasmid was electroporated into HEK293 cells ( CRL-1573 ^TM ). After electroporation, the obtained cells were transferred to DMEM medium (Gibco, 11995065) containing 10% FBS (Gibco, 15140-141) by volume and without antibiotics. The cells were then transferred to a 10×10 cm cell culture dish and cultured for 48 hours. The cells were then seeded into a 96-well cell culture plate at an average density of 0.51E+4 cells/well, and puromycin was added at a final concentration of 2 μg/mL as a screening pressure. After about 2 weeks, the cell lines that formed clones were picked for identification.

Flow cytometric identification of M1R-FL-EGFP-HEK293 overexpressing cells: The cells of the above cell lines in the logarithmic growth phase were digested and plated into 96-well plates, washed with FACS buffer (1×PBS buffer containing 2% FBS by volume), and then added with commercially available first antibody (Anti-Monkeypox virus/MPXV M1RAntibody (SAA0283)) diluted with PBS gradient and incubated at 4°C for 30 minutes; after washing, the prepared fluorescent second antibody Anti human IgGFc (abcam, 98596) was added and incubated at 4°C for 30 minutes; finally, the cells were detected by flow cytometry (Beckman, CytoFLEXAOO-1-1102). The test results showed that the M1R-FL-EGFP-HEK293 cell line with high expression of human M1R on the cell surface was obtained.

Example 3 Construction and screening of human phage display recombinant antibody library

In this example, a phage display antibody gene library was constructed, and the library was screened using M1R-ECD-His and M1R-ECD-mFc as screening antigens to obtain multiple antibody molecules that specifically bind to the M1R-ECD-His protein. In this example, the construction and screening methods of the human phage display recombinant antibody library refer to Example 2 of patent CN112250763B.

After three rounds of screening, the second and third rounds of positive clones were selected for ELISA screening. Finally, 207 positive clones that could bind to M1R-ECD-His protein were screened out of 444 clones. After sequencing analysis, 48 clones were selected to construct the full length sequence for the next experiment.

The specific implementation methods are as follows:

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

Clones NL-M1R-A139, NL-M1R-A185, NL-M1R-A198, and NL-M1R-A98 are preferred molecules. The CDR amino acid sequences of the obtained antibody Fab are shown in the sequence listing and sequence table. The CDR sequences were determined using the AbM definition method.

Example 4 Mouse immunization and immune library construction and screening

4.1 Immunization regimen

Two Balb/C mice and two C57 mice (Mouse#1 and Mouse#2) were immunized with M1R-ECD-His antigen by subcutaneous injection and intraperitoneal injection; two Balb/C mice and two C57 mice (Mouse#3 and Mouse#4) were cross-immunized with M1R-ECD-mFc and M1R-ECD-His antigen by subcutaneous injection and intraperitoneal injection; (Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.), immunized once every two weeks, for a total of 4 immunizations. One week after the fourth immunization, the mouse blood was collected for immune titer detection, and finally, M1R-ECD-mFc was used for booster immunization once more.

4.2 Detection of serum antibody titer in mice after immunization

ELISA plates were coated with 2 μg/mL M1R-ECD-His at 4°C overnight (30 μL/well), washed 3 times, and blocked with 5% skim milk (5% PBSM) at room temperature for 1 hour; washed 3 times, mouse serum diluted in 5% PBSM was added, and antibody 7D11-chimera (sequence: PMID: 17688903) was added as a positive control, and incubated at room temperature for 1 hour; washed 6 times, and 5% After incubation at room temperature for 1 hour with secondary antibodies Goat-anti-mouse-lgG (1+2a+2b+3)-HRP (Jackson, 115-035-164) or Goat-anti-human-Kappa+Lambda-HRP (Millipore, AP502P+AP506P) diluted with mouse serum in gradient dilutions of PBSM, the plate was washed 6 times and TMB was added for 5-20 minutes for color development. After terminating the color development reaction, the data was read by using an OD450 microplate reader. The results are shown in Tables 1 and 2. The titers of the sera of the 8 mice were all up to standard.

Table 1: Final immunization serum titer test (ELISA)

Table 2: Final immunization serum titer test (ELISA)

4.3 Construction of phage display antibody gene library

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

4.4 Cell-based screening of phage-displayed antibody gene libraries

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, B310KJ), then 2 mL of 4% paraformaldehyde (Sangong, E672002-0500) was added for fixation for 0.5 hours, and finally washed twice with PBS and used as the screening raw material. During screening, the phage display library was incubated with fixed M1R-FL-EGFP-HEK293 cells at room temperature for 1 hour, washed three times with 1×PBS, and then 2 mL of glycine-HCl (pH=2.0) was added and gently mixed for 10 minutes to elute the phage that specifically binds to human ROR1. The eluted supernatant was then infected with SS320 bacteria (Lucigen, 60512-1) in the logarithmic phase, and then cultured at 37°C and 220 rpm for 1 hour. VSCM13 helper phage was added, and the cells were allowed to stand for 30 minutes. The cells were further cultured at 37°C and 220 rpm for 1 hour, centrifuged and replaced in C ⁺ /K ⁺ 2-YT medium, and the phages finally obtained were used for the second round of screening. The screening was repeated many times, and 10 clones were randomly selected for sequence analysis in each round to evaluate the library. After 3 rounds of screening, the sequence enrichment in the library was obvious.

4.5 Screening of phage display antibody gene library by immunotube method

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

The immunotube screening is to coat the antigen protein M1R-ECD-His or M1R-ECD-mFc on the surface of an immunotube with high adsorption capacity, add the phage display antibody library to the immunotube and incubate, wash and elute with the antigen protein adsorbed on the surface of the immunotube, undergo 2-4 rounds of panning, and finally enrich the specific monoclonal antibody Fab for the antigen. In this embodiment, the monoclonal antibody Fab for M1R-ECD-His was enriched after 3 rounds of panning. The specific method refers to Example 2.4.2 in patent CN112250763B.

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

4.6 Selection of single clones

After three rounds of screening, the second and third rounds of positive clones were selected for ELISA screening. Finally, 200 positive clones that could bind to M1R-ECD-His protein were screened out of 354 clones. After sequencing analysis, the sequences of 6 clones were finally selected to construct the full length for the next experiment.

The specific implementation methods are as follows:

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

Clone M1R_M_PR_A139 is the preferred molecule. The CDR amino acid sequence of the obtained antibody Fab is shown in the sequence listing and sequence table. The CDR sequence was determined using the AbM definition method.

Example 5 Construction and screening of humanized backbone nanobody phage display library

In this example, the phage display library constructed and prefabricated by our company was used for antibody screening. The library dAb-15 used was a humanized framework nanoantibody library with a library capacity of 2.52×10 ¹¹ . The screening path used included: screening the library with M1R-ECD-His and M1R-ECD-mFc. Antibody molecules that specifically bind to M1R-ECD-His were obtained.

5.1 Phage display library construction

dAb-15 is a humanized framework nano antibody library. Based on the fully humanized nano antibody framework, random primers of different lengths are designed to amplify gene fragments in different CDR regions, respectively, and recombined to construct a humanized framework nano antibody library (the library construction method refers to Example 8.1 in patent CN112625136A). The fully synthetic phage display antibody library obtained by recombining three CDRs. Through gradient dilution plating, the library capacity of this library was measured to be 2.52×10 ¹¹ , that is, an antibody gene library of 2.52×10 ¹¹ antibody genes (the library capacity calculation method refers to Example 2.2 in patent CN112250763B). VSCM13 helper phage (purchased from Stratagene) was used to package it to obtain an antibody gene phage display library (the preparation of the antibody gene phage display library refers to Example 2.3 in patent CN112250763B).

5.2 Screening of phage display libraries

Based on the immunotube method, the above library was screened using M1R-ECD-His and M1R-ECD-mFc. The purpose of the screening was to obtain antibodies with relatively conservative binding sequences and good affinity activity.

5.2.1 Screening of antibody gene phage display library by immunotube method

The principle of immunotube screening is to coat M1R-ECD-His or M1R-ECD-mFc on the surface of an immunotube with high adsorption capacity, add the phage display antibody library to the immunotube and perform a panning process of incubation, washing and elution with the antigen protein adsorbed on the surface of the immunotube. After three rounds of panning, specific monoclonal antibodies against the antigen are finally enriched.

The specific implementation methods are as follows:

In the first round of screening, 1 mL of 100 μg/mL M1R-ECD-His or M1R-ECD-mFc was added to the immunotube and coated overnight at 4°C. The coating solution was discarded the next day, and 5% milk in PBS was added for blocking for 2 hours. After rinsing twice with PBS, the phage library of nanoantibody genes of a total of 200 alpacas was added and incubated for 2 hours. The tube was rinsed 8 times with PBST and then rinsed twice with PBS to remove non-specifically bound phages. Then 0.8 mL of 0.05% EDTA trypsin digestion solution was added to the immunotube to elute the phage that specifically binds to the target antigen. The phage was then infected with SS320 bacteria (Lucigen, 60512-1) in the logarithmic phase and allowed to stand at 37°C for 30 minutes, then cultured at 220 rpm for 1 hour, and then VSCM13 helper phage was added, allowed to stand for 30 minutes, and continued to be cultured at 220 rpm for 1 hour, centrifuged and replaced to C ⁺ /K ⁺ 2-YT medium, and continue to culture overnight at 30°C, 220rpm. The next day, phages were precipitated for subsequent 2-4 rounds of screening. The second and third rounds of phage screening were coated with antigens: M1R-ECD-His or M1R-ECD-mFc, and the antigen coating concentration decreased in turn, 30μg/mL and 10μg/mL respectively; in addition, the PBST rinsing intensity was gradually increased, and the number of PBST elutions was 10 and 14 times respectively.

The ELISA was performed on the eluted phage pools in each round by coating M1R-ECD-His to evaluate the enrichment effect, and 10 clones were randomly selected from the phage pools in each round for sequence analysis. The results showed that the antibody sequence was significantly enriched after the third round of screening, and each round had better enrichment. Therefore, the clones obtained in the second and third rounds were selected for ELISA positive clone screening.

5.3 Selection of monoclonal

After three rounds of screening, clones obtained in the second and third rounds were selected for ELISA screening of positive clones. Finally, 296 positive clones that could bind to M1R-ECD-His protein were screened out of 446 clones. 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 the preferred molecule. The CDR amino acid sequence of the obtained VHH is shown in the sequence listing and sequence table. The CDR sequence was determined using the AbM definition method.

Example 6 Construction, expression and purification of candidate antibodies

6.1 Plasmid construction

The VH coding sequence in the Fab sequence of the monoclonal antibody obtained by screening was connected to the coding sequence of the heavy chain constant region of human IgG1 (SEQID NO: 45) to obtain the heavy chain coding sequence of the antibody, the VL coding sequence in the Fab sequence was connected to the coding sequence of the Kappa type (SEQ ID NO: 47) or Lambda type (SEQ ID NO: 48) 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 was connected to the coding sequence of the Fc region (IgG1-CS mutant type) (SEQ ID NO: 46) of human IgG1 to obtain the coding sequence of VHH-Fc. The constructed coding sequences were respectively inserted into the eukaryotic expression vector plasmid GSV0, transformed into Escherichia coli DH5α, and cultured at 37°C overnight. The plasmid was extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain an endotoxin-free antibody plasmid for eukaryotic expression.

6.2 Expression and purification of candidate antibodies

The candidate antibody was expressed by the ExpiCHO transient transfection expression system (Thermo Fisher, A29133), and the specific method was as follows: On the day of transfection, the cell density was confirmed to be about 7×10 ⁶ to 1×10 ⁷ viable cells/mL, and the cell survival rate was >98%. At this time, the cells were adjusted to a final concentration of 6×10 ⁶ cells/mL with fresh ExpiCHO expression medium pre-warmed at 37°C. The plasmid constructed in Example 3.1 was diluted with OptiPROTM SFM pre-cooled at 4°C (1 μg of plasmid was added to 1 mL of the culture medium), and ExpiFectamineTM CHO was diluted with OptiPROTM SFM at the same time, and then the two were mixed in equal volumes and gently pipetted to mix to prepare ExpiFectamineTM CHO/plasmid DNA mixed solution, incubated at room temperature for 1-5 minutes, slowly added to the prepared cell suspension and gently shaken at the same time, and finally placed in a cell culture shaker and cultured at 37°C and 8% CO ₂ .

18-22h after transfection, add ExpiCHOTMEnhancer and ExpiCHOTMFeed to the culture medium, and place the shake flask in a 32°C shaker and 5% CO ₂ to continue culturing. On the 5th day after transfection, add the same volume of ExpiCHOTMFeed, and gently mix the cell suspension while slowly adding it. 7 days after transfection, the cell culture supernatant expressing the target protein was centrifuged at 15000g for 10min, and the resulting supernatant was affinity purified with MabSelect SuRe LX (GE, 17547403), and then the target protein was eluted with 100mM sodium acetate (pH3.0), followed by neutralization with 1M Tris-HCl, and finally the resulting protein was exchanged into PBS buffer through an ultrafiltration concentration tube (Millipore, UFC901096).

Example 7 Identification of Physicochemical Properties of Candidate Antibodies

Preparation of non-reducing solution: 1 μg of the candidate antibody of Example 6 and the quality control product IPI (i.e., Ipilimumab) were added to 5×SDS loading buffer and 40 mM iodoacetamide, heated in a 75°C dry bath for 10 min, cooled to room temperature, and centrifuged at 12000 rpm for 5 min to obtain the supernatant.

Preparation of reducing solution: 2 μg of the candidate antibody in Example 6 and the quality control product IPI were added with 5×SDS loading buffer and 5 mM DTT, heated in a 100°C dry bath for 10 min, cooled to room temperature, and centrifuged at 12,000 rpm for 5 min to obtain the supernatant.

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

The results are shown in Table 3. The purity of the antibodies was greater than 95%.

Table 3. Physical and chemical test results of candidate antibodies

Example 8 Affinity detection of candidate antibodies at the ELISA level

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

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

96-well ELISA plates (30 μL/well) were coated with 2 μg/mL M1R-ECD-His at 4°C overnight. The next day, the plates were washed 3 times with PBST and blocked with 5% skim milk for 2 hours. After washing the plates 3 times with PBST, gradient dilutions (3.0, 0.33, 0.11, 0.037, 0.012, 0.004, 0.0014, 0.0002 μg/mL) of each antibody and positive control antibody 7D11-chimera (sequence: PMID: 17688903) were added and incubated for 1 hour. After that, the plates were washed 3 times with PBST and the secondary antibody Goat-Anti-human Fc-HRP (abcam, ab97225) was added and incubated for 1 hour. After the incubation, the plates were washed 6 times with PBST and TMB (SurModics, TMBS-1000-01) was added for color development. According to the color development results, 2 M stop solution was added to terminate the reaction, and the absorbance was read at OD450 using a microplate reader (Molecular Devices, SpecterMax 190).

The results are shown in Figure 2 and Table 4: The antibody molecules NL-M1R-A139, NL-M1R-A185, NL-M1R-A198, and NL-M1R-A98 have strong binding activity with the antigen protein M1R-ECD-His.

Table 4. Binding data of candidate antibodies in the full human library to M1R-ECD-His

Protein Name Library Source _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 ability of candidate antibodies from the mouse immune library to M1R-ECD-His

Coat 96-well ELISA plates (30 μL/well) with 2 μg/mL M1R-ECD-His at 4°C overnight. The next day, wash the plates three times with PBST and block with 5% skim milk for 2 hours. Wash the plates three times with PBST, add gradient dilutions (3.0, 0.33, 0.11, 0.037, 0.012, 0.004, 0.0014, 0.0002 μg/mL) of each antibody and positive control antibody 7D11-chimera and incubate for 1 hour. After that, wash the plates three times with PBST, add secondary antibody Goat-Anti-human Fc-HRP (abcam, ab97225) and incubate for 1 hour. After incubation, wash the plates six times with PBST and add TMB (SurModics, TMBS-1000-01) for color development. According to the color development results, 2 M stop solution was added to terminate the reaction, and the absorbance was read at OD450 using a microplate reader (Molecular Devices, SpecterMax 190).

The results are shown in FIG3 and Table 5: The antibody molecule M1R_M_PR_A139 has a strong binding activity with the antigen protein M1R-ECD-His.

Table 5. Binding data of mouse immunization candidate antibodies to M1R-ECD-His

Protein Name Library Source _EC50 (μg/mL) M1R_M_PR_A139 Mouse immune library 0.002317

8.3 ELISA-based detection of the binding ability of candidate antibodies in the nanobody library to M1R-ECD-His

Coat 96-well ELISA plates (30 μL/well) with 2 μg/mL M1R-ECD-His at 4°C overnight. The next day, wash the plates three times with PBST and block with 5% skim milk for 2 hours. Wash the plates three times with PBST, add gradient dilutions (3.0, 0.33, 0.11, 0.037, 0.012, 0.004, 0.0014, 0.0002 μg/mL) of each antibody and positive control antibody 7D11-chimera and incubate for 1 hour. After that, wash the plates three times with PBST, add secondary antibody Goat-Anti-human Fc-HRP (abcam, ab97225) and incubate for 1 hour. After incubation, wash the plates six times with PBST and add TMB (SurModics, TMBS-1000-01) for color development. According to the color development results, 2 M stop solution was added to terminate the reaction, and the absorbance was read at OD 450 using a microplate reader (Molecular Devices, SpecterMax 190).

The results are shown in FIG4 and Table 6: The antibody molecule M1R_N_PR_A013 has a strong binding activity with the antigen protein M1R-ECD-His, with an EC ₅₀ of 0.008817 μg/mL.

Table 6. Binding data of candidate antibodies in the nanobody library to M1R-ECD-His

Protein Name Library Source _EC50 (μg/mL) M1R_N_PR_A013 Nanobody Library 0.008817

Example 9 ELISA double antibody sandwich method antibody pairing

In this example, the antibodies against the M1R-ECD-His antigen protein were paired based on the ELISA double antibody sandwich method.

Coat a 96-well ELISA plate (30 μL/well) with 2 μg/mL of antibody at 4°C overnight. The next day, wash the plate three times with PBST and block with 5% skim milk for 2 h. Wash the plate three times with PBST, add 2 μg/mL of M1R-ECD-His and incubate for 1 h. Wash the plate three times with PBST, add a gradient dilution (10, 3.33333, 1.11111, 0.37037, 0.12346, 0.04115, 0.01372, 0.00457 μg/mL) of biotinylated detection antibody and incubate for 1 h. After that, wash the plate three times with PBST, add the secondary antibody NeutrAvidin-HRP (Thermo, 31001) and incubate for 1 h. After incubation, the plate was washed 6 times with PBST and TMB (SurModics, TMBS-1000-01) was added for color development. According to the color development results, 2M stop solution was added to terminate the reaction, and the absorbance was read at OD450 using a microplate reader (Molecular Devices, SpecterMax 190).

The results are shown in Figure 5: The two pairs of paired antibodies, M1R_M_PR_A139 and M1R_N_PR_A013, and M1R_M_PR_A139 and NL-M1R-A198, have better effects in detecting M1R-ECD-His antigen. In Figure 5, the antibody M1R_M_PR_A139 was coated on the ELISA plate, and the liquid phase was biotin-labeled with different candidate antibodies. It can be seen from the figure that when M1R_M_PR_A139 was coated on the ELISA plate and the liquid phase was biotin-labeled with M1R_N_PR_A013 or NL-M1R-A198, the detection effect was better, and its EC ₅₀ was 0.05359μg/mL and 0.02592μg/mL, respectively.

Example 10 Detection of M1R monkeypox virus protein using colloidal gold detection card

In this example, the antibody pairs M1R_M_PR_A139 and M1R_N_PR_A013, M1R_M_PR_A139 and NL-M1R-A198 selected in Example 9 for detecting the M1R-ECD-His antigen protein were used to prepare colloidal gold detection cards to test their sensitivity in detecting the M1R monkeypox virus protein.

10.1 Preparation of colloidal gold test card

In this embodiment, according to the method described in the embodiment of Chinese patent application CN102747040A, the above paired antibodies were used to prepare colloidal gold detection cards, and various quality tests were passed. In the detection card, antibody M1R_M_PR_A139 (1.71 mg/ml) was used as a capture antibody, and antibody M1R_N_PR_A013 (2.76 mg/ml) or NL-M1R-A198 (0.74 mg/ml) was used as a detection antibody.

10.2 Sensitivity determination

In this embodiment, the monkeypox virus M1R protein was diluted 2 times from 1 μg/mL to 650 pg/mL. The gradient diluted M1R protein was added to the sample wells of the colloidal gold detection card (left lane of the card) at a volume of 120 μL per well, and the color was developed for 20 minutes, wherein the C line was the quality control line and the T line was the detection line. Three parallel groups were set and 9 blank controls (only diluent was added). As shown in Figure 6, the detection sensitivity of the detection card for the monkeypox virus M1R protein can reach 10 ng/mL.

The left side of Figure 6 shows the M1R_M_PR_A139 and M1R_N_PR_A013 groups, and the right side shows the M1R_M_PR_A139 and NL-M1R-A198 groups.

Exemplary sequences

Claims (11)

1. An antibody or antigen binding fragment thereof that targets a monkey poxvirus, the antibody comprising a heavy chain variable region and a light chain variable region or comprising a heavy chain variable region but no 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 an LCDR1, an LCDR2 and an LCDR3 which are respectively shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6; 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 an LCDR1, an LCDR2 and an LCDR3 which are respectively shown in SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14; or,

the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown in SEQ ID NO. 17, SEQ ID NO. 18 and SEQ ID NO. 19 respectively; the light chain variable region comprises an LCDR1, an LCDR2 and an LCDR3 which are respectively shown in SEQ ID NO. 20, SEQ ID NO. 21 and SEQ ID NO. 22; or,

the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown in SEQ ID NO. 25, SEQ ID NO. 26 and SEQ ID NO. 27 respectively; the light chain variable region comprises an LCDR1, an LCDR2 and an LCDR3 which are respectively shown in SEQ ID NO. 28, SEQ ID NO. 29 and SEQ ID NO. 30; 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; the light chain variable region comprises an LCDR1, an LCDR2 and an LCDR3 which are respectively shown in SEQ ID NO. 36, SEQ ID NO. 37 and SEQ ID NO. 38;

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.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region has an amino acid sequence as shown in SEQ ID No. 7 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID No. 7, and the light chain variable region has an amino acid sequence as shown in SEQ ID No. 8 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID No. 8; or,

the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO. 15 or has 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 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 has 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 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 has 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 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 has 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 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 has 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 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 has 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 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 has 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 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 has 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 with SEQ ID NO 40; or,

the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO. 44 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO. 44.

3. The antibody or antigen-binding fragment thereof of claim 2, wherein the antibody is a full-length antibody, fab ', F (ab') 2, fv, VHH, or multispecific antibody, targeting the monkey poxvirus M1R protein;

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 kappa chain or lambda chain of a human antibody or a variant thereof;

more preferably, 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; when the antibody is a VHH, 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.

4. An antibody combination comprising one or more antibodies or antigen-binding fragments thereof according to 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 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; 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, and 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 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; the second antibody comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having amino acid sequences set forth in SEQ ID NO. 41, SEQ ID NO. 42 and SEQ ID NO. 43, respectively, but not a light chain variable region.

5. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1-3, or the combination of antibodies of claim 4.

6. A recombinant expression vector comprising the nucleic acid of claim 5;

preferably, the recombinant expression vector is a plasmid, cosmid, phage or viral vector;

more preferably, the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector or an adeno-associated viral vector.

7. A transformant comprising the recombinant expression vector of claim 6 in a host cell;

Preferably, the host cell is a prokaryotic cell or a eukaryotic cell;

more preferably, 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; the mammalian cells are, for example, HEK293 cells.

8. A method of making an antibody or antigen-binding fragment thereof that targets M1R, comprising culturing the transformant of claim 7, and obtaining the antibody or antigen-binding fragment thereof that targets M1R from the culture.

9. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-3, or the antibody combination of claim 4, and a pharmaceutically acceptable carrier.

10. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-3, or the antibody combination of claim 4, the nucleic acid of claim 5, the recombinant expression vector of claim 6, the transformant of claim 7, or the pharmaceutical composition of claim 9;

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

11. Use of an antibody or antigen binding fragment thereof according to any one of claims 1-3, an antibody combination according to claim 4, a nucleic acid according to claim 5, a recombinant expression vector according to claim 6, a transformant according to claim 7, a pharmaceutical composition according to claim 9, or a kit according to claim 10 for the preparation of a medicament for the diagnosis, prevention and/or treatment of a viral infection;

preferably, the viral infection is a poxvirus infection;

more preferably, the poxvirus infection is a monkey poxvirus infection.