Antibody combined with C5aR2 membrane protein and preparation method thereof

Abstract

The invention provides an antibody combined with a C5aR2 membrane protein, and also provides a preparation method and application of the antibody. The C5aR2 membrane protein or the epitope thereof is displayed by adopting VLP for the first time, the full-length membrane protein and the epitope of the membrane protein are subjected to post-translational modification with important functions through mammalian cell expression, the full-length membrane protein and the epitope of the membrane protein are used as antigen protein for immunization, and the monoclonal antibody with high-level binding affinity with C5aR2 is obtained through screening. The antibody provided by the invention can be applied to various detections such as ELISA and FACS, and has the potential of developing antibody drugs.

Description

Technical Field

[0001] The present invention relates to the field of antibodies, and in particular, to an antibody binding to C5aR2 membrane protein and a preparation method thereof. Background Art

[0002] C5aR2 (also known as C5L2 or GPR77, uniprot: Q9P296) is a member of the complement receptor subfamily in the G protein-coupled receptor (GPCR) superfamily. C5aR1 and C5aR2 are two subtypes of C5aR complement receptors. Both are co-expressed on neutrophils, macrophages, astrocytes and other cells, and are receptor proteins for the inflammatory mediator complement C5a. C5aR1 and C5aR2 have similar affinities to C5a, and C5aR2 has a higher binding affinity to C5a-des-Arg than C5aR1. C5aR1 is a typical GPCR that mediates downstream signaling pathways through G protein-coupled activation, or mediates G protein-independent β-arrestin signaling. C5aR2 is an atypical GPCR that does not exhibit G protein-coupled activity and only signals through β-arrestins. The third and seventh transmembrane regions of C5aR2 do not contain the conserved DRY and NPXXY motifs, but are replaced by D131LC and N287PLMF. Its intracellular third domain is a truncated form. These structural features are likely to have an important impact on its function. C5aR2 is a hot topic in the development of drugs for the treatment of inflammatory diseases. Other studies have shown that in the field of tumor treatment, blocking antibodies targeting C5aR2 can reduce the number of tumor stem cells and enhance tumor chemotherapy sensitivity [1,2].

[0003] Due to the low expression of GPCR membrane protein, it is difficult to prepare C5aR2 antigen protein for immunization and antibody screening. VLP displays full-length membrane protein or VLP displays specific membrane protein epitopes, becoming excellent immune antigens. Envelope VLP displays full-length membrane protein, and membrane protein has a natural conformation. VLP has the function of immune adjuvant, which can stimulate the host to produce an immune response against membrane protein, which is conducive to screening and obtaining membrane protein-specific antibodies. Non-enveloped VLP displays important epitopes of membrane protein, which is conducive to activating the host to produce an immune response against specific epitopes, thereby screening and obtaining functional antibodies. The development of antibodies that bind to C5aR2 membrane protein can become a tool for studying GPCR and also has the potential for drug development. Summary of the invention

[0004] The object of the present invention is to provide an antibody binding to C5aR2 membrane protein and a method for preparing the same.

[0005] In a first aspect of the present invention, an anti-C5aR2 antibody or an antigen-binding fragment thereof is provided, wherein the anti-C5aR2 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region selected from the group consisting of:

[0006] (Z1) the heavy chain variable region comprises the following three CDRs: H-CDR1 as shown in SEQ ID NO:7; H-CDR2 as shown in SEQ ID NO:8; H-CDR3 as shown in SEQ ID NO:9; and

[0007] The light chain variable region comprises the following three CDRs: L-CDR1 as shown in SEQ ID NO: 13; L-CDR2 as shown in GIS; L-CDR3 as shown in SEQ ID NO: 14; or

[0008] (Z2) the heavy chain variable region comprises the following three CDRs: H-CDR1 as shown in SEQ ID NO: 17; H-CDR2 as shown in SEQ ID NO: 18; H-CDR3 as shown in SEQ ID NO: 19; and

[0009] The light chain variable region comprises the following three CDRs: L-CDR1 shown in SEQ ID NO:23; L-CDR2 whose sequence is shown in LVS; and L-CDR3 shown in SEQ ID NO:24.

[0010] In another preferred example, the heavy chain variable region and the light chain variable region further comprise a FR region.

[0011] In another preferred example, the heavy chain variable region comprises a murine or human FR region, and / or the light chain variable region comprises a murine or human FR region.

[0012] In another preferred embodiment, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 90% (preferably at least 95%, 96%, 97%, 98%, 99%) sequence identity thereto; and / or

[0013] The light chain variable region of the antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 12, or an amino acid sequence having at least 90% (preferably at least 95%, 96%, 97%, 98%, 99%) sequence identity thereto.

[0014] In another preferred example, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:6, and / or a light chain variable region with an amino acid sequence as shown in SEQ ID NO:12.

[0015] In another preferred embodiment, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 16, or an amino acid sequence having at least 90% (preferably at least 95%, 96%, 97%, 98%, 99%) sequence identity thereto; and / or

[0016] The light chain variable region of the antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 22, or an amino acid sequence having at least 90% (preferably at least 95%, 96%, 97%, 98%, 99%) sequence identity thereto.

[0017] In another preferred example, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 16, and / or a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 22.

[0018] In another preferred embodiment, the antibody further comprises a heavy chain constant region and / or a light chain constant region.

[0019] In another preferred embodiment, the heavy chain constant region and / or light chain constant region is of murine or human origin.

[0020] In another preferred embodiment, the heavy chain constant region is derived from mouse heavy chain IgG1 or IgG2a.

[0021] In another preferred embodiment, the light chain constant region is derived from mouse kappa (κ) chain.

[0022] In another preferred example, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 10 or 20.

[0023] In another preferred example, the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:15.

[0024] In another preferred embodiment, the antibody or antigen-binding fragment thereof includes: monoclonal antibody, polyclonal antibody, diabody, single-chain antibody (scFv), Fab, Fab', F(ab')2 antibody.

[0025] In another preferred embodiment, the antibody is a recombinant antibody.

[0026] In another preferred embodiment, the antibody is a recombinant monoclonal antibody, which comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO:1, and a light chain with an amino acid sequence as shown in SEQ ID NO:2.

[0027] In another preferred example, the recombinant monoclonal antibody comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO:3, and a light chain having an amino acid sequence as shown in SEQ ID NO:4.

[0028] In another preferred embodiment, the antibodies include animal-derived antibodies (eg, mouse-derived antibodies of different subtypes), chimeric antibodies (eg, human-mouse chimeric antibodies), and humanized antibodies.

[0029] In another preferred embodiment, the antibody binds to human C5aR2 and cynomolgus monkey C5aR2, and has human / monkey cross-species binding activity.

[0030] In another preferred embodiment, the antibody competes with the C5a ligand for binding to C5aR2, thereby blocking the binding of the C5a ligand to C5aR2.

[0031] In a second aspect of the present invention, a recombinant antibody is provided, wherein the recombinant antibody has:

[0032] (i) the sequence of the anti-C5aR2 antibody or antigen-binding fragment thereof according to the first aspect of the present invention; and

[0033] (ii) a signal peptide that promotes secretory expression of the antibody and / or a tag sequence for purification and detection.

[0034] In another preferred embodiment, the recombinant antibody has a signal peptide that promotes secretory expression of the antibody.

[0035] In another preferred embodiment, the signal peptide has an amino acid sequence as shown in SEQ ID NO: 5, 11 or 21.

[0036] In another preferred embodiment, the tag sequence is selected from: FLAG, Myc, His tag, etc.

[0037] In another preferred example, the recombinant antibody comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO:1, and a light chain with an amino acid sequence as shown in SEQ ID NO:2.

[0038] In another preferred example, the recombinant antibody comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO:3, and a light chain with an amino acid sequence as shown in SEQ ID NO:4.

[0039] In the third aspect of the present invention, a polynucleotide molecule is provided, which encodes a polypeptide selected from the group consisting of:

[0040] (1) the anti-C5aR2 antibody or antigen-binding fragment thereof according to the first aspect of the present invention; or

[0041] (2) The recombinant antibody according to the second aspect of the present invention.

[0042] In another preferred example, the polynucleotide molecule comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 25-28.

[0043] In the fourth aspect of the present invention, an expression vector is provided, which contains the polynucleotide molecule as described in the third aspect of the present invention.

[0044] In another preferred embodiment, the vector includes a eukaryotic cell expression vector and a prokaryotic cell expression vector.

[0045] In another preferred embodiment, the vector is a mammalian cell expression vector.

[0046] In another preferred embodiment, the expression vector is selected from the following group: DNA, RNA, viral vector, plasmid, transposon, other gene transfer system, or a combination thereof.

[0047] In the fifth aspect of the present invention, a host cell is provided, which contains the vector as described in the fourth aspect of the present invention, or the polynucleotide molecule as described in the third aspect of the present invention is integrated into its genome.

[0048] In another preferred embodiment, the host cells include eukaryotic cells (such as mammalian cells) and prokaryotic cells.

[0049] In a sixth aspect of the present invention, an antibody conjugate is provided, wherein the antibody conjugate comprises:

[0050] (a) the anti-C5aR2 antibody or antigen-binding fragment thereof according to the first aspect of the present invention or the recombinant antibody according to the second aspect of the present invention; and

[0051] (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, an enzyme, a cytokine, a radionuclide, and a nanoparticle / nanorod.

[0052] In another preferred embodiment, the detectable marker includes a fluorescent marker or a chemiluminescent marker.

[0053] In another preferred embodiment, the radioactive nuclide includes: diagnostic isotope; and / or therapeutic isotope.

[0054] In another preferred embodiment, the drug is a cytotoxic drug.

[0055] In another preferred embodiment, the cytotoxic drug is selected from the following group: anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or a combination thereof.

[0056] In another preferred embodiment, the antibody conjugate is an antibody-drug conjugate (ADC).

[0057] In a seventh aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0058] (a) the anti-C5aR2 antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the recombinant antibody according to the second aspect of the present invention, or the antibody conjugate according to the sixth aspect of the present invention; and

[0059] (b) a pharmaceutically acceptable carrier.

[0060] In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

[0061] In another preferred embodiment, the pharmaceutical composition is used to prepare a drug for preventing and / or treating a disease or condition associated with C5aR2.

[0062] In another preferred embodiment, the disease or condition associated with C5aR2 includes inflammatory disease, cancer or tumor.

[0063] In an eighth aspect of the present invention, there is provided a use of the anti-C5aR2 antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the recombinant antibody according to the second aspect of the present invention, or the antibody conjugate according to the sixth aspect of the present invention for preparing:

[0064] (1) a detection reagent, a detection plate or a detection kit for detecting C5aR2 molecules;

[0065] (2) A drug for preventing and / or treating a disease or condition associated with C5aR2.

[0066] In another preferred embodiment, the disease or condition associated with C5aR2 includes inflammatory disease, cancer or tumor.

[0067] In another preferred embodiment, the detection includes flow cytometry (FACS), cell immunofluorescence detection, ELISA detection, and protein immunoblot (WB) detection.

[0068] In another preferred embodiment, the use is diagnostic and / or non-diagnostic, and / or therapeutic and / or non-therapeutic.

[0069] In a ninth aspect of the present invention, there is provided a method for producing the anti-C5aR2 antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or the recombinant antibody according to the second aspect of the present invention, comprising the steps of:

[0070] (s1) culturing the host cell according to the fifth aspect of the present invention under conditions suitable for producing the antibody or antigen-binding fragment, thereby obtaining a culture containing the antibody or antigen-binding fragment;

[0071] (s2) isolating or recovering the antibody or antigen-binding fragment from the culture; and

[0072] (s3) Optionally, purifying and / or modifying the antibody or antigen-binding fragment obtained in step (b).

[0073] In the tenth aspect of the present invention, a method for detecting C5aR2 protein in a sample is provided, the method comprising the steps of:

[0074] (1) contacting a sample with the anti-C5aR2 antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, the recombinant antibody as described in the second aspect of the present invention, or the antibody conjugate as described in the sixth aspect of the present invention;

[0075] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of C5aR2 protein in the sample.

[0076] In another preferred embodiment, the sample includes: an in vitro tissue sample or a cell sample.

[0077] In another preferred embodiment, the aspect is an in vitro method.

[0078] In another preferred embodiment, the method is a non-diagnostic and non-therapeutic method.

[0079] In the eleventh aspect of the present invention, a method for screening anti-C5aR2 antibodies is provided, comprising the steps of:

[0080] (S1) using VLP to multivalently display C5aR2 protein or C5aR2 protein epitope, and expressing it in mammalian cells to obtain C5aR2 antigen-VLP for immunization;

[0081] (S2) immunizing a test animal with the C5aR2 antigen-VLP prepared in step (S1), and collecting antiserum from the immunized animal;

[0082] (S3) detecting the binding of the collected antiserum to the C5aR2 antigen-VLP. If the binding is positive, taking the spleen of the immunized animal to prepare hybridoma clones;

[0083] (S4) using the C5aR2 antigen-VLP to perform preliminary screening on hybridoma clones, thereby obtaining antigen-binding positive hybridoma clones;

[0084] (S5) using empty VLPs without C5aR2 antigen to further perform negative screening on clones that are positive for antigen binding, and screen out positive hybridoma clones that only bind to C5aR2 antigen;

[0085] (S6) The positive hybridoma clones that only bind to the C5aR2 antigen are limitedly diluted to a monoclonal state, and the binding of the monoclonal hybridoma cell supernatant to the C5aR2 antigen is repeatedly verified, thereby obtaining a monoclonal hybridoma cell line that can produce anti-C5aR2 antibodies that specifically bind to the C5aR2 antigen.

[0086] In another preferred embodiment, the C5aR2 antigen-VLP is an enveloped VLP (humanC5aR2 / VLP) displaying the full-length human C5aR2, or a non-enveloped VLP (human C5aR2NTD-VLP) displaying the N-terminal sequence of human C5aR2.

[0087] In another preferred example, the N-terminal sequence of human C5aR2 has an amino acid sequence as shown in SEQ ID NO:29.

[0088] In another preferred embodiment, the mammalian cells are selected from: HEK293 cells and CHO cells.

[0089] In another preferred embodiment, the test animal is selected from: mice, rats, rabbits, goats, monkeys, etc.

[0090] In another preferred embodiment, the detection in step (S3) is ELISA detection.

[0091] In another preferred embodiment, the screening in steps (S4) and (S5) are performed by indirect ELISA method.

[0092] In the twelfth aspect of the present invention, a method for preventing and / or treating a disease or condition associated with C5aR2 is provided, the method comprising: administering to a subject in need thereof an anti-C5aR2 antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention, a recombinant antibody as described in the second aspect of the present invention, an antibody conjugate as described in the sixth aspect of the present invention, or a pharmaceutical composition as described in the seventh aspect of the present invention.

[0093] In another preferred embodiment, the subject in need thereof includes a human or non-human mammal.

[0094] In another preferred embodiment, the disease or condition associated with C5aR2 includes inflammatory disease, cancer or tumor.

[0095] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figure 1 The Elisa test of antiserum obtained by immunization with C5aR2 antigen and antigen protein is shown. A shows the binding of antiserum obtained by immunization with full-length human C5aR2 / VLP to two antigens, full-length human C5aR2 / VLP and human C5aR2 NTD-VLP; B shows the binding of antiserum obtained by immunization with human C5aR2 NTD-VLP to two antigens.

[0097] Figure 2 The binding of 17A2D9 and 30F10E9 recombinant antibodies to human and cynomolgus monkey C5aR2 is shown. A shows that 17A2D9 and 30F10E9 recombinant antibodies can bind to human C5aR2; B shows that 17A2D9 and 30F10E9 recombinant antibodies can bind to cynomolgus monkey C5aR2.

[0098] Figure 3 The binding of 17A2D9 and 30F10E9 recombinant antibodies, and C5a ligand to human C5aR2NTD-VLPs, respectively, is shown.

[0099] Figure 4 The results of the competitive binding of 17A2D9 and 30F10E9 recombinant antibodies to C5a ligand and human C5aR2NTD-VLP are shown.

[0100] Figure 5 The results of FACS flow cytometry detection of recombinant monoclonal antibodies binding to Hela cells and competing with C5a ligands are shown. A shows the results of 17A2D9 recombinant antibodies binding to Hela cells and competing with C5a ligands; B shows the results of 30F10E9 recombinant antibodies binding to Hela cells and competing with C5a ligands. DETAILED DESCRIPTION

[0101] After extensive and in-depth research, a large number of screening and verification, the inventors finally obtained two antibodies that bind to the membrane protein C5aR2, and for the first time proposed a method for preparing antibodies that specifically bind to the C5aR2 membrane protein by using VLP to display the C5aR2 membrane protein or its epitope as an antigen to immunize animals. The present invention uses envelope VLPs that display the full-length human C5aR2 membrane protein and non-enveloped VLPs that display the N-terminal sequence of the membrane protein C5aR2 for immunization, respectively, to obtain two high-affinity mouse monoclonal antibodies 17A2D9 and 30F10E3 that bind to different epitopes of the C5aR2 membrane protein. Both antibodies have cross-species binding activity of human / monkey C5aR2 membrane proteins, and can be used for Elisa and FACS to detect membrane proteins. Among them, the mouse monoclonal antibody 30F10E3 can competitively block the binding of the ligand C5a to the receptor human C5aR2 membrane protein.

[0102] On this basis, the present invention has been completed.

[0103] the term

[0104] In order to more easily understand the present invention, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can be changed. It should also be understood that the terms used herein are intended only to describe specific embodiments, and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0105] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0106] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0107] The three-letter and one-letter codes for amino acids used in the present invention are as described in J. biol. chem, 243, p3558 (1968).

[0108] As used herein, the term "optional" or "optionally" means that the event or situation described subsequently may occur but need not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable region of a specific sequence may have but need not have, and may be 1, 2 or 3.

[0109] "Sequence identity" as used herein refers to the degree of identity between two nucleic acid or amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions or deletions. The sequence identity between the sequences described herein and the sequences with which they are identical may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

[0110] As used herein, the term "non-enveloped VLP multivalent display" or "non-enveloped VLP display" refers to fusing the antigen epitope to the VLP (Virus Like Particle) capsid monomer subunit through structural design, expressing it in mammalian cells, and the VLP monomers self-assembling to form VLP particles and displaying the antigen epitope on the VLP surface.

[0111] As used herein, the term "enveloped VLP multivalent display" or "enveloped VLP display" refers to the expression of enveloped VLP (Enveloped VLP) in mammalian HEK293 cells. After the protein subunits are assembled into particles, the particles bud from the cells, bringing out the full-length membrane protein recombinantly expressed on the cell surface, and displaying it on the particle surface to form membrane protein / VLP (lipoparticles) (see CN117003876B). The C5aR2 / VLP prepared by this method directly displays multi-molecule, conformationally intact C5aR2 membrane proteins on the surface of the VLP without the need for additional surfactants. It is a way to obtain membrane proteins, and VLP has the function of an immune adjuvant. C5aR2 / VLP is suitable for immunization as a membrane protein antigen.

[0112] Antibody

[0113] As used herein, the term "antibody" or "immunoglobulin" is a heterotetramer formed by two light chains (L) and two heavy chains (H). The N-terminus of each heavy chain is a variable region (VH), connected to the heavy chain constant region. The N-terminus of each light chain is a variable region (VL), connected to the light chain constant region.

[0114] As used herein, the term "variable" means that the variable region in an antibody is different in a specific sequence, forming the affinity and specificity of a specific antibody for binding to a specific antigen. The variable region of an antibody includes a complementary determining region (CDR) or a hypervariable region and a framework region (FR) whose sequence is relatively conservative. The primary sequence of the heavy chain and light chain variable regions is composed of 4 FR sequences and 3 CDR sequences arranged at intervals (see Kabat et al., NIH Publ. No. 91-3242, Volume I, 647-669 pages (1991)). The sequence and spatial structural conformation of the heavy chain and light chain variable regions determine the specific binding of the antibody to the antigen epitope. The constant region of the antibody does not directly participate in the binding of the antibody to the antigen, but has an impact on the performance of the capture antibody and the detection antibody.

[0115] The "light chain" of vertebrate antibodies (immunoglobulins) can be classified into one of the categories of κ and λ according to the amino acid sequence of their constant region. According to the amino acid sequence of their heavy chain constant region, immunoglobulins can be divided into different categories, mainly 5 types of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and antibody subtypes (isotypes), such as mouse IgG including IgG1, IgG2a, IgG2b subtypes. The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known to those in the art.

[0116] As used herein, the term "monoclonal antibody (mAb)" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical. Monoclonal antibodies are highly specific for a single antigenic determinant (epitope). The modifier "monoclonal" indicates the characteristic of the antibody, being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring any particular method to produce the antibody.

[0117] The present invention also includes monoclonal antibodies having the corresponding amino acid sequence of the monoclonal antibody against C5aR2, monoclonal antibodies having the variable region chain of the monoclonal antibody against C5aR2, and other proteins or protein conjugates and fusion expression products having these chains. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a light chain and a heavy chain containing a variable region (complementarity determining region, CDR), as long as the variable region is identical to the variable region of the light chain and heavy chain of the present invention or has at least 90% homology, preferably at least 95% homology.

[0118] As known to those skilled in the art, antibody conjugates and fusion expression products include: fluorescent or luminescent markers, radioactive markers, enzymes capable of producing detectable products, gold nanoparticles / nanorods and other molecules that can be used for detection are combined with the anti-C5aR2 antibody or its antigen-binding fragment to form a conjugate; or therapeutic drugs, toxins, radionuclides and other therapeutic molecules are combined with the anti-C5aR2 antibody or its antigen-binding fragment to form a conjugate.

[0119] The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of a full-length antibody can be used to perform the antigen-binding function of an antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment of an antibody" include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond on the hinge region; (iii) a scFv fragment consisting of the VH and VL domains of a single arm of an antibody.

[0120] The present invention includes not only complete monoclonal antibodies, but also antibody fragments having binding activity, such as Fab or (Fab')2 fragments; antibody heavy chains; antibody light chains or scFv.

[0121] The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-consecutive amino acids in a unique spatial conformation.

[0122] The terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen.

[0123] As used herein, the term "antigenic determinant" refers to a discrete three-dimensional site on an antigen that is recognized by the antibodies or antigen-binding fragments of the present invention.

[0124] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0125] In the present invention, antibodies include mouse antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies can be prepared using DNA recombination techniques well known in the art. The term "mouse antibody" in the present invention refers to a monoclonal antibody against C5aR2 membrane protein prepared according to the knowledge and skills in the art.

[0126] In the present invention, the antibodies may be monospecific, bispecific, trispecific, or more multispecific.

[0127] As used herein, the term "heavy chain variable region" and "VH" are used interchangeably. The term "light chain variable region" and "VL" are used interchangeably.

[0128] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242).

[0129] In one aspect of the present invention, an anti-C5aR2 antibody or an antigen-binding fragment thereof is provided. The present invention obtains two anti-C5aR2 antibodies by mouse immune screening, one of which is a monoclonal antibody with a clone number of 17A2D9 obtained by immunizing mice with envelope VLPs of the full-length human C5aR2 membrane protein, and the other is a monoclonal antibody with a clone number of 30F10E3 obtained by immunizing mice with non-enveloped VLPs displaying the N-terminal sequence of the membrane protein C5aR2.

[0130] Heavy chain amino acid sequence of the recombinant 17A2D9 antibody (SEQ ID NO: 1):

[0131] MEWSWIFLFLLSGTAGVHS EVQLQQSGPELVKPGASVKMSCKAS GYTFTSYV IHWVKQKPGQGLEWIGY INPYNDGT NYNEKFKGKATLTSDKSSSTAFMELSSLTSEDSAVYYC AISPYYGNPFAYWGQGTRVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQIS WFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0132] Among them, the bold underlined part at the N-terminus is the signal peptide sequence (SEQ ID NO: 5); the middle part is the heavy chain variable region (SEQ ID NO: 6), and the underlined parts are the CDRs of the heavy chain variable region: H-CDR1 (SEQ ID NO: 7), H-CDR2 (SEQ ID NO: 8) and H-CDR3 (SEQ ID NO: 9), which are divided according to the IMGT rules; the italic bold part is the heavy chain constant region (mouse IgG2a, SEQ ID NO: 10).

[0133] Light chain amino acid sequence of the recombinant 17A2D9 antibody (SEQ ID NO: 2):

[0134] MNLPVHLLVLLLFWIPASRG DVVVTQTPLSLPVSFGNQVSISCRSS QSLANSYGNTY LSWYLHKPGQSPQLLIY GIS NRFSGVPDRFSGSGSGTDFTLKISAIKPEDLGIYYC LQTQ FGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0135] Among them, the bold underlined part at the N-terminus is the signal peptide sequence (SEQ ID NO: 11); the middle part is the light chain variable region (SEQ ID NO: 12), and the underlined parts are the CDRs of the light chain variable region: L-CDR1 (SEQ ID NO: 13), L-CDR2 (GIS) and L-CDR3 (SEQ ID NO: 14), which are divided according to the IMGT rules; the italic bold part is the light chain constant region (mouseIg kappa, SEQ ID NO: 15).

[0136] Heavy chain amino acid sequence of the recombinant 30F10E3 antibody (SEQ ID NO: 3):

[0137] MEWSWIFLFLLSGTAGVHS QVQLKESGPGLVAPSQSLSITCTVS GFSLMDYG VSWIRQPPGKGLEWLGV IUsQ NYNSALKSRLNIREDKSKSQVVLKMSSLQTDDTAMYYC AWGFLY WGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFV DDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0138] Among them, the bold underlined part at the N-terminus is the signal peptide sequence (SEQ ID NO: 5); the middle part is the heavy chain variable region (SEQ ID NO: 16), and the underlined parts are the CDRs of the heavy chain variable region: H-CDR1 (SEQ ID NO: 17), H-CDR2 (SEQ ID NO: 18) and H-CDR3 (SEQ ID NO: 19), which are divided according to the IMGT rules; the italic bold part is the heavy chain constant region (mouse IgG1, SEQ ID NO: 20).

[0139] Light chain amino acid sequence of the recombinant 30F10E3 antibody (SEQ ID NO: 4):

[0140] MRVLAELLGLLLFCFLGVRC DVVMTQTPLTLSVTFGQPASISCKSS QSLLHSDGRTY LNWLLQRPGQSPKRLIY LVS KLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC WQH FGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0141] Among them, the bold underlined part at the N-terminus is the signal peptide sequence (SEQ ID NO: 21); the middle part is the light chain variable region (SEQ ID NO: 22), and the underlined parts are the CDRs of the light chain variable region: L-CDR1 (SEQ ID NO: 23), L-CDR2 (LVS) and L-CDR3 (SEQ ID NO: 24), which are divided according to the IMGT rules; the italic bold part is the light chain constant region (mouseIg kappa, SEQ ID NO: 15).

[0142] The function of the antibody of the present invention is determined by the light chain and heavy chain variable region sequences and the antibody structural conformation, and can specifically bind to the C5aR2 membrane protein. Using the antibody variable region gene or complementary determining region (CDR) gene, different forms of genetically engineered antibodies can be transformed and produced in any expression system using prokaryotic and eukaryotic cells.

[0143] In the present invention, the terms "antibody of the present invention", "protein of the present invention", or "polypeptide of the present invention" are used interchangeably and refer to an antibody that specifically binds to the C5aR2 membrane protein, such as a protein or polypeptide having a heavy chain (such as the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 25) and a light chain (such as the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 26); or a protein or polypeptide having a heavy chain (such as the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 27) and a light chain (such as the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 28).

[0144] "Fragments", "derivatives" and "analogs" of antibodies refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substituent group in one or more amino acid residues, or (iii) polypeptides formed by coupling a mature polypeptide with another compound (such as a compound for chemiluminescence, such as an acridinium ester), or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a tag protein sequence or other fusion protein sequence used to purify or detect the polypeptide). These fragments, derivatives and analogs are well known to those skilled in the art.

[0145] The antibody of the present invention refers to a polypeptide having C5aR2 membrane protein binding activity and comprising the above CDR region. The term also includes variant forms of polypeptides having the same function as the antibody of the present invention and comprising the above CDR region. These variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions, insertions and / or substitutions, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. The term also includes active fragments and active derivatives of the antibodies of the present invention.

[0146] The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, and polypeptides or proteins obtained by using antiserum against the antibody of the invention.

[0147] The present invention also includes fragments of the antibodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibodies of the present invention, preferably at least about 60 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0148] Polynucleotide molecules, vectors and host cells

[0149] The present invention also provides a polynucleotide molecule encoding the above-mentioned antibody or its fragment or its fusion protein. The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide can be the same as the coding region sequence shown in SEQ ID NO:25-28 or a degenerate variant. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence that encodes an amino acid sequence identical to the polypeptide of the present invention, but differs from the coding region sequence shown in SEQ ID NO:25-28.

[0150] Heavy chain nucleotide sequence of recombinant 17A2D9 antibody (SEQ ID NO: 25)

[0151]

[0152] Light chain nucleotide sequence of recombinant 17A2D9 antibody (SEQ ID NO: 26)

[0153] ATGAATCTTCCCGTGCACCTGCTTGTATTGCTGCTTTTCTGGATACCCGCATCAAGGGGAGACGTGGTGGTCACCCAAACACCGTTGTCCCTTCCAGTTTCCTTTGGGAACCAAGTGAGCATCAGCTGTCGCTCATCCCAAAGTCTGGCAAACTCATACGGCAACACTTACCTTTCATGGTATCTTCATAAGCCCGGACAATCTCCACAGCTTTTGATCTATGGAATCTCCAACAGGTTTAGCGGCGTGCCTGATCGATTTTCAGGATCTGGCTCAGGGACCGACTTCACACTGAAGATCAGCGCCATAAAGCCCGAAGACCTCGGAATTTATTACTGTCTCCAGGGAACTCATCAGCCGTGGACTTTTGGAGGGGGAACAAAACTGGAGATTAAACGCGCCGATGCCGCTCCTACAGTGAGCATCTTTCCTCCTTCCTCCGAGCAGCTGACAAGCGGCGGCGCCAGCGTGGTGTGTTTCCTGAACAACTTCTATCCTAAGGACATCAATGTGAAGTGGAAGATCGACGGCAGCGAGAGACAGAACGGCGTGCTGAACTCCTGGACCGACCAGGATTCCAAGGACTCCACCTACTCCATGTCCTCCACACTGACCCTGACCAAGGATGAGTACGAGAGGCACAACAGCTACACATGCGAGGCCACACACAAGACCTCCACCAGCCCTATCGTGAAGAGCTTCAATAGAAACGAGTGC

[0154] Heavy chain nucleotide sequence of recombinant 30F10E3 antibody (SEQ ID NO: 27)

[0155]

[0156] Light chain nucleotide sequence of recombinant 30F10E3 antibody (SEQ ID NO: 28)

[0157] ATGCGCGTTCTTGCTGAGCTGCTCGGCCTCCTGCTGTTCTGCTTCTTGGGCGTCAGGTGTGACGTCGTGATGACACAGACTCCTCTGACACTGTCAGTGACGTTCGGACAGCCCGCTTCCATTTCATGTAAGTCCTCCCAGAGTTTGTTGCACAGCGACGGTAGGACTTACCTGAATTG GTTGCTGCAAAGACCAGGTCAGTCTCCTAAGCGACTGATATACCTGGTGTCTAAGCTCGATAGCGGTGTCCCCGATAGATTTACTGGATCAGGTAGTGGCACCGACTTTACCCTGAAGATCTCTCGCGTGGAGGCAGAAGATCTGGGCGTCTACTACTGCTGGCAGGGCACTCACTTCC CTCACACTTTTGGAGGGGGAACAAAACTGGAGATTAAACGCGCCGATGCCGCTCCTACAGTGAGCATCTTTCCTCCTTCCTCCGAGCAGCTGACAAGCGGCGGCGCCAGCGTGGTGTGTTTCCTGAACAACTTCTATCCTAAGGACATCAATGTGAAGTGGAAGATCGACGGCAGCGAG AGACAGAACGGCGTGCTGAACTCCTGGACCGACCAGGATTCCAAGGACTCCACCTACTCCATGTCCTCCACACTGACCCTGACCAAGGATGAGTACGAGGCACAACAGCTACACATGCGAGGCCACACACAAGACCTCCACCAGCCCTATCGTGAAGAGCTTCAATAGAAACGAGTGC

[0158] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only a mature polypeptide; a coding sequence of a mature polypeptide and various additional coding sequences; a coding sequence of a mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0159] The term "polynucleotide encoding a polypeptide" may be a polynucleotide encoding the polypeptide, or may be a polynucleotide further comprising additional coding and / or non-coding sequences. The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can usually be obtained by PCR amplification, recombination or artificial synthesis. In addition, the heavy chain or light chain can be fused with a protein or tag sequence (such as a fluorescent protein, a Flag tag) to form a fusion protein.

[0160] Once the antibody sequence is obtained, recombinant antibodies can be prepared using bioengineering methods. Usually, the gene encoding the antibody sequence is cloned into a vector, and then the expression vector is transferred into cells for expression, and the cells or expression supernatant are harvested and purified to obtain recombinant antibodies. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form.

[0161] At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing plasmids (or other expression vectors) known in the art. In addition, mutations can also be introduced into the antibody sequence of the present invention by chemical synthesis.

[0162] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0163] Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0164] Transforming host cells with recombinant DNA to recombinantly express antibodies is a conventional technique well known to those skilled in the art. The recombinantly expressed antibodies can be separated and purified by conventional techniques well known to those skilled in the art, which will not be described in detail here.

[0165] Pharmaceutical composition

[0166] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment, or its fusion protein, or its immunoconjugate, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the properties of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intraperitoneal, intravenous, or topical administration.

[0167] The pharmaceutical composition of the present invention can be directly used to bind to C5AR2 protein molecules, and thus can be used to treat C5aR2-related diseases, such as inflammatory diseases, cancer or tumors, etc. In addition, other therapeutic agents can also be used simultaneously.

[0168] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned antibody (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 milligrams / kg body weight per day. In addition, the antibody or immunoconjugate of the present invention can also be used with other therapeutic agents.

[0169] In one embodiment of the invention, when using a pharmaceutical composition, a safe and effective amount of the antibody or immunoconjugate of the invention is administered to a mammal, wherein the safe and effective amount is usually at least about 10 μg / kg body weight, and in most cases does not exceed about 50 mg / kg body weight, preferably the dosage is about 10 μg / kg body weight to about 10 mg / kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

[0170] application

[0171] The antibodies of the present invention have wide biological and clinical application values, and their applications involve multiple fields such as diagnosis and treatment of diseases related to C5aR2, basic medical research, biological research, etc. A preferred application is for clinical diagnosis and targeted treatment of C5aR2-related diseases, such as diagnosis and treatment of inflammation-related diseases; or diagnosis and treatment of cancer or tumors.

[0172] Method for preparing antibody of the present invention

[0173] In one aspect of the present invention, a method for screening and preparing anti-C5aR2 antibodies is provided, the method comprising the steps of preparing VLP multivalently displaying C5aR2 antigen (C5aR2 antigen-VLP, such as the envelope VLP (human C5aR2 / VLP) displaying the full-length human C5aR2 in the present invention, or the non-enveloped VLP (human C5aR2 NTD-VLP) displaying the N-terminal sequence of human C5aR2), using C5aR2 antigen-VLP to immunize animals for subsequent screening of monoclonal hybridoma cells, thereby obtaining antibodies capable of binding to the C5aR2 membrane protein.

[0174] The method of the present invention displays the C5aR2 membrane protein antigen epitope on the surface of VLP and expresses it using mammalian cells, so that the antigen or antigen epitope has post-translational modification, and VLP has the function of immune adjuvant, so that the prepared C5aR2 antigen-VLP can more effectively activate immune animals to produce antibodies.

[0175] The main advantages of the present invention include:

[0176] 1. The present invention uses VLP to display membrane proteins or membrane protein epitopes as antigens for immunization to stimulate host immune responses. The present invention displays human C5aR2 membrane proteins or their epitopes multivalently on VLPs and expresses them in mammalian cells, so that the antigens or antigenic epitopes have post-translational modifications; and VLPs have the function of immune adjuvants, and the antigenic epitopes they display can produce better immune responses.

[0177] 2. The present invention uses VLP to display membrane proteins or membrane protein epitopes as antigen screening, and uses VLP without antigen display for reverse screening, thereby obtaining high-affinity specific functional antibodies.

[0178] 3. The mouse monoclonal antibodies 17A2D9 and 30F10E3 screened and prepared by the present invention have human / monkey cross-species activity against C5aR2 receptor membrane protein.

[0179] 4. The mouse monoclonal antibody 17A2D9 of the present invention is obtained by immunization and screening of the full-length C5aR2 membrane protein VLP; the mouse monoclonal antibody 30F10E3 is obtained by immunization and screening of the N-terminal sequence VLP displaying C5aR2, both of which can specifically bind to the C5aR2 membrane protein; and 30F10E3 has the function of competing with the C5a ligand for blocking.

[0180] 5. The mouse monoclonal antibodies 17A2D9 and 30F10E3 of the present invention can be used for Elisa, FACS detection of C5aR2 membrane protein and cell sorting. In addition, the mouse monoclonal antibodies 17A2D9 and 30F10E3 of the present invention have the potential to be used as therapeutic antibodies to treat C5aR2-related diseases after humanization.

[0181] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. Experiments for which specific conditions are not specified in the embodiments or test examples of the present invention are generally carried out under conventional conditions or under conditions recommended by the raw material / commodity manufacturer; reagents for which specific sources are not specified are conventional reagents purchased on the market.

[0182] Example 1 C5aR2 antigen immunization and antiserum detection

[0183] 1.1 C5aR2 antigen immunization: Envelope VLPs displaying the full-length human C5aR2 membrane protein and non-enveloped VLPs displaying the N-terminal sequence (1-38aa, MGNDSVSYEYGDYSDLSDRPVDCLDGACLAIDPLRVAP, SEQ ID NO: 29) of the membrane protein C5aR2 (uniport: Q9P296) were used for immunization. The C5aR2 VLP antigens were expressed by HEK293 mammalian cells. For the first immunization, 50ul of C5aR2 VLP antigen (estimated total protein concentration 0.5mg / ml) was mixed evenly with an equal volume of aluminum hydroxide adjuvant and injected into 6-week-old female Balb / C mice at multiple points in the muscle. The dose of booster immunization was the same as that of the first immunization, and each immunization was separated by two weeks, for a total of five immunizations.

[0184] 1.2 Antiserum titer detection: The plates were coated with full-length human C5aR2 / VLP and human C5aR2 NTD-VLP antigens (2.5ug / ml), the antiserum was diluted 300 times, and then added to the Elisa plate to bind the antigen in a 3-fold gradient dilution. Figure 1 Center A shows the binding of antiserum obtained by immunization with full-length human C5aR2 / VLP to two antigens. Figure 1 B shows the binding of antiserum obtained by immunization with human C5aR2NTD-VLP to two antigens. Both antigen immunizations yielded good antiserum titers, which can be used to further screen specific antibodies that bind to membrane proteins.

[0185] Example 2 Screening of antibodies binding to C5aR2 and antibody sequencing

[0186] 2.1 Antibody screening after immunization with full-length human C5aR2 / VLP

[0187] Before mouse cell fusion, 50ul full-length human C5aR2 / VLP (envelope VLP, estimated total protein concentration 0.5mg / ml) was diluted to 300ul with 1×PBS and injected intraperitoneally for booster immunization. Mouse spleen was mixed with SP2 / 0 cells, and cell fusion was performed with PEG1500 as a fusion agent. After 10 days of culturing the fused cells, 100ul / well (2.5ug / ml) of full-length human C5aR2 / VLP was coated on a polyvinyl chloride Elisa plate, and antigen-binding positive hybridoma polyclones were screened by indirect ELISA. VLPs that do not display membrane proteins were used for counter-screening, and 20ul / ml was coated on a polyvinyl chloride plate. Hybridoma polyclones were negatively screened by indirect ELISA, and hybridoma strains that bind to VLPs were removed to obtain polyclonal hybridoma strains that bind to membrane proteins. After limiting dilution to a monoclonal state, they were expanded and frozen. The supernatant of monoclonal hybridoma cells was repeatedly verified by indirect ELISA, and the 17A2D9 monoclonal hybridoma cell line was screened to be able to specifically bind to human C5aR2 membrane protein.

[0188] 2.2 Antibody Screening Using Human C5aR2 NTD-VLP Immunization

[0189] Before mouse cell fusion, 50ul human C5aR2 NTD-VLP (non-enveloped VLP, estimated total protein concentration 0.5mg / ml) was diluted to 300ul with 1XPBS and injected intraperitoneally for booster immunization. Mouse spleen was mixed with SP2 / 0 cells, and cell fusion was performed using PEG1500 as a fusion agent. After 10 days of culturing the fused cells, human C5aR2 NTD-VLP 100ul / well (2.5ug / ml) was coated on a polyvinyl chloride Elisa plate, and antigen-binding positive hybridoma polyclones were screened by indirect ELISA. Non-enveloped VLP that does not display the NTD epitope was used for counter-screening, and the Elisa plate was coated with 20ul / ml. Hybridoma polyclones were negatively screened by indirect ELISA, and hybridoma strains that bind to VLP were removed to obtain polyclonal hybridoma strains that bind to the membrane protein NTD. Further, human C5aR2 NTD-VLP 100ul / well (2.5ug / ml) was coated on the Elisa plate, and 50ul of the screened positive polyclonal supernatant was mixed with 50ul 20nM biotin-labeled ligand C5a and added to the Elisa plate to compete for binding to humanC5aR2 NTD-VLP. At the same time, the binding of biotin-labeled ligand C5a alone to human C5aR2NTD-VLP was used as a control to screen out 30F10 polyclones that could competitively block the binding of ligands to human C5aR2NTD-VLP. 30F10 polyclones were then limitedly diluted to a monoclonal state and then expanded and frozen. The monoclonal hybridoma cell supernatant was repeatedly verified by indirect ELISA, and the 30F10E3 monoclonal hybridoma cell line was obtained, which could competitively block the binding of ligand C5a to receptor human C5aR2.

[0190] 2.3 Hybridoma monoclonal antibody sequencing and recombinant antibody expression preparation

[0191] The monoclonal hybridoma cell line was cultured to a cell density of 1E+07cell / ml, and hybridoma monoclonal antibody sequencing was performed (Baiying Biotechnology). The heavy chain of the 17A2D9 antibody is IgG2a (SEQ ID NO: 1), and the light chain is mouse Ig kappa (SEQ ID NO: 2). The heavy chain of the 30F10E3 antibody is IgG1 (SEQ ID NO: 3), and the light chain is mouse Ig kappa (SEQ ID NO: 4). Gene synthesis encoding signal peptides, variable regions and constant regions of antibody heavy chain and light chain gene sequences (SEQ ID NO: 25 and 26; SEQ ID NO: 27 and 28) were constructed into mammalian cell expression vectors, respectively. The expression vector can be any commercial mammalian cell expression vector such as pTT5 and pCDNA3.1. The recombinant plasmid was transfected into Expi293 mammalian cells, and the antibody was secreted and expressed. The cell expression supernatant was purified by protein A affinity to obtain the recombinant anti-human C5aR2 antibody.

[0192] Example 3 Recombinant monoclonal antibodies 17A2D9 and 30F10E3 have human / monkey C5aR2 cross-species activity

[0193] Human C5aR2 / VLP (envelope VLP) and cynomolgus monkey (Cyno) C5aR2 / VLP (envelope VLP) were coated on Elisa plates at 2.5ug / ml, respectively. Recombinant 17A2D9 and 30F10E9 monoclonal antibodies (120nM) were diluted 3-fold and combined with C5aR2 / VLP for Elisa.

[0194] The results are as follows Figure 2 As shown in Figure A, the 17A2D9 and 30F10E9 recombinant antibodies can bind to human C5aR2. Figure 2 Middle B shows that the 17A2D9 and 30F10E9 recombinant antibodies can bind to cynomolgus monkey C5aR2 (uniport: F7H5Z8), indicating that both 17A2D9 and 30F10E9 antibodies have cross-reactivity between humans and cynomolgus monkeys.

[0195] Example 4 Competitive Binding of Recombinant Monoclonal Antibodies 17A2D9 and 30F10E3 to C5a Ligand

[0196] Human C5aR2 NTD-VLP (non-enveloped VLP) was coated at 2.5ug / ml, and recombinant 17A2D9, 30F10E9 monoclonal antibodies (120nM) and biotin-labeled ligand C5a (100nM) were combined with C5aR2 NTD-VLP in 3-fold gradient dilutions for Elisa. Figure 3It showed that both recombinant antibodies and the ligand C5a could bind to human C5aR2 NTD, and the recombinant 30F10E3 monoclonal antibody bound to the C5aR2 N-terminal epitope with high affinity.

[0197] Human C5aR2 NTD-VLP (non-enveloped VLP) was coated at 1ug / ml, and recombinant 17A2D9 and 30F10E9 monoclonal antibodies (120nM) were diluted two-fold and mixed with 50nM biotin-labeled ligand C5a, and then added to the coated Elisa plate to bind to human C5aR2 NTD-VLP for Elisa. Figure 4 The results showed that the recombinant 30F10E3 monoclonal antibody could competitively block the binding of the ligand C5a to the human C5aR2 NTD, while the recombinant 17A2D9 monoclonal antibody had no effect on the C5a ligand binding to the N-terminal epitope, indicating that 17A2D9 and 30F10E3 bind to different epitopes.

[0198] Example 5 FACS flow cytometric detection of the binding of recombinant monoclonal antibodies to Hela cells and their competitive binding with C5a ligands

[0199] Hela cells overexpress C5aR2 membrane protein. Hela cells were prepared into single cell suspension (3% FBS, PBS) with a cell density of 2E+06 / ml. Antibodies 17A2D9, 30F10E3, and biotin-labeled C5a ligand were diluted to 0.1uM and combined with Hela cells, and then allowed to stand at 4°C for 30 minutes. FITC-labeled goat anti-mouse (binding to mouse monoclonal antibodies 17A2D9 and 30F10E3) and SA-FITC (binding to biotin-labeled C5a ligand) were added and incubated at 4°C for 30 minutes. Hela cells were washed three times with 1×PBS and then gently blown to form single cells. FACS analysis was performed as antibody-cell binding and ligand-cell binding experimental groups. The other group was the antibody-ligand competitive binding group. The 17A2D9 and 30F10E3 recombinant antibodies were mixed with the biotin-labeled C5a ligand to a final concentration of 0.1uM, mixed with Hela cells, and allowed to stand at 4°C for 30 minutes. SA-FITC was then added and allowed to stand at 4°C for 30 minutes. The Hela cells were washed three times with PBS and then gently blown to form single cells, and analyzed on a FACS machine.

[0200] Figure 5 Curve 1 in both A and B is Hela cells (negative control); Figure 5 Curve 2 in center A shows binding of 17A2D9 to Hela cells; Figure 5 Curve 2 in center B shows binding of 30F10E3 to Hela cells; Figure 5 The curve 3 of A in the middle, Figure 5Curve 3 in B shows that the biotin-labeled C5a ligand binds to Hela cells (positive control); Figure 5 Curve 4 in A shows that the 17A2D9 recombinant antibody cannot block the binding of the ligand C5a to the C5aR2 receptor on Hela cells; Figure 5 Curve 4 in B shows that the 30F10E3 recombinant antibody can competitively block the binding of biotin-labeled C5a ligand to the C5aR2 receptor on Hela cells.

[0201] References

[0202] 1.Xaria

[0203] 2.Megan Cul ly.,Fibroblast subtype provides niche for cancer stemcells.Nature Reviews Cancer.18,136(2018).

[0204] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. An anti-C5aR2 antibody or an antigen-binding fragment thereof, characterized in that: The anti-C5aR2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region selected from the group consisting of: (Z1) the heavy chain variable region comprises the following three CDRs: H-CDR1 as shown in SEQ ID NO:7; H-CDR2 as shown in SEQ ID NO:8; H-CDR3 as shown in SEQ ID NO:9; and The light chain variable region comprises the following three CDRs: L-CDR1 as shown in SEQ ID NO: 13; L-CDR2 as shown in GIS; L-CDR3 as shown in SEQ ID NO: 14; or (Z2) the heavy chain variable region comprises the following three CDRs: H-CDR1 as shown in SEQ ID NO: 17; H-CDR2 as shown in SEQ ID NO: 18; H-CDR3 as shown in SEQ ID NO: 19; and The light chain variable region comprises the following three CDRs: L-CDR1 shown in SEQ ID NO:23; L-CDR2 whose sequence is shown in LVS; and L-CDR3 shown in SEQ ID NO:

24.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The heavy chain variable region of the antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 90% (preferably at least 95%, 96%, 97%, 98%, 99%) sequence identity thereto; and / or The light chain variable region of the antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 12, or an amino acid sequence having at least 90% (preferably at least 95%, 96%, 97%, 98%, 99%) sequence identity thereto.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein: The heavy chain variable region of the antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 16, or an amino acid sequence having at least 90% (preferably at least 95%, 96%, 97%, 98%, 99%) sequence identity thereto; and / or The light chain variable region of the antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 22, or an amino acid sequence having at least 90% (preferably at least 95%, 96%, 97%, 98%, 99%) sequence identity thereto.

4. A recombinant antibody, characterized in that: The recombinant antibody has: (i) the sequence of the anti-C5aR2 antibody or antigen-binding fragment thereof according to claim 1; and (ii) a signal peptide that promotes secretory expression of the antibody and / or a tag sequence for purification and detection.

5. The recombinant antibody according to claim 4, characterized in that The recombinant antibody comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO:1, and a light chain having an amino acid sequence as shown in SEQ ID NO:

2.

6. The recombinant antibody according to claim 4, characterized in that The recombinant antibody comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO:3, and a light chain having an amino acid sequence as shown in SEQ ID NO:

4.

7. A polynucleotide molecule encoding a polypeptide selected from the group consisting of: (1) the anti-C5aR2 antibody or antigen-binding fragment thereof according to claim 1; or (2) The recombinant antibody according to claim 4.

8. An antibody conjugate, characterized in that: The antibody conjugate comprises: (a) the anti-C5aR2 antibody or antigen-binding fragment thereof according to claim 1 or the recombinant antibody according to claim 4; and (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, an enzyme, a cytokine, a radionuclide, and a nanoparticle / nanorod.

9. Use of the anti-C5aR2 antibody or antigen-binding fragment thereof according to claim 1, the recombinant antibody according to claim 4, or the antibody conjugate according to claim 8 for preparing: A detection reagent, a detection plate or a detection kit for detecting C5aR2 molecules.

10. A method for screening anti-C5aR2 antibodies, comprising the steps of: (S1) using VLP to multivalently display C5aR2 protein or C5aR2 protein epitope, and expressing it in mammalian cells to obtain C5aR2 antigen-VLP for immunization; (S2) immunizing a test animal with the C5aR2 antigen-VLP prepared in step (S1), and collecting antiserum from the immunized animal; (S3) detecting the binding of the collected antiserum to the C5aR2 antigen-VLP. If the binding is positive, taking the spleen of the immunized animal to prepare hybridoma clones; (S4) using the C5aR2 antigen-VLP to perform preliminary screening on hybridoma clones, thereby obtaining antigen-binding positive hybridoma clones; (S5) using empty VLPs without C5aR2 antigen to further perform negative screening on clones that are positive for antigen binding, and screen out positive hybridoma clones that only bind to C5aR2 antigen; (S6) The positive hybridoma clones that only bind to the C5aR2 antigen are limitedly diluted to a monoclonal state, and the binding of the monoclonal hybridoma cell supernatant to the C5aR2 antigen is repeatedly verified, thereby obtaining a monoclonal hybridoma cell line that can produce anti-C5aR2 antibodies that specifically bind to the C5aR2 antigen.

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